WO2009108935A2 - Method and apparatus for impregnating porous biomaterials with bioactive agents - Google Patents

Method and apparatus for impregnating porous biomaterials with bioactive agents Download PDF

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Publication number
WO2009108935A2
WO2009108935A2 PCT/US2009/035731 US2009035731W WO2009108935A2 WO 2009108935 A2 WO2009108935 A2 WO 2009108935A2 US 2009035731 W US2009035731 W US 2009035731W WO 2009108935 A2 WO2009108935 A2 WO 2009108935A2
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WO
WIPO (PCT)
Prior art keywords
calcium phosphate
bone
implant
cavity
chamber
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PCT/US2009/035731
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French (fr)
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WO2009108935A3 (en
Inventor
Mark Shuster
Hugo Pedrozo
Kevin Brand
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Osteotherapeutics, L.L.C.
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Application filed by Osteotherapeutics, L.L.C. filed Critical Osteotherapeutics, L.L.C.
Publication of WO2009108935A2 publication Critical patent/WO2009108935A2/en
Publication of WO2009108935A3 publication Critical patent/WO2009108935A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A61L24/0015—Medicaments; Biocides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A61L24/0036—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/02—Surgical adhesives or cements; Adhesives for colostomy devices containing inorganic materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/12—Phosphorus-containing materials, e.g. apatite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
    • A61L2300/602—Type of release, e.g. controlled, sustained, slow
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions

Definitions

  • TITLE METHOD AND APPARATUS FOR IMPREGNATING POROUS BIOMATERIALS WITH BIOACTIVE AGENTS
  • the present invention generally relates to systems and methods for the preparation of biocompatible porous materials suitable for use as pharmaceutical carriers. More specifically, the invention relates to systems and methods for impregnating a porous biomaterial with pharmaceutical composition.
  • the present invention refers to the use of injectable calcium phosphate cements (CPCs) as growth pharmaceutical carriers for bioactive/therapeutic compositions.
  • CPCs calcium phosphate cements
  • the growth factors/cement composites are injectable and have selected setting times and compression strength which render them suitable for use as bone void filler.
  • the growth factors/cement composite can also deliver growth factors to defect site and stimulate bone healing.
  • Calcium phosphate ceramics particularly hydroxyapatite (HA) and beta-tricalcium phosphate (TCP), have been reported to promote bone ingrowth and are reabsorbed by the host tissue. Moreover, they have been used as carriers for the sustained release of various therapeutic agents, such as growth factors (see, e.g., Uchida et al, J. Orthoptera Res. 10:440 (1992), Matsumoto et al, Biomaterials 25:3807 (2004), Paul et al, J. Biomater. Appl. 17:253 (2003)). Proteins can be favorably adsorbed on the calcium phosphate ceramics because of electrostatic interactions and delivered into a bony defect where they serve as osteoinductive agents.
  • HA hydroxyapatite
  • TCP beta-tricalcium phosphate
  • Injectable calcium phosphate cements are a desirable and less invasive means to treat bony defects caused by trauma or abnormal metabolic bone conditions such as osteoporosis.
  • growth factors to such preparations has been unsuccessful to date.
  • the major issues associated with injectable formulations of calcium phosphate are entrapment of the growth factor resulting in no release and aggregation or denaturation of the growth factor within the setting solution that renders the protein biologically inactive. Decreasing the interaction between the growth factor and the hydroxyapatite surface and increasing the surface area by using porogens or biodegradable polymers increases the release rate and enables controlled release kinetics of growth factors.
  • U.S. Patent Application Serial No. 2005/0170070 by Layrolle et al. entitled "Method for applying a bioactive coating on a medical device” describes ceramic coatings containing bioactive agents formed on the surfaces of medical devices made of inorganic, metallic or organic materials, and methods and systems for making same. The coatings are deposited on the implant surface by passing the implant through a stream of a coating solution in a reactor system.
  • U.S. Patent Application Serial No. 2005/0031704 by Ahn et al. entitled “Tricalcium phosphates, their composites, implants incorporating them, and method for their production” describes bioceramics, particularly tricalcium phosphate bioceramics, composites incorporating these materials, and methods for their production.
  • the surface of a calcium phosphate powder such as TCP or hydroxyapatite may contain therapeutic compositions (e.g., nucleic acids, proteins, or antibiotics) for drug delivery.
  • U.S. Patent Application Serial No. 20050119761 by Matsumoto et al. entitled “Porous calcium phosphate ceramic and method for producing same” describes sintered calcium phosphate ceramics with macroporosity for use in medical applications. The ceramic is capable of binding polypeptides.
  • U.S. Patent Application Serial No. 20040091544 by Ruff et al. entitled “Coated dibasic calcium phosphate” describes dibasic calcium phosphate coatings as pharmaceutical carriers for sustained release of orally administered peptides.
  • U.S. Patent No. 5,769,897 to Harle entitled “Synthetic bone,” discloses a method of manufacturing synthetic bone. Further, it discloses a vacuum vessel for treating, storing and transporting the artificial bone material according to the invention prior to implantation.
  • U.S. Pat. No. 5,037,377 to Alonso entitled “Means for improving biocompatibility of implants, particularly of vascular grafts,” discloses a means for improving biocompatibility of implants.
  • a method of applying soluble collagen to vascular grafts is achieved by vacuum impregnation and impregnation under pressure. The grafts are kept in a vessel and which is evacuated to obtain a pressure differential of 120mmHg (0.84atm).
  • a porous biomaterial e.g., a medical implant
  • a liquid pharmaceutical carrier medium carrying one or more bioactive compositions.
  • a device suitable for perioperatively impregnating a biomaterial with a pharmaceutical agent is provided for.
  • the device may be operated at a point of care setting.
  • the device may be operated with a single-hand motion.
  • a system for impregnating a porous biomaterial with a bioactive composition may include a cassette portion.
  • the cassette portion may include a body; a cavity within the body, the cavity being sized to accept a porous biomaterial; and an opening from the cavity to a surface of the body.
  • the cassette portion may include a barrel of a syringe.
  • the system may include a sealing portion operatively couplable to the cassette portion.
  • the sealing portion may be configured such that, when coupled to the cassette portion, the opening of the cavity may be sealed to form a substantially airtight chamber disposed in the cassette body.
  • a sealing portion may include a plunger of a syringe.
  • the system may further include an actuator operatively coupled to the sealing portion.
  • the actuator may be configured to reversibly apply a vacuum to the chamber.
  • the actuator may include a squeeze trigger.
  • the system may further include a porous biomaterial configured to reside within the chamber formed by coupling the cassette potion to the sealing portion. The volume of the porous biomaterial may be less than the volume of the chamber.
  • the system may further include a pharmaceutical carrier liquid.
  • the pharmaceutical carrier liquid may include a physiologically acceptable liquid.
  • the pharmaceutical carrier liquid may further include a bioactive composition.
  • the volume of pharmaceutical carrier liquid may be sufficient to occupy at least a portion of the chamber volume not occupied by the porous biomaterial.
  • the volume of pharmaceutical carrier liquid may be sufficient to allow at least a portion of the surface of a porous biomaterial residing in the chamber to contact the pharmaceutical carrier liquid.
  • a method of applying a pharmaceutical composition to a porous biomaterial may include providing a system comprising a cassette portion, the cassette portion comprising a body; a cavity within the body, the cavity being sized to accept a porous biomaterial; and an opening from the cavity to a surface of the body; a sealing portion operatively couplable to the cassette portion, the sealing portion being configured such that, when coupled to the cassette portion, the opening of the cavity is sealed to form a substantially airtight chamber disposed in the cassette body; and an actuator operatively coupled to the sealing portion, the actuator being configured to reversibly apply a vacuum to the chamber when engaged; providing a porous biomaterial to the cavity of the system; providing a pharmaceutical carrier liquid comprising a bioactive composition to the cavity of the system; coupling the cassette portion to the sealing portion, thereby sealing the porous biomaterial and the pharmaceutical carrier liquid in the chamber; and applying a vacuum to the chamber by engaging the actuator.
  • a vacuum applied to the chamber may be sufficient to achieve a chamber pressure in the range of 0.1 to about 1.0 atm below ambient pressure. In an embodiment, a vacuum may be maintained for up to about lminute. In an embodiment, the step of applying a vacuum to the chamber by engaging the actuator is performed up to about 3 times or more.
  • FIGS. IA-B show S.E.M. images (taken at 10,000-fold magnification) of implant surfaces having nanoporous nanocrystalline calcium phosphate material made by soaking the implant in;
  • Fig. IA Hank's Balanced Salt Solution (with Ca and Mg) for 3 days;
  • Fig. IB phosphate buffered saline for 5 days;
  • FIG. 2 is a graph depicting the enhanced rate of growth factor (BMP) release from hardened CPC materials in the presence of amino acids
  • FIG. 3 is a graph depicting the enhanced rate of growth factor release from hardened CPC materials in the presence of certain polymer and amino acid/polymer combinations. While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will herein be described in detail. It should be understood, however, that the drawing and detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the intention is to cover all modifications, equivalents and alternatives falling within the spirit and scope of the present invention as defined by the appended claims.
  • a material, composition or object that is “bioresorbable,” generally refers to a biocompatible material, composition or object that has the ability to be gradually integrated into a host.
  • the term generally refers to the ability of at least a portion of the prosthetic bone implant to gradually be replaced by natural bone, such replacement typically occurring naturally by the physiological process of bone remodeling.
  • materials that may be considered to be bioresorbable include certain calcium phosphate ceramics (including but not limited to, for example, hydroxyapatite), collagen, tendon, and grafted bone material.
  • biodegradable generally refers to a substance or a composition that can be at least partially broken down in the body or by microorganisms into a simpler substance. While not necessarily being mutually exclusive, the term “biodegradable,” is generally not used interchangeably with “bioresorbable.” Whereas a biodegradable material may be broken down into its constituent materials, such materials, unlike a bioresorbable material, are not integrated into the host and are released locally or systemically.
  • biodegradable polymer is generally employed herein to denote a (generally biocompatible, non-toxic) polymeric material that gradually degrades or erodes in vivo to smaller polymer units and/or its substituent monomers. Such degradation may be the result of, for example, enzymatic, chemical and/or physical processes.
  • biodegradable polymers suitable for the methods and compositions contemplated herein herein include, for example, poly(lactides), polycaprolactic acid, poly(glycolides), poly(lactide-co- glycolides), poly(gamma-glutamic acid)- sulfonate, polyketals, poly(lactic acid)s, poly(glycolic acid)s, polycarbonates, polyesters, polyesteramides, polyanydrides, poly(amino acids) e.g., polypeptides, polyorthoesters, poly(dioxanone)s, poly(alkylene alkylate)s, copolymers or polyethylene glycol and polyorthoester, biodegradable polyurethane, chitosan, or blends or copolymers thereof.
  • poly(lactides) polycaprolactic acid
  • poly(glycolides) poly(lactide-co- glycolides)
  • biocompatible polymer systems such as hydrogels.
  • polymer systems associated with a particular polymer including but not limited to those systems incorporating, for example, various modifying agents, additives, cross-linkers, and the like.
  • biodegradable polymer e.g., PLGA which breaks down into lactic acid and glycolic acid
  • terms such as "pharmaceutical carrier,” “pharmaceutical composition,” “pharmaceutical formulation,” “pharmaceutical preparation,” or the like generally refer to formulations that are adapted to deliver a prescribed dosage of one or more pharmacologically active compounds to a cell, a group of cells, an organ or tissue, an animal or a human.
  • the determination of an appropriate prescribed dosage of a pharmacologically active compound to include in a pharmaceutical composition in order to achieve a desired biological outcome is within the skill level of an ordinary practitioner of the art.
  • a pharmaceutical composition may be provided as sustained-release or timed-release formulations.
  • Such formulations may release a bolus of a compound from the formulation at a desired time, or may ensure a relatively constant amount of the compound present in the dosage is released over a given period of time.
  • Terms such as “sustained release,” “controlled release,” or “timed release” and the like are widely used in the pharmaceutical arts and are readily understood by a practitioner of ordinary skill in the art.
  • Pharmaceutical preparations may be prepared as solids, semi-solids, gels, hydrogels, liquids, solutions, suspensions, emulsions, aerosols, powders, or combinations thereof.
  • a pharmaceutical preparation may be one or more carriers, preservatives, flavorings, excipients, coatings, stabilizers, binders, solvents and/or auxiliaries that are, typically, pharmacologically inert. It will be readily appreciated by an ordinary practitioner of the art that, included within the meaning of the term are pharmaceutically acceptable salts of compounds. It will further be appreciated by an ordinary practitioner of the art that the term also encompasses those pharmaceutical compositions that contain an admixture of two or more pharmacologically active compounds, such compounds being administered, for example, as a combination therapy.
  • subject generally refers to a mammal, and in particular to a human.
  • terapéuticaally effective amount and “effective amount” are synonymous unless otherwise indicated, and mean an amount of a compound of the present invention that is sufficient to improve the condition, disease, or disorder being treated. Determination of a therapeutically effective amount, as well as other factors related to effective administration of a compound of the present invention to a patient in need of treatment, including dosage forms, routes of administration, and frequency of dosing, may depend upon the particulars of the condition that is encountered, including the patient and condition being treated, the severity of the condition in a particular patient, the particular compound being employed, the particular route of administration being employed, the frequency of dosing, and the particular formulation being employed. Determination of a therapeutically effective treatment regimen for a patient is within the level of ordinary skill in the medical or veterinarian arts.
  • an effective amount may be the amount that is recommended by the U.S. Food and Drug Administration, or an equivalent foreign agency.
  • the amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the mammalian host treated and the particular mode of administration.
  • prophylactically effective amount is meant an amount of a pharmaceutical composition that will substantially prevent, delay or reduce the risk of occurrence of the biological or physiological event in a cell, a tissue, a system, animal or human that is being sought by a researcher, veterinarian, physician or other caregiver.
  • pharmaceutically inert generally refers to a compound (e.g., additives, excipients, binders, vehicles, and the like) that is substantially free of any pharmacologic or "drug-like" activity.
  • bone substitute material generally refers to any biocompatible composition, including but not limited to natural bone graft, particulate or crushed bone, demineralized bone matrix, ceramics, polymer systems, composites, or mixtures thereof, that is suitable for use in medical/dental applications characterized by replacement, augmentation, or filling of a hardened calcified tissue (e.g., bone, teeth, enamel and the like).
  • a hardened calcified tissue e.g., bone, teeth, enamel and the like.
  • the term refers to calcium phosphate-based ceramic materials (such as cements) that form hardened structures having physical and chemical properties approximating those of natural bone mineral.
  • CPC calcium phosphate cement
  • CPC particles may have an average diameter in the range of about 0.1 ⁇ m to about 500 ⁇ m, in the range of about 0.5 ⁇ m to about 10 ⁇ m, or in the range of about 0.05 ⁇ m to about 50 ⁇ m.
  • a variety of CPCs are known in the art and detailed descriptions thereof, as well as their method of manufacture may be found, for example, in the disclosures of U.S. Patent Nos.
  • CPC particles may refer to particles of one or more of alpha- tricalcium phosphate ( ⁇ -TCP), beta-tricalcium phosphate ( ⁇ -TCP), tetracalcium phosphate (TTCP), monocalcium phosphate monohydrate (MCPM), monocalcium phosphate anhydrous (MCPA), dicalcium phosphate dihydrate (DCPD), dicalcium phosphate anhydrous (DCPA), octacalcium phosphate (OCP), calcium dihydrogen phosphate, calcium dihydrogen phosphate hydrate, acid calcium pyrophosphate, anhydrous calcium hydrogen phosphate, calcium hydrogen phosphate hydrate, calcium pyrophosphate, calcium triphosphate, calcium phosphate tribasic, calcium polyphosphate, calcium metaphosphate, anhydrous tricalcium phosphate, tricalcium phosphate hydrate, and amorphous calcium phosphate.
  • ⁇ -TCP alpha- tricalcium phosphate
  • ⁇ -TCP beta-tricalcium phosphate
  • the hardened material formed using CPC particles may be substantially composed of an apatite material, e.g., hydroxyapatite.
  • CPCs may optionally include one or more additives that affect the physico-chemical properties of hardened cements made therewith.
  • additives which may find use in certain embodiments include those additives selected from the group consisting of sodium phosphate (Na 3 PO 4 ), disodium hydrogen phosphate (Na 2 HPO 4 ), sodium dihydrogen phosphate (NaH 2 PO 4 ), disodium hydrogen phosphate dodecahydrate (Na 2 HPO 4 - 12H 2 O), disodium hydrogen phosphate heptahydrate (Na 2 HPO 4 -7H 2 0), sodium phosphate dodecahydrate (Na 3 PO4 12H 2 O), orthophosphoric acid (H 3 PO 4 ), calcium sulfate (CaSO 4 ), Ca 4 (PO 4 ) 2 O, CaHPO 4 -2H 2 O, CaHPO 4 , Ca 8 H 2 (PO 4 ) 6 -5H 2 O, alpha-Ca 3 (PO 4 ) 2 , beta-Ca 3 (PO 4 ) 2 , Ca 2 P 2 O 7 , and Ca 2 H 2 P 2 O 8 , (NH), sodium phosphate
  • setting liquid generally refer to liquid compositions that, when contacted with cement particles, allow cementing reactions to occur between said particles.
  • Exemplary setting liquids suitable for use in the presently disclosed embodiments include, though are not limited to, acidic solutions, basic solutions, solutions having substantially physiological pH (e.g., 6.0-8.5) or substantially pure water.
  • Suitable acidic solutions may include solutions containing nitric acid (HNO 3 ), hydrochloric acid (HCl), phosphoric acid (H 3 PO 4 ), carbonic acid (H 2 CO 3 ), sodium dihydrogen phosphate (NaH 2 PO 4 ), sodium dihydrogen phosphate monohydrate (NaH 2 PO ⁇ H 2 O), sodium dihydrogen phosphate dihydrate, sodium dihydrogen phosphate dehydrate, potassium dihydrogen phosphate (KH 2 PO 4 ), ammonium dihydrogen.phosphate (NH 4 H 2 PO 4 ), malic acid, acetic acid, lactic acid, citric acid, malonic acid, succinic acid, glutaric acid, tartaric acid, oxalic acid and their mixture.
  • HNO 3 nitric acid
  • HCl hydrochloric acid
  • H 3 PO 4 phosphoric acid
  • carbonic acid H 2 CO 3
  • sodium dihydrogen phosphate NaH 2 PO 4
  • sodium dihydrogen phosphate monohydrate NaH 2 PO
  • Suitable basic solutions may include solutions containing ammonia, ammonium hydroxide, alkali metal hydroxide, alkali earth hydroxide, disodium hydrogen phosphate (Na 2 HPO 4 ), disodium hydrogen phosphate dodecahydrate, disodium hydrogen phosphate heptahydrate, sodium phosphate dodecahydrate (Na 3 PO 4 * 12H 2 O), dipotassium hydrogen phosphate (K 2 HPO 4 ), potassium hydrogen phosphate trihydrate (K 2 HP ⁇ 4 # 3H 2 O), potassium phosphate tribasic (K 3 PO 4 ), diammonium hydrogen phosphate ((NH 4 ) 2 HPO 4 ), ammonium phosphate trihydrate ((NH 4 ) 3 P ⁇ 4 # 3H 2 O), sodium hydrogen carbonate (NaHCO 3 ), sodium carbonate Na2CO 3 , and their mixture.
  • solutions having substantially physiological pH include solutions containing phosphate ions, Tris-buffered solution, HEPES -buffered solutions, Hank's
  • injectable self-setting calcium phosphate cement paste generally refers to a composition resulting contacting calcium phosphate cement particles with a setting solution in a ratio such that the resulting mixtures is sufficiently viscous to allow the injection thereof through a standard medical syringe. Such a mixture may typically be obtained by mixing the CPC particles and the setting solution in a ratio of about 0.1 mg/ml to about 20 mg/ml.
  • self-setting calcium phosphate cement pastes suitable for use herein are described in U.S. patent application No. 20030216777 entitled "Method of enhancing healing of interfacial gap between bone and tendon or ligament," U.S. patent application No.
  • porogen generally refers to any particulate non-toxic biocompatible material that may be incorporated into an injectable CPC formulation and that, upon hardening of said CPC formulation to a hardened CPC, is gradually removed from the hardened CPC matrix (by virtue of its bioresorbability, biodegradability, solubility etc.) leaving a void therein.
  • a porogen may have any 3-dimensional shape, e.g., substantially spherical, substantially cuboidal, substantially cylindrical, substantially pyramidal, substantially ovoid, or irregular in shape. Typically, a dimension of a porogen will be in the range of about 0.01 ⁇ m to about 1 mm.
  • Porogens suitable for use in the present embodiments include crystalline materials (e.g., salts such), polypeptides, and polymer compositions.
  • microspheres generally refers to particles that are substantially spherical or ovoid in shape and have an average mid- sectional diameter that is less than about 2 mm, less than about 1 mm, less than about 0.5 mm, less than about 250 ⁇ m, less than about 100 ⁇ m, less than about 50 ⁇ m, less than about 20 ⁇ m, less than about 10 ⁇ m, less than about 54 ⁇ m, less than about 1 ⁇ m or less than about 0.5 ⁇ m.
  • microfibers generally refers to fibers that are that have an average mid-sectional diameter that is less than about 2 mm, less than about 1 mm, less than about 0.5 mm, less than about 250 ⁇ m, less than about 100 ⁇ m, less than about 50 ⁇ m, less than about 20 ⁇ m, less than about 10 ⁇ m, less than about 54 ⁇ m, less than about 1 ⁇ m or less than about 0.5 ⁇ m.
  • interconnected porosity generally refers to pores or cavities in the body or matrix of the subject prosthetic bone implants that are in fluid communication and that form a continuous network of pores capable of conveying liquids or gases, or materials dissolved therein.
  • the amount of interconnected porosity of the subject implants is related to the bioresorbability thereof.
  • pore throat diameter generally refers to the size or diameter of the openings between adjacent pores, or between a pore and the implant surface.
  • non-dispersible when used in the context of the presently described calcium phosphate cements, generally refers to a physical property of the cement whereby a paste made by combining the cement powder with a setting liquid resists dispersion in an aqueous environment. The ability of a calcium phosphate cement paste to resist dispersion may be related to the surface structure of its constituent particles.
  • nanocrystalline generally refers to a ceramic material whose polycrystalline grain structure is reduced from the micron range to the nanometer range. The surface of a nanocrystalline ceramic has physico-chemical properties that distinguish its polycrystalline counterpart and may make it more receptive to binding certain molecules and ions. Nanocrystalline calcium phosphate may be formed through the crystallization of amorphous calcium phosphate.
  • nanoporous generally refers to a porous material (i.e. a calcium phosphate ceramic) whose average pore diameter is in the nanometer range (typically between 1 to 1000 nm).
  • wicking generally refers to the ability of a porous calcium phosphate article to convey liquid by capillary action.
  • cortical portion when used in the context of the subject prosthetic bone implants, generally refers to a portion of the prosthetic bone implant that functions in a load-bearing capacity and whose function and structure are substantially similar to that of naturally occurring cortical or compact bone.
  • the terms "cancellous portion,” or “cancellous” when used in the context of the subject prosthetic bone implants generally refer to portions of the subject prosthetic bone implants that are more porous than the cortical portions, and whose structure and function of which are substantially similar to that of naturally occurring trabecular or spongy bone. Due to its high degree of porsity, a cancellous portion has a relatively high surface area and can support tissue ingrowth and infiltration of body fluids and cells. A cancellous portion may also increase the wicking profile of a prosthetic bone implant.
  • apatite generally refers to a group of phosphate minerals, (typically to hydroxyapatite, fluorapatite, and chlorapatite) having the general chemical formula Ca 5 (PO 4 ) S X, where X is OH, F, or Cl.
  • hydroxyapatite sometimes referred to as “HA” or “HAp,” as used herein generally refers to a form of apatite with the formula Ca 5 (PO 4 ) S (OH), but is more typically represnted as Ca 10 (PO 4 ) 6 (OH) 2 to denote that the crystal unit cell comprises two molecules. Hydroxyapatite is the hydroxylated member of the complex apatite group.
  • hydroxyapatite may be altered by replacing the OH ion with other anions (e.g., fluoride, chloride or carbonate). Additionally, HAp has a relatively high affinity for peptides, making it an ideal carrier for the delivery and/or sustained release of polypeptides over long periods of time in situ.
  • Materials that are refered to herein as "apatitic,” are generally those materials that have apatite as the major phase (i.e., materials that are substantially comprised of apatite).
  • porous biomaterial when used in the context of an implantable medical article (e.g., a material suitable for implantation in a calcified tissue) generally refers to a hardened calcium phosphate cement (CPC) having 2 vol% porosity or greater.
  • CPC calcium phosphate cement
  • crystalline is an art-recognized term that is used to describe a mineral composition having relatively a well-defined crystal structure, with a unique arrangement of atoms within the component crystals. There are at least 7 art-recognized crystals systems. Pure hydroxyapatite typically crystallizes in the hexagonal crystal system, although alternate crystal structures may be realized by altering the composition of the mineral.
  • amorphous when used in the context of mineral compositions, generally refers to a relatively unstructured, non-crystalline form of a mineral that is capable of acting as a seed and support for the growth of crystals thereon.
  • terms such as “bioactive composition,” “bioactive agent” or the like generally refer to compositions or agents that are capable of inducing or affecting a biological response action in a biological system, e.g. by inducing or affecting a therapeutic or prophylactic effect, an immune response, tissue growth, cell growth, cell differentiation, cell proliferation, etc.
  • a bioactive composition/agent may be provided by means of a suitable pharmaceutical delivery vehicle.
  • the delivery vehicle would typically be optimized to stably accommodate an effective dosage of one or more compounds having biological activity.
  • the determination of the effective dose of a bioactive compound that should be included in a bioactive composition to achieve a desired biological response is dependent on the particular compound, the magnitude of the desired response, and the physiological context of the composition.
  • the determination of the effective dose range of a particular bioactive compound or composition is within the skill level of an ordinary practitioner of the art.
  • Components of bioactive compositions may include, by way of non-limiting example, growth factors, bone proteins, analgesics, antibiotics, or other pharmacologically active compounds.
  • a composition that is referred to as being “physiologically acceptable” is a composition that is non-toxic, biocompatible and whose physical and chemical features (e.g., pH, osmolarity, temperature, and the like) fall within a range that is substantially unlikely to induce or be the cause of adverse physiological responses (e.g., inflammation, hypersensitivity, toxicity, and the like).
  • a "physiologically acceptable" aqueous solution will typically have a pH in the range of about 6.0 to about 8.5, in the range of about 7.0 to 8.0, or in the range of about 7.2 to about 7.6.
  • Such a solution will typically have an osmolarity in the range of about 200 to about 500 mOsmol/L, about 250 to about 350 mOsmol/L or about 280 to about 310 mOsmol/L.
  • physiological acceptable salts is generally meant to encompass those salts, as well as aqueous solutions made therefrom, having the chemical and biological properties described above.
  • excipient is a term of the pharmaceutical arts that generally refers to a pharmacologically inert substance or composition that serves as a delivery vehicle or carrier medium for a drug or bioactive composition.
  • An excipient may include one or more binders, stabilizers, fillers, lubricants, preservatives and the like.
  • compositions or agents that serve as excipient are known in the art and include, by way of non-limiting example only, certain polymers, small carbohydrates, amphiphilic molecules.
  • agents that may serve as excipient in the presently disclosed embodiments may include, for example amino acids (e.g. glutamic acid), physiological acceptable salts, sodium phosphates, and small polypeptides. Nevertheless, it will readily be appreciated by an ordinary practitioner of the art that various other excipients may be employed during the practice of the invention without departing from the spirit and scope of the embodiments described herein.
  • an osteoinductive composition generally refers to a composition that induces and/or supports the formation, development and growth of new bone, and/or the remodeling of existing bone.
  • An osteoinductive composition typically includes one or more osteogenic agents.
  • An "osteogenic agent,” as used herein, is an agent that can elicit, facilitate and/or maintain the formation and growth of bone tissue. Many osteogenic agents function, at least in part, by stimulating or otherwise regulating the activity of osteoblast and/or osteoclasts.
  • osteogenic agents include certain polypeptide growth factors, such as, osteogenin, Insulin-like Growth Factor (IGF)-I, IGF-II, TGF- ⁇ l, TGF- ⁇ 2, TGF- ⁇ 3, TGF- ⁇ 4, TGF- ⁇ 5, osteoinductive factor (OIF), basic Fibroblast Growth Factor (bFGF), acidic Fibroblast Growth Factor (aFGF), Platelet-Derived Growth Factor (PDGF), vascular endothelial growth factor (VEGF), Growth Hormone (GH), growth and differentiation factors (GDF)-5 through 9, osteogenic protein- 1 (OP-I) and any one of the many known bone morphogenic proteins (BMPs), including but not limited to BMP-I, BMP-2, BMP-2A, BMP-2B, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-8b, BMP-9, BMP-IO, BMP-Il, BMP- 12, BMP-13, BMP-14
  • An osteoinductive composition may include one or more agents that support the formation, development and growth of new bone, and/or the remodeling thereof.
  • Typical examples of compounds that function in such a supportive manner include, though are not limited to, extracellular matrix-associated bone proteins (e.g., alkaline phosphatase, osteocalcin, bone sialoprotein (BSP) and osteocalcin in secreted phosphoprotein (SPP)-I, type I collagen, fibronectin, osteonectin, thrombospondin, matrix-gla-protein, SPARC, alkaline phosphatase and osteopontin).
  • the term "growth factor” generally refers to a factor, typically a polypeptide, which affects some aspect of the growth and/or differentiation of cells, tissues, organs, or organisms.
  • BMP bone morphogenic protein
  • TGF- ⁇ superfamily polypeptide growth factors belonging to the TGF- ⁇ superfamily. While BMPs are widely expressed in a variety of tissues, many function, at least in part, by affecting the formation, maintenance, structure or remodeling of bone and/or other calcified tissues. Members of the BMP family are potentially useful as therapeutics. For example, BMP-2 has been shown in clinical studies to be of use in the treatment of a variety of bone-related conditions.
  • bone protein generally refers to a polypeptide factor that supports the growth, remodeling, mineralization or maintenance of calcified tissues. Bone proteins are typically components of extracellular matrix (ECM), or associate with cells and structures that form ECM structures. Exemplary though non-limiting bone proteins that may find use in the embodiments provided for herein may include alkaline phosphatase, osteocalcin, bone sialoprotein (BSP) and osteocalcin in secreted phosphoprotein (SPP)-I, type I collagen, type IV collagen, fibronectin, osteonectin, thrombospondin, matrix-gla-protein, SPARC, alkaline phosphatase and osteopontin. One or more bone proteins may be included in an osteoinductive composition.
  • antibiotic generally refers to a naturally occurring, synthetic or semi- synthetic chemical substance that, when diluted in an aqueous medium, kills or inhibits the growth of microorganisms and can cure or treat infection.
  • the term “analgesic” is used in reference to a pharmacologically active agent or composition that alleviates pain without causing loss of consciousness
  • the term “polypeptide” generally refers to a naturally occurring, recombinant or synthetic polymer of amino acids, regardless of length or post-translational modification (e.g., cleavage, phosphorylation, glycosylation, acetylation, methylation, isomerization, reduction, farnesylation, etc%), that are covalently coupled to each other by sequential peptide bonds.
  • a "large” polypeptide is typically referred to in the art as a “protein” the terms “polypeptide” and “protein” are often used interchangeably.
  • portion as used herein in the context of a polypeptide (as in “a portion of a given polypeptide/polynucleotide”) generally refers to fragments of that molecule. The fragments may range in size from three amino acid or residues to the entire molecule minus one amino acid. Thus, for example, a polypeptide "comprising at least a portion of the polypeptide sequence” encompasses the polypeptide defined by the sequence, and fragments thereof, including but not limited to the entire polypeptide minus one amino acid.
  • a polypeptide may be made using recombinant means, or may be isolated from its natural source (i.e., partially purified).
  • the term "recombinant,” when used in reference to a polypeptide, generally refers to a protein, or a fragment thereof, that is made and/or at least partially purified using recombinant DNA technology. Techniques for the production of recombinant proteins are widely known in the art. Briefly, a nucleotide sequence encoding the protein (or portion thereof) that is to be expressed is inserted in-frame into an expression vector (e.g., a viral, bacterial, fungal, yeast, plant, insect or mammalian expression vector) that includes DNA sequences that are required for transcription of the encoding nucleotide sequence and the subsequent translation of the RNA resulting therefrom. The expression vector is then introduced into an appropriate cell system in culture.
  • an expression vector e.g., a viral, bacterial, fungal, yeast, plant, insect or mammalian expression vector
  • the recombinant protein produced by cultured cells may be at least partially purified using a variety of techniques.
  • General guidance in techniques used for the production and purification of recombinant proteins may be found, for example, in Sambrook, J. et al. Molecular Cloning: A Laboratory Manual. 2 nd , Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, which is incorporated herein by reference.
  • the term "at least partially purified" when used in the context of a polypeptide or a composition containing a polypeptide generally refers to an expressed polypeptide that is at least partially free from other cellular material, or culture medium that was present during the production thereof.
  • autogenic bone generally refers to bone material derived from a substantially genetically identical reference source (i.e., the same subject or a genetic clone thereof). Typically, autogenic bone refers to material derived from bone of the same subject in which the material will ultimately be used.
  • allogenic bone generally refers to bone material that is derived from a genetically distinct source belonging to the same species as the subject in which the bone material will ultimately be used.
  • xenogenic bone generally refers to bone material that is derived from a different species as the subject in which the bone material will ultimately be used.
  • the following embodiments provide for porous, bioresorbable calcium phosphate bone replacement compositions which harden to materials having high compressive strength (> 20 MPa) and which also function as pharmaceutical carriers for bioactive compositions.
  • the presently described embodiments are further directed methods of making same.
  • the bone replacement compositions will typically be made using calcium phosphate cement (CPC) pastes that self -harden to compositions that are substantially apatitic in nature.
  • CPC calcium phosphate cement
  • Calcium phosphate cements suitable for use with the presently described embodiments, including their method of manufacture and use may include without limitation, those disclosed in U.S. Patent Nos. 6,379,453 and 6,840,995 to Lin et al., entitled “PROCESS FOR PRODUCING FAST SETTING, BIORESORBABLE CALCIUM PHOSPHATE CEMENT”; U.S. Patent Appl. Publ. No. 2004/0031420 by Lin et al., entitled “CALCIUM PHOSPHATE CEMENT, USE AND PREPARATION THEREOF”; U.S. Patent No. 6,960,249 to Lin et al.
  • TETRACALCIUM PHOSPHATE HAVING CALCIUM PHOSPHATE WHISKER ON SURFACE AND PROCESS FOR PREPARING THE SAME
  • calcium phosphate cements may be formed from acidic calcium phosphates (e.g., calcium phosphates having a calcium to phosphorous ratio of less than 1.33), basic calcium phosphates (e.g., calcium phosphates having a calcium to phosphorous ratio of greater than 1.33) or combinations of acidic and basic calcium phosphates.
  • acidic calcium phosphates e.g., calcium phosphates having a calcium to phosphorous ratio of less than 1.33
  • basic calcium phosphates e.g., calcium phosphates having a calcium to phosphorous ratio of greater than 1.33
  • the presently described CPCs may optionally include one or more bioactive compositions dispersed or dissolved therein, such as are described in detail below.
  • CPCs made using calcium phosphate particles having whiskers on the surface of the particles, such as are disclosed in the above-cited references and incorporate by reference herein. Without being bound by any particular mechanism of action, it is believed that the whiskers described in these references increase the surface area of cement particles and allow for improved cementing reactions to occur, resulting in hardened materials having improved compressive strength. Additionally, and by virtue of their ability to form interlocking complexes with the whiskers of adjacent particles, surface whiskers advantageously allow a CPC paste to be non-dispersive in aqueous solutions.
  • whiskers comprising TTCP may be formed on the surface of TTCP particles by soaking the particles in an aqueous phosphate solution having basic pH.
  • aqueous phosphate solution having basic pH typically at a pH of about 8.0
  • crystalline TTCP that is exposed to alkaline solutions (typically at a pH of about 8.0) for a period of several minutes (e.g. typically bout 5 minutes), may result in the dissolution of a portion of the calcium phosphate material into the aqueous surrounding.
  • the loss of the calcium phosphate material into the aqueous solution may contribute to the formation of TTCP crystals on the surface of TTCP particles (e.g. etching).
  • TTCP particles e.g. etching
  • the etching seen during formation of the whiskers described above and in the above- cited references follows the grain boundaries of the calcium phosphate crystals.
  • a portion of the dissolved calcium may react with dissolved phosphate ions in the aqueous surroundings to form amorphous calcium phosphate precipitate. This precipitate may further contribute to the size and shape of calcium phosphate whiskers.
  • whiskered TTCP particles may be contacted with a setting solution and heated to result in a hardened apatitic cement suitable for use as an injectable bone filler material, or for use in the manufacture of prosthetic bone implants.
  • Modified calcium phosphate cement compositions suited for use in the presently described embodiments may be chosen according certain chemical and/or physical properties that are advantageous for therapeutic use. It is desirable that the constituent CPCs used herein have the ability to harden into biomaterials having relative high compressive strength. For example, a CPC composition may generally be chosen such that a hardened cement made therefrom has a compressive strength of > 30 MPa, > 50 MPa, or > 100 MPa.
  • CPC compositions may be chosen such that, when mixed with an appropriate setting solution, a paste having sufficient viscosity so as to allow the paste to be injected through a syringe or other aperture to a site within a body or a mold will be formed.
  • the preceding two parameters are, at least in part, related to the density of whiskers on the surface of constituent calcium phosphate particles, and to the density of particles comprising the paste.
  • the density of surface whiskers will typically be in a range such that the resulting material has the desired characteristics of being non-dispersive and able to withstand high compressive forces, while allowing the paste to remain injectable. Typically, such characteristics may be realized when the density of surface whiskers is > 2.0/ ⁇ m 2 and less than 100/ ⁇ m 2 .
  • a paste made therefrom should have a setting time and working time that is greater than 1 minute and less than 45 minutes.
  • 2005/0069479 discloses that heating a TTCP paste to between about 5O 0 C to 35O 0 C for at least one minute, results in a CPC paste having a working time and setting time of between about 8 to 45 minutes and about 9.5 minutes to about one hour, respectively.
  • Calcium phosphate prosthetic bone implants discloses that heating a TTCP paste to between about 5O 0 C to 35O 0 C for at least one minute, results in a CPC paste having a working time and setting time of between about 8 to 45 minutes and about 9.5 minutes to about one hour, respectively.
  • porous biomaterials suitable for use with the presently described embodiments may include, though are not limited to, those disclosed in U.S. Patent Application Publication Nos. 2005/0029701 by Lin et al. entitled “METHOD OF MAKING A MOLDED CALCIUM PHOSPHATE ARTICLE”; 2005/0184417; 2005/0186354; 2005/0186449; 2005/0184418; and 2005/0186353, by Lin et al., entitled "METHOD FOR
  • apatite comprising the implant body may be made in the absence of a sintering step.
  • the lack of a sintering step serves, at least in part, to preserve micro- and nano-sized pores present in the matrix of the calcium phosphate material.
  • the presence of such porosity in the finished CPC article advantageously allows the article to have a wicking profile that encourages the penetration thereof by body fluids as well as infiltration of the implant by cells (e.g. osteoblasts, osteoclasts supportive cells) when compared to implants that are made from conventional sintered CPC.
  • the hardened CPC will typically be at least partially porous (e.g., as a "porous block” or a “porous biomaterial"), and may accommodate porosity up to about 90-vol%.
  • porosity of a calcium phosphate implant is directly related to its bioresorbability, and inversely related to its compressive strength. The relationship between porosity, bioresorbability and compressive strength of an implanted may be exploited to configure an implant having both high compressive strength (typically > 50 MPa and up to 170 MPa), and high bioresorbability.
  • the subject prosthetic bone implants may be adapted to withstand compressive forces equal to or in excess of those typically exerted on naturally occurring bone may be accomplished by coupling hardened calcium phosphate articles having different porosities to each other in configurations that are optimally suited for implantation of the implant in or near a bone of a subject.
  • a dense CPC block will be less than 40 % by volume and will function in a load bearing capacity, whereas a porous CPC block will be 20- 90% by volume.
  • the porosity of the calcium phosphate matrix may be controlled by altering one or more process and or composition parameters during manufacture of the implant.
  • the porosity of an implant may be readily controlled by, for example, including a pore forming powder or changing the ratio of a pore forming agent in the CPC.
  • the porosity of the implant may be constant throughout the calcium phosphate matrix.
  • prosthetic bone implants particularly suited to presently described embodiments are described in the following U.S. Patent Applications: U.S. Patent Application Serial No. 10/780,728 by Lin et al., entitled "METHOD FOR MAKING A
  • an implant may be adapted to have varying porosity throughout the calcium phosphate matrix.
  • the implant may optionally be configured to functionally and structurally mimic the configuration of natural occurring bone, with a denser, load bearing cortical portion, and one or more porous cancellous portions integrally disposed therein. Such a configuration may optimize penetration of body fluids and tissue ingrowth into the implant body.
  • an implant may have a load bearing cortical portion having at least two opposite surfaces and a cancellous portion integrally disposed in the cortical portion and being exposed through the two opposite sides. Both the cancellous portion and the cortical portion may be formed from hardened calcium phosphate cement.
  • the cancellous portion may have a porosity that is greater than the porosity of the cortical portion.
  • the porosity of the cancellous portion may be at least about 20% by volume.
  • the cortical portion may also be formed from a porous calcium phosphate cement. The cortical portion may have a porosity of less than about 40% by volume.
  • certain embodiments may be directed to implants having improved bioresorbability properties.
  • Improved bioresorbability may be realized, at least in part, by including an additional layer of nano- and micro-sized porosity to the surface of the implant.
  • the outer porous layer will be at least 100 ⁇ m in thickness. Implants that incorporate such an outer porous layer will exhibit improved wicking profiles, and may allow body fluids, vascularization and cellular infiltration of the implant from the exterior of the implant. Such may be readily achieved by coupling a porous component to the exterior surface of the implant during the manufacture thereof.
  • an exterior porous layer may be formed on the surface of the implant by subjecting the implant surface to a treatment that forms a layer of porous calcium phosphate material on the surface thereof. Such treatments are described in detail below.
  • the bioresorbability of a calcium phosphate implant is related the size and interconnectedness of pores distributed throughout the matrix or body of the implant. Ideally, the pores will be large enough to allow body fluid wicking and osteoblast infiltration. Typically, infiltration of osteoblasts is facilitated when at least a portion of the pores have openings and/or pore throat diameters of approximately 100 ⁇ m or larger. Pore throat diameter and pore opening diameter ranges may be from 100-500 ⁇ m to about 100-300 ⁇ m, respectively. CPC implants having defined porosity
  • pore size, pore throat diameter, and the degree of interconnected porosity may be manipulated by incorporating within a CPC paste one or more porogens having defined size and/or shape.
  • porogens that are particularly contemplated for use herein include those porogens that may be removed from the hardened CPC material after the setting thereof, and which, when removed, leave a space or void in the hardened CPC matrix.
  • Exemplary porogens that may find use in the presently described embodiments include, though are not limited to, water-soluble materials (e.g., crystalline salts) and/or biodegradable polymers such as are disclosed above and incorporated herein.
  • Porogens incorporated into a CPC paste may be varied in size and/or shape, and the proportions thereof may be selected such that a desired degree and configuration of interconnected porosity is achieved.
  • porogens may be substantially spherical or ovoid in shape.
  • porogen microspheres having average diameter of less than about 5 mm; less than about 2.5 mm; less than about lmm; less than about 750 ⁇ m; less than about 500 ⁇ m; less than about 300 ⁇ m; less than about 200 ⁇ m; less than about 100 ⁇ m; less than about 50 ⁇ m; less than about 20 ⁇ m; less than about 10 ⁇ m; less than about 5 ⁇ m; less than about 2.5 ⁇ m; less than about 1 ⁇ m; less than about 0.5 ⁇ m; or less than about 0.1 ⁇ m.
  • porogens may be substantially cuboidal in shape.
  • porogens may be substantially pyramidal in shape.
  • porogen microparticles having average particle volume of less than about 0.5 mm 3 ; less than about 0.25 mm 3 ; less than about 0.1 mm 3 ; less than about 0.075 mm 3 ; less than about 0.05 mm 3 ; less than about 0.030 mm 3 ; less than about 0.020 mm 3 ; less than about 0.010 mm 3 ; less than about 0.005 mm 3 ; less than about 0.002 mm 3 ; less than about 0.001 mm 3 ; less than about 0.0005 mm 3 ; less than about 0.00025 mm 3 ; less than about 0.0001 mm 3 ; less than about 0.00005 mm 3 ; or less than about 0.00001 mm 3 .
  • porogens may be configured as fibers.
  • porogen microfibers having average cross-sectional diameter of less than about 5 mm; less than about 2.5 mm; less than about lmm; less than about 750 ⁇ m; less than about 500 ⁇ m; less than about 300 ⁇ m; less than about 200 ⁇ m; less than about 100 ⁇ m; less than about 50 ⁇ m; less than about 20 ⁇ m; less than about 10 ⁇ m; less than about 5 ⁇ m; less than about 2.5 ⁇ m; less than about 1 ⁇ m; less than about 0.5 ⁇ m; or less than about 0.1 ⁇ m.
  • porogens of varying geometry may improve the interconnected porosity of an implant.
  • the relative degree of interconnected porosity of an implant may be manipulated by varying the ratio of porogen having different particle size and geometry (e.g. combination of spherical and cuboidal particles).
  • Certain embodiments may incorporate microfiber porogens therein.
  • the porogen includes mixtures of one or more salts, KCl, whose crystal structure is substantially spherical in shape may be used to incorporate microspherical pores in the matrix of a hardened CPC material, and NaCl, whose crystal structure is substantially cuboidal in shape may be used to incorporate microcuboidal pores in the matrix of a hardened CPC material.
  • porogens may be employed to result in a hardened CPC matrix having pores of defined density, interconnectedness and geometry without departing from the spirit and scope of the present invention.
  • porogens may be formulated so as to incorporate one or more therapeutically active compositions therein.
  • spherical salt particles will have less adverse effect on the mechanical strength of the implant, but do not allow maximum interconnected porosity.
  • the lack of interconnected communication between adjacent pores in the implant body may be remedied by including non- spherical salt crystals therein.
  • the degree of interconnected porosity may be further manipulated by varying the ratio of spherical to non-spherical salt crystals.
  • the ratio of spherical to non-spherical salt crystals comprising the pore forming powder will be from about 9:1 to about 1:4, or from 3:4 to about 1:4.
  • the ratio of spherical to non- spherical salt crystals comprising the pore forming powder will be about 1:1.
  • interconnected porosity and pore size may be influenced by the average particle size of constituent particles comprising the hardened cement.
  • CPC particles having an average diameter between about 0.1 ⁇ m to about 500 ⁇ m are used to form the implants.
  • the ratio of porogen to CPC powder dry weight ratio
  • Using higher ratios may adversely affect the compressive strength of the resulting implant.
  • microcavities and or internal voids may be created in the body of the subject implants by suspending one or more porogens therein.
  • the density of porogen particles is such that they do not substantially touch adjacent particles.
  • the particles may function as drug reservoirs when the drug is loaded therein.
  • the microcavities formed in this manner may serve as reservoirs for bioactive compositions, thus increasing the elution time and or effective treatment time of a pharmaceutical agent.
  • the porogens may be removed from the hardened calcium phosphate implant by soaking the implant in an aqueous solution, as set forth in above-cited references.
  • the porogens may alternatively be removed by allowing the degradation, dissolution or resorption thereof after the biocomposite material is in place in a calcified tissue.
  • incorporating one or more bioactive agents into a prosthetic implant may enhance the biocompatibility and/or bioresorbability of the implant.
  • Constituents of the bioactive composition may be selected to impart certain advantageous therapeutic or physiological properties on the implant.
  • bioactive agents may include one or more osteoinductive compounds.
  • osteoinductive compounds include osteogenic compounds.
  • Numerous osteogenic compounds are known to practitioners of ordinary skill in the art including any one of a number of polypeptide growth factors known for their ability to induce the formation or remodeling of bone.
  • osteogenic compounds suitable for inclusion in the presently described embodiments include, but are not limited to, osteogenin, Insulin-like Growth Factor (IGF)-I, IGF-II, GDF-5 through GDF-9, Transforming Growth Factor (TGF)- ⁇ l, TGF- ⁇ 2 , TGF- ⁇ 3 , TGF- ⁇ 4, TGF- ⁇ 5, osteoinductive factor (OIF), basic Fibroblast Growth Factor (bFGF), acidic Fibroblast Growth Factor (aFGF), Platelet-Derived Growth Factor (PDGF), vascular endothelial growth factor (VEGF), Growth Hormone (GH), and osteogenic protein- 1 (OP-I).
  • IGF Insulin-like Growth Factor
  • IGF-II Insulin-like Growth Factor
  • IGF-II Insulin-like Growth Factor
  • GDF-5 GDF-5 through GDF-9
  • TGF Transforming Growth Factor
  • TGF Transforming Growth Factor
  • TGF Transforming Growth Factor
  • growth factors belonging to the Bone Morphogenic Protein (BMP) family of growth factors which include, but are not limited to, BMP-I, BMP-2A, BMP- 2B, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-8b, BMP-9, BMP-IO, BMP-Il, BMP- 12, BMP- 13, BMP- 14, BMP- 15, or combinations thereof, may be especially suited for inclusion in the subject implants.
  • BMP Bone Morphogenic Protein
  • bioactive agents may include one or more compounds that support the formation, development and growth of new bone, and/or the remodeling thereof.
  • Typical examples of compounds that function in such a supportive capacity include, though are not limited to, bone matrix proteins (e.g., alkaline phosphatase, osteocalcin, bone sialoprotein (BSP) and osteocalcin in secreted phosphoprotein (SPP)-I, type I collagen, type IV collagen, fibronectin, osteonectin, thrombospondin, matrix-gla-protein, SPARC, alkaline phosphatase and osteopontin).
  • a peptide or peptide fragment may contain the amino acid sequence Arg-Gly-Asp, which has been shown to bind to and enhance the recruitment of osteoblasts.
  • Bioactive agents may, in some embodiments, further include pharmacologically active compounds that do not act locally to stimulate bone growth and healing, but that may nonetheless be therapeutically advantageous in certain applications, such as, for example, antibiotic and or analgesic agents.
  • exemplary analgesic agents suitable for use herein include, but are not limited to, norepinephrine, bupivacaine, ropivacaine, 2-chloroprocaine, lidocaine, mepivacaine, ropivacaine, mepivacaine, benzocaine, tetracaine, dibucaine, cocaine, prilocaine, dibucaine, procaine, chloroprocaine, prilocaine, mepivacaine, etidocaine, tetracaine, xylocaine, morphine, fentanyl, alphaxalone and active analogs, 5-alpha-pregnane-3 alpha-21-diol-20-one (tetrahydro-deoxycorticosterone
  • antibiotic agents include, but are not limited to, tylosin tartrate, tylosin, oxytetracycline, tilmicosin phosphate, ceftiofur hydrochloride, ceftiofur sodium, sulfadimethoxine cefamandole, tobramycin, penicillin, cefoxitin, oxacillin, vancomycin, cephalosporin C, cephalexin, cefaclor, cefamandole, ciprofloxacin, bisphosphonates, isoniazid, ethambutol, pyrazinamide, streptomycin, clofazimine, rifabutin, fluoroquinolones, ofloxacin, sparfloxacin, rifampin, azithromycin, clarithromycin, dapsone, tetracycline, erythromycin, ciprofloxacin, doxycycline, ampicillin, ampho
  • the amount of a pharmacologically active agent to include in the subject bioactive coating compositions may typically vary with the identity of the agent, the physiological context in which the agent is being employed, and the magnitude of the desired response.
  • Typical dosages of pharmacologically active agents that will be loaded onto the calcium phosphate carrier may be in the range of 2 ng/m 3 to 1 mg/m 3 , according to the volume of pharmaceutical carrier used to deliver the bioactive agent.
  • General guidance in determining effective dose ranges for pharmacologically active compounds may be found, for example, in the publications of the International Conference on Harmonisation and in REMINGTON'S PHARMACEUTICAL SCIENCES, chapters 27 and 28, pp. 484-528 (Mack Publishing Company 1990), which is incorporated by reference as though fully set forth herein.
  • Bioactive agents may be coupled to the implant by way of a pharmaceutically acceptable carrier.
  • Desirable characteristics for pharmaceutical carriers employed in the presently described embodiments include at least one of i) biocompatibility; ii) bioresorbability; iii) ability of the carrier to stably store the bioactive agents and/or allow its sustained release to surrounding tissues and cells. Such characteristics may be realized using a thin (10-50 ⁇ m in thickness) crystalline hydroxyapatite layer formed on the surface of the implant.
  • a bioactive composition may be coupled, added or incorporated to a calcium phosphate- based pharmaceutical carrier (e.g., CPC paste or hardened CPC material).
  • a calcium phosphate- based pharmaceutical carrier e.g., CPC paste or hardened CPC material.
  • a method whereby a layer of crystalline calcium phosphate is formed on the surface a calcium phosphate prosthetic bone implant by co- precipitating apatite and one or more bioactive agents from a physiologically acceptable aqueous calcium phosphate solution.
  • the co-precipitated bioactive agents will be stably integrated and dispersed within the matrix of said crystalline calcium phosphate surface layer.
  • bioactive agents are gradually released from the crystalline calcium phosphate layers of the subject implants in a sustained manner.
  • Calcium phosphate layers produced using current art-recognized techniques are typically composed of large, partially molten HAp particles. HAp produced synthetically under these conditions is prone to delamination and is poorly degraded in situ.
  • the calcium phosphate layers of the present embodiments in addition to being bioresorbable and biocompatible, are produced under physiological conditions and thus have the additional advantage of being able to integrally accommodate bioactive molecules, such as osteogenic agents, that typically cannot withstand harsh processing treatments (e.g., elevated temperature pressure, osmotic conditions and pH).
  • the bioactive molecules may be co-precipitated with the inorganic mineral components that will form the crystalline calcium phosphate.
  • the bioactive agents are incorporated into the crystal structure of the precipitated mineral coating, rather than being merely deposited upon the surface of the implant and or the coating.
  • the elution profile of the integrated bioactive agent is more constant and sustained rather than being a single burst (as when superficially adsorbed).
  • the reduced elution rate advantageously prolongs the osteoinductive and healing potential of therapeutics agents acting locally at the implantation site.
  • the crystalline coating may involve the nucleation and growth of HAp crystals on the surface of a calcium phosphate prosthetic bone implant.
  • the subject implants do not require a pre-treatment process to deposit a nucleating layer on the implant surface, although such a pre-treatment step may be performed if desired.
  • formation of the crystalline coating may include contacting the implant with a coating composition that includes a source of calcium and a source of phosphate. Contacting the implant with the coating composition may include fully or partially immersing the implant in the coating composition. Typically, this step will be carried out at a temperature that is within physiologic range (e.g., between about 2O 0 C to about 45 0 C, between about 25 0 C to about 37 0 C, or at about 37 0 C).
  • the implant will be contacted with the coating composition for a period of time sufficient to allow the precipitation of crystalline calcium phosphate on the surface of the implant.
  • a layer crystalline calcium phosphate mineral that is at least 0.5 to about 100 ⁇ m thick, between 20 to about 50 ⁇ m thick, or about 40 ⁇ m thick, will be allowed to form on the surface of the implant. Layers of such thickness will typically be achieved in less than 100 hours at 37 0 C, or more typically, in less than about 48 hours at 37 0 C.
  • the thickness of a calcium phosphate mineral layer may be monitored using techniques widely familiar to practitioners, such as densitometry, reflectometry, scanning electron microscopy, spectroscopy, or the like.
  • the coating composition will contain amounts of calcium and phosphate that are sufficient to precipitate crystalline HAp at physiological temperature and pH.
  • concentration of calcium ions in the coating composition may range from 0.5 to 10 mM, or from 0.5 to 5 mM.
  • concentration of phosphate ions in the coating composition may range from 0.5 to 6 mM, or from 0.5 to 3 mM.
  • Sodium chloride, or any suitable salt may be added to maintain the ionic strength of the coating composition. Typically the ionic strength of the solution should be between 100 mM to 200 mM sodium chloride, and more typically 150 mM.
  • the size of HAp crystals may be controlled by varying the amount of crystal growth inhibitors in the coating composition (e.g., magnesium and carbonate), with crystal size being inversely proportion to the concentration of crystal growth inhibitors present in the solution.
  • the concentration of magnesium should be less than 7.5 mM, more typically less than 2.5 mM, and most typically less than 0.5 mM.
  • HAp crystals ideally form when the concentration of carbonate ions is less than 25 mM, more typically less than 10 mM, and most typically less than 5 mM.
  • precipitation of HAp crystals will occur at a substantially physiological pH
  • An appropriate buffer like tris (amino-ethane) or HEPES (N- [2- hydroxyethyl] piperazine-N'-[4-ethanesulfonic acid]) is preferably used to maintain the desired pH.
  • Suitable buffers to maintain a desired pH are known from the art. The relationship between temperature, pH and calcium phosphate solubility per se is known in the art. The skilled practitioner will be able to derive suitable conditions from the guidelines described above.
  • the bioactive agents that are to be co-precipitated with HAp crystals will be solubilized in the coating composition.
  • concentration of the one or more bioactive agents in the solution will be in a concentration range of 0.1 mg/1 to 10 g/1, in the range of 0.1- 1000 mg/1, in the range of 0.1-500 mg/1, or in the range of 0.1-20 mg/1.
  • concentrations, pH ranges and temperatures may choose to use particular concentrations, pH ranges and temperatures to form the crystals.
  • Most preferably calcium and phosphate are among the inorganic ions used to incorporate bioactive agents into an implant.
  • a coating composition for depositing crystalline HAp on the surface of a calcium phosphate implant will typically be buffered at a pH in the range of 6 to 8.
  • the pH of the coating composition may depend upon the isoelectric point (pi) of a bioactive agent that is to be incorporated into the coating. Co-precipitation of bioactive agent with inorganic crystals is related to electrostatic interactions. For chargeable compounds, and in particular for amphoteric compounds, the efficiency of incorporation depends on the pi of the bioactive agent and pH of the coating composition. The pi of a compound can be measured by isoelectric focusing using polyacrylamide gel electrophoresis. In some embodiments, the bioactive agent is charged at the pH at which the bioactive agent is incorporated into the implant, because this positively affects the amount of bioactive agent that is incorporated.
  • BMP-2 has a IEP of 9.2. Accordingly the protein has a positive charge below 9.2 and negative charge above 9.2. At a pH of 7.4 for the coating composition, the protein is positively charged and thereby interacts with anions (such as phosphate) in solution. The interaction of the protein with the anions, enhances co-precipitation thereof with HAp crystals growing on the implant surface. For instance, a concentration of BMP-2 in a coating composition of 5 mg/L may lead to an incorporation of 5 ⁇ g/mg of coating at pH 7.4. BMP-7, however, has an IEP of 7.7. At a pH of 7.4, the efficiency for incorporation is low due to insufficient difference between IEP and coating pH.
  • anions such as phosphate
  • the incorporation of BMP-7 is only 0.25 ⁇ g/mg coating at pH 7.4 for 5 mg/1 of BMP-7 in coating solution.
  • a lower pH for coating solution may be selected (e.g. 6.7).
  • the difference between pH and pi for each bioactive agent in the composition should be at least about 1 pH unit for optimal co-precipitation of bioactive agents with the growing inorganic layer.
  • co-precipitation is preferably performed at a pH below pi
  • acidic amphoteric compound (pI ⁇ 7.0) co- precipitation is preferably performed at a pH higher than pi.
  • a pH close to 6 or close to 8 is preferred.
  • therapeutic agent such as osteogenic compounds and analgesic compounds may be precipitated on an outer layer of the implant, while therapeutic agents such a bone proteins or antibiotics may be deposited first.
  • the pH of the calcium phosphate solution typically has less influence on the incorporation rate of uncharged bioactive agents. In general physiological pH, around 7.4 is suitable for this purpose.
  • including one or more bioactive agents, in particular one or more osteoinductive agents, in the coating may stimulate cell activity and cell differentiation near an implant. Accordingly, the subject coated implants may regenerate or repair bone tissue more efficiently and more rapidly than implants which do not contain bioactive agents.
  • the release of bioactive agent(s) is related to the rate of coating degradation. After implantation, the mineral coating is remodeled or degraded by osteoclastic activity, leading to a gradual release of the bioactive agent(s), around the implanted medical device.
  • an optimal concentration of bioactive agent(s) can be maintained around the medical device, and burst-release of bioactive agent(s), which may lead to unwanted side effects and premature cessation of therapeutic activity of the implant may be avoided.
  • the degradation of the coating and release of the bioactive agent(s) may be monitored by measuring the calcium and or bioactive agent(s) release under physiological conditions as a function of time.
  • Methods to monitor levels of these compounds are known in the art and include monitoring via a calcium-ion selective electrode, chromatography or enzyme- linked immunosorbant assay to measure the elution profiles of polypeptide factors.
  • a growth factor incorporated into a crystalline calcium phosphate layer as described herein will have an elution profile at physiological pH (about 7.4) that roughly corresponds to the dissolution rate of the calcium phosphate matrix in which it is incorporated.
  • the surface of the prosthetic bone implant with an initial layer (e.g., an amorphous mineral) of inorganic compounds, such as with an initial layer comprising calcium and phosphate.
  • the amorphous layer may be obtained by contacting the implant surface with an aqueous calcium phosphate pre- coat solution under high nucleation conditions to obtain a thin and amorphous calcium phosphate layer.
  • the optional amorphous layer may act as a seed to enhance the ability of more highly structured crystalline HAp to be precipitated on the implant surface. In some applications, including the optional amorphous layer may improve the stability and the activity of the crystalline HAp coating and the bioactive agent(s) incorporated therein.
  • the implant may be pre-coated for a period of time sufficient to deposit an amorphous layer of calcium phosphate material at least 1 ⁇ m in thickness (typically, between 12-24 hours).
  • the composition of the inorganic components of the pre-coat solution may be chemically similar to that found in body fluids.
  • the concentration of calcium ions in the pre-coat solution may range from 0.5 to 20 mM, or from 8 to 12.5 mM.
  • the concentration of phosphate in the pre-coat solution may range from 0.5 to 10 mM, or from 2.5 to 5 mM.
  • the concentrations of calcium and phosphate may have to be adjusted to maintain a desired pH.
  • the solubility of calcium phosphate is inversely proportional to pH, that is, as pH increases the solubility of calcium phosphate decreases. For example, at 37 0 C, and at a pH of 6.7, calcium phosphate is more soluble than at physiological pH (about 7.4). Concentrations of calcium and phosphate, in some embodiments, will be between 4 mM to 15 mM for calcium and 2 mM to 20 mM for phosphate.
  • magnesium ions is thought to inhibit the deposition of crystalline calcium phosphate mineral coatings.
  • the presence of magnesium has been found to inhibit or reduce the crystal growth of the coating during deposition from the calcium phosphate solution, resulting in an amorphous calcium phosphate layer that may act as a seed to enhance formation of crystalline HAp subsequently precipitated thereon.
  • Optimum control of crystal growth leads to a uniform, strong and wear resistant coating.
  • Magnesium and carbonate ions may be present in the pre-coat solution at concentrations below 10 and 25 mM, respectively. The quantity of magnesium and carbonate, both inhibitors of crystal growth may be adjusted for optimal formation and attachment of the optional amorphous pre-coat layer.
  • apatite crystals are to be formed it is desirable to produce apatite crystals of submicrometer dimensions ( ⁇ 1 microns), which may result in a mechanically stronger coating.
  • increasing the magnesium and carbonate ion concentration may decrease the average crystal size.
  • bioactive compositions may be coupled to prosthetic bone implants by first forming a layer of nanoporous HAp nanocrystals on the surface of at least a portion of the implant.
  • Nanoporous HAp nanocrystals may also be formed on the surface of a calcium phosphate implant surfaces using any art-recognized technique.
  • the nanocrystalline HAp surface will be highly porous and have a surface area in the range of about 25 m 2 /g to about 150 m 2 /g.
  • the surface area of the nanocrystalline HAp coating the subject implants will be directly proportional to amount of bioactive composition that can be coupled to the implant.
  • the surface area of the nanocrystalline HAp coating is inversely related to the elution rate of the bioactive composition when implanted in a subject.
  • FIG. 1 shows SEM images (at 10,000 fold magnification) of the surface of calcium phosphate implants having a surface layer of nanoporous HAp nanocrystals according to some embodiments. Individual particles of calcium phosphate are cemented to each other, and a layer of nanoporous HAp nanocrystals is formed thereon.
  • a layer of nanoporous HAp nanocrystals that is well suited for prolonged retention and slow elution of bioactive agents may be formed on the surface of a CaP implant by contacting the portion of the implant that is to be coated with an aqueous solution containing a source of phosphate ions.
  • the solution may contain a source of calcium ions.
  • the implant will be soaked in the solution for a period of time that is sufficient to form nanocrystalline HAp on the implant surface.
  • the implant may be soaked for up to 8 days. After soaking, the implant may be rinsed with the solution, with water, or with an appropriate physiological buffer.
  • the implant may be dried and stored under sterile conditions for use in a point-of-care setting.
  • the nanoporous nanocrystalline HAp layer will have a surface area of between about 25 m 2 /g to about 150 m 2 /g, or between about 60 m 2 /g to about 100 m 2 /g.
  • the increased surface area of the prosthetic bone implants significantly increases the drug binding capacity of the implant (i.e. results in a greater amount of bioactive composition to be coupled thereto).
  • the physical and chemical properties of surface nanoporous HAp nanocrystals may be by altered by including one or more additives in the aqueous solution.
  • the additives may include, for example, inhibitors of crystal formation, such as magnesium and/or carbonate ions (as described extensively above).
  • the morphology of nanoporous HAp nanocrystals may be regulated.
  • the physical and chemical properties of surface nanoporous HAp nanocrystals may also be determined by the amount of time that the implant is left in contact with the aqueous solution.
  • the implant will be contacted with the aqueous solution for a period of time ranging from between 1 to 8 days.
  • the amount of time that the implant is to be contacted with the aqueous solution is dependent on factors such as the chemical composition of the solution, and the surface area that is desired.
  • FIG. 1 demonstrates the dependence of the surface area nanocrystalline HAp on chemical composition and incubation time.
  • FIGs. IA and IB each show an SEM image of 10,000-fold magnification of the surface of a hardened calcium phosphate cement that has undergone the indicated treatment.
  • the image depicted in FIG. IA corresponds to a CaP sample that has been immersed in Hank's Balanced Salt Solution (HBSS, with calcium and magnesium) for 3 days.
  • the image depicted in FIG. IB corresponds to a CaP sample that has been immersed in Phosphate Buffered Saline (PBS) for 5 days. Nanophase nanocrystalline HAp is formed under both sets of conditions.
  • HBSS Hank's Balanced Salt Solution
  • PBS Phosphate Buffered Saline
  • deposition of nanophase HAp nanocrystals on the surface of the subject implants may be performed using techniques such as ion- spray or sol-gel surface chemistry techniques. Formation of nanophase HAp nanocrystals typically occurs under physiologically unfavorable conditions and may be performed in the absence of bioactive agents whose stabilities are intolerant to such conditions.
  • the implant and nanophase HAp coating may be prepared and packaged under ascetic conditions.
  • Bioactive agents may be loaded onto the surface thereof in a point-of-care setting by immersing the coated prosthetic bone implant in a sterile, physiologically buffered aqueous solution containing the dissolved bioactive composition. After loading onto the implant, the implant is delivered to its desired site in the body. Due to its high surface area and affinity for polypeptides, in particular BMPs, the elution rate of bioactive agents from the nanophase HAp is similar to the dissolution rate of the HAp crystals.
  • Bioactive compositions may be loaded onto the CaP subject bone implant by soaking the implant in an aqueous composition including a bioactive agent. This soaking step may be performed in addition to co-precipitating a drug onto the surface of an implant as described above. Alternatively, loading a bioactive agent onto the implant surface by performing a soaking step may be suited to situations where the implant was manufactured under conditions that would destabilize, degrade, or otherwise adversely affect the function of the drug. The soaking step may be performed without limitation with regard to strength, composition, pH or temperature of the soaking solution. Charging the implant by performing a soaking step may be suited to situation where activation of the drug must be performed under conditions that are adverse to the precipitation and/or formation of crystalline HAp on implant surfaces.
  • Methods for the use of injectable CPC cements as pharmaceutical carriers may include the use thereof for delivery of bioactive compositions with controlled release profile (e.g., multimodal, exhibiting an initial burst followed by gradual release or slow release profile over longer time). Certain structures of the cement may be capable of being substantially or completely reabsorbed by the host tissue. In one embodiment, the release can range from 2% to 40% of the initial load within 24 hrs.
  • bioactive compositions that modulate the affinity of a component thereof (e.g., one or more growth factors) for a CPC material may be included in one or more components used to in fabrication of a CPC paste.
  • pores and or interconnected porosity in accordance with the procedures set forth above and incorporated herein may facilitate controlled release of a bioactive composition from a CPC matrix during use.
  • the pores may be made prior to injection or created slowly as a result of the dissolution, degradation or resorption of one or more of the porogens, in conjunction with the dissolution, degradation or resorption of the hardened CPC material in the presence of said porogen.
  • controlled release of a bioactive composition from a CPC matrix may be facilitated by a combination of the two preceding embodiments to produce a heterogeneous injectable construct with biphasic desorption profile, release by passive dissolution of the soluble phase and long-term release by active resorption of the cement.
  • an excipient for bone growth factor such as BMPs and TGF-beta
  • an excipient may include a mixture of one or more of sodium phosphate, amino acids, bone growth factors, polymers, sugars and carbohydrates.
  • any bioactive agent that facilitates or stimulates new bone growth may be delivered to a calcified tissue in accordance with the embodiments described herein to produce an excipient combined with injectable calcium phosphate cement as a carrier.
  • certain osteoinductive agents may be included in an injectable CPC bone substitute material.
  • Osteoinductive agents that may find use according to such embodiments may include one or more bone morphogenetic proteins e.g. BMP-2, BMP-7, BMP-9, GDF-5, GDF-6, and GDF-7, one or more transforming growth factors (e.g., TGF-beta), one or more IGFs (e.g., IGF-I, and IGF-2), or various portions and/or combinations thereof.
  • autologous bone marrow e.g., derived from the subject who will be receiving it
  • bone-derived TGF-beta e.g., insulin-like growth factors, platelet-derived growth factor and BMP2, or any of the bioactive agents disclosed herein, may be combined with the injectable bone substitute materials.
  • various excipients e.g., amino acids and/or polypeptides
  • polypeptide growth factors e.g., BMPs
  • a calcium phosphate matrix may be included in the subject bone substitute compositions.
  • FIG. 2 the effect of various excipients on growth factor release kinetics from CPC biomaterials is shown.
  • Hardened CPC articles were manufactured in accordance with the present disclosure and the growth factor BMP was equally loaded on the articles.
  • a portion of the hardened articles contained no additional excipient (control; shown in FIG. 2 as closed diamonds), a portion further contained the amino acid glutamate (GIu; shown in FIG.
  • allowing the leaching out of various water-soluble porogens may generate pores.
  • Hardened CPC particles made therefrom may typically have compressive strengths of about 5.5 MPa or more after incubation thereof in a Hank's solution for about 24 hours. The cumulative release after 24 hrs is up to 46%.
  • the biodegradable polymers may be mixed with cement powder or setting solution and act as porogen. Without being bound by any particular theory or mechanism of action, it is believed that the degradation of such polymers in situ results in an at least partially acidic micro-environment (e.g., pH ⁇ about 7). Acidic conditions in the vicinity of the degrading porogen may enhance localized dissolution of the calcium phosphate mineral matrix, resulting in a porous bioceramic matrix that releases the growth factors from the cement over the time.
  • Polymers such as polylactic acid, polylactic acid-polyethylene glycol block copolymer and their derivatives are used to fabricate microspheres or microfibers and combined with calcium phosphate cement to deliver growth factors.
  • the biodegradable polymers may be provided to the subject bone replacement material in combination with one or more additional excipients, such as, for example, molecules that decrease the binding affinity of apatite fro peptide growth factors (e.g., certain amino acids such as alanine).
  • additional excipients such as, for example, molecules that decrease the binding affinity of apatite fro peptide growth factors (e.g., certain amino acids such as alanine).
  • FIG. 3 the effect of various excipients on growth factor release kinetics from CPC biomaterials is shown.
  • Hardened CPC articles were manufactured in accordance with the present disclosure and the growth factor BMP was equally loaded on the articles.
  • a portion of the hardened articles contained no biodegradable polymer porogen (control; shown in FIG. 3 as closed triangles)
  • a portion contained the biodegradable polymer poly lactic acid (PLA; shown in FIG.
  • a bone substitute material suitable for use as a pharmaceutical carrier medium may include an injectable paste having calcium phosphate cement (CPC) particles, an excipient comprising a physiologically acceptable aqueous phosphate solution and an osteoinductive composition.
  • CPC calcium phosphate cement
  • a bone substitute material paste may further include one or more porogens mixed therein.
  • Exemplary porogens may include one or more biodegradable and/or bioresorbable compositions such as salts, polymers, biopolymers, and the like. Porogens may be provided in combination with CPC particles and/or mixed with the physiologically acceptable setting solution.
  • porogens may include certain biodegradable polymers, the degradation of which results in an acidic (e.g., pH ⁇ about 7.0) local micro-environment that encourages localized dissolution of hardened CPC matrix, in addition to creating voids (i.e., pores) in the space occupied by the biodegradable/bioresorbable polymer composition or other porogen.
  • the inclusion of such biodegradable/bioresorbable porogens may advantageously result in a porous bioceramic matrix that locally releases the bioactive composition from the hardened cement over the time, thereby delivering a therapeutic dose of said bioactive composition.
  • a bone substitute material paste may further include one or more porogens in a ratio such that the hardened CPC may develop a predetermined porosity (i.e., >90 vol%, >70 vol.%, >50 vol.%, >30 vol.%, >15 vol.%, >5 vol.%, or > 1 vol.%).
  • Porogens may include crystals of physiologically acceptable salts, biodegradable polymers, or combinations thereof.
  • Biodegradable polymers suitable for applications described herein may include, though are not limited to, natural or synthetic polypeptides, polylactic acid, chitosan, polylactic acid- polyethylene glycol block copolymer and their derivatives.
  • suitable biodegradable polymers may include polyesters, poly(L-lactic acid), poly(D,L-lactic acid), poly(glycolic acid), polycaprolactone, block copolymers and copolymers thereof.
  • at least a portion of the porogen may be formulated as microspheres.
  • at least a portion of the porogen may be formulated as microfibers.
  • the dry weight ratio of porogen to CPC particles in bone substitute material paste may be up to about 1:1.
  • the presently disclosed bone substitute materials may be formulated to act as a pharmaceutical carrier for one or more bioactive agents.
  • the bioactive agent may, in turn be formulated to include an osteoinductive composition, a growth factor composition, an antibiotic composition, and analgesic composition, or combinations thereof.
  • a bioactive composition may include at least one growth factor from the TGF- ⁇ superfamily of growth factors, at least one growth factor from the BMP family of growth factors, at least one growth factor from the GDF family of growth factors, at least one growth factor from the IGF family of growth factors, or their combination.
  • a bioactive composition may include BMP-2, BM-4, BMP-12, or their combination.
  • a bioactive composition may include at least a portion of one or more polypeptides, including but not limited to at least a portion of a polypeptide growth factor, at least a portion of one or more TGF- ⁇ superfamily growth factors, at least a portion of one or more BMP growth factors, or various combinations thereof.
  • a polypeptide for use in an osteoinductive composition as described herein may be at least partially purified.
  • Source materials for at least partial purification of the polypeptides as described herein may include natural source material (e.g., natural bone, bone marrow, cultured cells), or recombinant material (e.g., protein whose expression is facilitated and or enhanced by way of a suitable viral, bacterial, yeast, insect, plant or mammalian protein expression system including a suitable expression vector).
  • natural source material e.g., natural bone, bone marrow, cultured cells
  • recombinant material e.g., protein whose expression is facilitated and or enhanced by way of a suitable viral, bacterial, yeast, insect, plant or mammalian protein expression system including a suitable expression vector.
  • one or more of the bone growth factors may be derived from autogenic bone, allogenic bone, xenogenic bone, or from recombinant sources.
  • CPC particles and a physiologically acceptable aqueous setting solution comprising an excipient may be provided in a ratio sufficient to form an injectable paste.
  • the excipient may include one or more of amino acids, physiological acceptable salts, sodium phosphates, and polypeptides.
  • the injectable calcium phosphate cement bone substitute compositions may harden to form a calcium phosphate material.
  • the hardened material may be soaked in a physiologically acceptable aqueous solution to promote the dissolution/degradation of at least a portion of the porogen embedded throughout the hardened matrix, thereby creating a network of pores therein.
  • the hardened material may be implanted into a site in the body (e.g., a tooth or a bone). Over time, body fluids in contact with the hardened calcium phosphate material/porogen composite may penetrate the matrix thereof and allow dissolution/degradation of the porogen, thereby creating a network of pores therein.
  • the injectable calcium phosphate cement bone substitute compositions may further include physiologically acceptable excipient.
  • the injectable calcium phosphate cement bone substitute compositions may be a paste that is made by a process of providing a solid phase that includes calcium phosphate cement (CPC) particles in combination with at least one porogen; and contacting said solid phase with a liquid phase; and further with an osteoinductive composition.
  • the osteoinductive composition may be included as a component of the solid phase.
  • the osteoinductive composition may be included as a component of the liquid phase.
  • the osteoinductive composition may be included as a component of both the solid phase and the liquid phase.
  • the ratio of the solid phase to liquid phase of the composition may be sufficient to form a paste that can readily be injected using syringe to a site on a bone or to a mold in order to make an implantable structure.
  • the ratio of solid phase to liquid phase may be in the range of about 0.1 mg/ml to about 20 mg/ml.
  • the injectable calcium phosphate cement bone substitute compositions may be delivered to a bone defect or to a mold and form a hardened bioresorbable calcium phosphate material.
  • certain structures of the hardened CPC may be substantially reabsorbed by or released around the site of its application.
  • porogens within the hardened calcium phosphate matrix may gradually be removed from the hardened material, leaving pores and/or an interconnected network of porosity dispersed throughout the matrix.
  • bioactive compositions incorporated in the hardened calcium phosphate material and/or in the porogens may be gradually released to surrounding tissue.
  • any bioactive agent that facilitates or stimulates new bone growth may be delivered to a bone defect or to a mold and form a hardened bioresorbable calcium phosphate material.
  • certain structures of the hardened CPC may be substantially reabsorbed by or released around the site of its application.
  • porogens within the hardened calcium phosphate matrix may gradually be removed from the hardened material, leaving pores and/or an interconnected network of porosity dispersed
  • bioactive compositions may include osteoinductive compositions such as bone morphogenetic proteins e.g. BMP-2, BMP-7, BMP-9, growth differentiation factors (GDF)-5, GDF-6, and GDF- 7, platelet-derived growth factors (PDGFs), transforming growth factors (e.g., TGF- ⁇ ), insulin- like growth factors (IGF)-I, and IGF-2, autogenic or allogenic bone or bone marrow, bone- derived TGF- ⁇ , IGF, and BMP2.
  • bone morphogenetic proteins e.g. BMP-2, BMP-7, BMP-9, growth differentiation factors (GDF)-5, GDF-6, and GDF- 7, platelet-derived growth factors (PDGFs), transforming growth factors (e.g., TGF- ⁇ ), insulin- like growth factors (IGF)-I, and IGF-2, autogenic or allogenic bone or bone marrow, bone- derived TGF- ⁇ , IGF, and BMP2.
  • BMP-2, BMP-7, BMP-9 growth differentiation
  • amino acids or polypeptides may be employed to decrease the binding affinity between polypeptide factors and the hardened calcium phosphate cement matrix.
  • cumulative release of components of a bioactive composition from the hardened CPC material approximately 24 hrs after the introduction thereof to a bone defect may be up to about 2.5%.
  • composite (e.g., growth factor/CPC) bone substitute materials may have a compressive strength of up to about 150 MPa after incubation in a physiologic solution.
  • the porosity in the hardened CPC matrix may be realized, at least in part, by the dissolution, degradation, or resorption of porogens found therein.
  • the hardened CPC bone substitute materials may be adapted to have a compressive strength up to about 150 MPa after incubation of said materials in a physiologically acceptable aqueous solution.
  • the cumulative release profile of therapeutic compositions therefrom after 24 hrs may be up to about
  • bioactive agents may be loaded onto a porous biomaterial using systems and/or devices that allow an operator to control the amount of a bioactive agent that is delivered to a porous CPC matrix.
  • systems and/or devices that allow an operator to control the amount of a bioactive agent that is delivered to a porous CPC matrix.
  • a device for that allows for the perioperative addition of a bioactive composition to a porous biomaterial.
  • addition of the bioactive composition to a porous biomaterial in accordance with the presently described embodiments may optionally be performed in advance of a surgical procedure, or may alternatively be performed immediately prior to implantation of an implant in a subject (e.g., at a point-of-care setting).
  • a device suitable for the perioperative addition of a bioactive composition to a porous biomaterial may include a cassette portion operatively coupled to an actuator.
  • the cassette portion may include a body having a cavity with an opening to the surface of the body. No limitations are place on the size, dimensions or configuration of the cassette portion, or of the cavity formed therein, except that the cavity is capable of accepting the porous implant that is to be impregnated, and that the volume of the cavity is larger than the volume of the porous implant.
  • the cassette portion may be made from any suitable rigid, semi-rigid, pliable or resilient material that is able to withstand a pressure differential of up to about 1 atm between the cavity and the outside surface of the cassette, such as, for example, metals, metal alloys, metal polymer composites, polymers materials, and/or thermoplastic materials.
  • the cassette portion may be injection molded from a suitable biocompatible thermoplastic material.
  • the cassette portion may further include a sealing portion.
  • the sealing portion may be coupled to the cassette portion or may be removable from the cassette portion.
  • the sealing portion may be configured such that the shape thereof is substantially complementary to at least a portion of the cavity or of the opening connecting the cavity to the surface of the body of the cassette portion.
  • the sealing portion may be configured such that, when the sealing portion is in operation, a substantially airtight seal may be formed between the sealing portion and the cavity, thereby producing a sealed chamber.
  • the sealing portion may be operatively coupled to an actuator. No limitation is placed on the means by which the sealing portion and the actuator may be coupled, except that the coupling thereof will generally not interfere with the placement and/or retention of a porous biomaterial article and/or the liquid in which it is suspended in the cavity of the cassette portion, nor with the ability of the sealing portion to form an at least partially airtight seal with the cavity of the cassette portion.
  • An actuator may include, without limitation, any system or device that enables an operator apply a vacuum to the porous biomaterial and liquid carrier medium residing within the cavity of the cassette portion.
  • an actuator may include a means (e.g., a squeeze trigger, a plunger, a piston, or the like) for the reversible operation thereof.
  • the actuator may, according to some embodiments, be operable using a single hand motion.
  • an actuator may include a squeeze trigger operatively coupled to the sealing portion.
  • a porous biomaterial article onto which a bioactive composition is to be loaded may be provided to the cavity of the cassette portion.
  • the dimensions of the porous biomaterial article will generally be smaller than the dimensions of the chamber formed when the sealing portion is operatively coupled to the cassette portion, such that when the porous biomaterial article is placed in the cavity and the cavity is sealed by the sealing portion to form the chamber, at least a portion of the chamber volume will not be occupied by the porous biomaterial article.
  • a liquid carrier composition may be provided to cavity.
  • the liquid carrier composition may include any physiologically acceptable solution that stably carries bioactive agents dispersed therein, in combination with one or more bioactive agents.
  • a sufficient volume of said liquid carrier composition will be provided such that the porous article is at least partially suspended therein, and further such that at least a portion of the surface of the porous article is in contact with said liquid carrier composition.
  • the volume of liquid carrier composition provided thereto will typically not exceed the volume of the chamber that is not occupied by the porous biomaterial article when the device is in use.
  • the order in which the article and the liquid carrier composition are provided to the cavity is arbitrary.
  • the device may then be sealed by coupling the cassette portion to the sealing portion thereby sealing the porous article suspended in the liquid carrier composition within the chamber.
  • the actuator may then be engaged such that a vacuum is achieved within the chamber.
  • the pressure within the chamber may be ⁇ about 0.1 atm, ⁇ about 0.2 atm, ⁇ about 0.5 atm, or less than about 1 atm.
  • the vacuum may be maintained for at least about 5 seconds, at least about 10 seconds, at least about 20 seconds, at least about 30 seconds, or at least about 1 minute, after which time, the actuator is released, and the pressure of the chamber return to ambient pressure. Without being bound to any specific mechanism, the vacuum created within the chamber displaces air trapped inside the porous network of the biomaterial.
  • the air that was displaced from the pores attempts to refill the pores, however, since the pharmaceutical carrier liquid is surrounding the biomaterial it is driven into pores that were once occupied by air.
  • the preceding steps may be repeated any number of times until a desired volume of the liquid carrier composition occupies the porous network of the biomaterial. Typically, such may be achieved after about three wet-vacuum cycles.
  • a device that functions in accordance with the preceding description may allow for deep impregnation of pharmaceutical agents to porous implants that would otherwise not have been driven to the core of the biomaterial under normal atmospheric loading.
  • Porous calcium phosphate cement coupons were made by the following procedure.
  • An injectable paste of calcium phosphate cement was prepared by mixing 0.6 g of whiskered
  • TTCP powder made according to the procedures set forth in U.S. Patent Appl. Publ. No. 2004/0003757 with concentrated (NfLO 2 HPO 4 solution in water at a liquid to solid ratio of 0.3 for 1 min.
  • the paste was then thoroughly mixed with a mixture (1:1) of NaCl and KCl salt particles (pore forming powder).
  • the amount of salt mixed with the paste was equal to the dry weight of the salt used to make the paste.
  • the resulting paste mixture was filled into a cylindrical stainless steel mould having a diameter of 12 mm and compressed with a gradually increased pressure up to about 45 MPa and the cement was allowed to harden.
  • the hardened material was immersed in distilled water at 37 0 C for 48 hours and dried in air for 24 hours.
  • Example 1 The dried material made in Example 1 was immersed in Hank's balanced salt solution IX, HyQ ®HBSS cell culture reagents without Phenol Red, 0.1 ⁇ m sterile filtered; HyClone,
  • Example 1 The dried material made in Example 1 was immersed in phosphate buffered saline (PBS) for 5 days, rinsed with distilled water and then dried in air for 24 hours.
  • PBS phosphate buffered saline
  • the hardened CPC discs made in Examples 2 and 3 were gold coated and the surface morphology of nanocrystalline HAp was examined using scanning electron microscopy.

Abstract

Systems and methods suitable for loading/impregnating a porous biomaterial matrix with pharmaceutical agents are provided for herein.

Description

TITLE: METHOD AND APPARATUS FOR IMPREGNATING POROUS BIOMATERIALS WITH BIOACTIVE AGENTS
BACKGROUND OF THE INVENTION 1. Field of the Invention
The present invention generally relates to systems and methods for the preparation of biocompatible porous materials suitable for use as pharmaceutical carriers. More specifically, the invention relates to systems and methods for impregnating a porous biomaterial with pharmaceutical composition. 2. Description of the Relevant Art
The present invention refers to the use of injectable calcium phosphate cements (CPCs) as growth pharmaceutical carriers for bioactive/therapeutic compositions. A phosphate-based setting solution containing excipients such as, biopolymers, amino acids, or carbohydrates, is used to formulate bone growth factors to provide gradual and controlled release of the growth factors for therapeutic tissue repair. The growth factors/cement composites are injectable and have selected setting times and compression strength which render them suitable for use as bone void filler. The growth factors/cement composite can also deliver growth factors to defect site and stimulate bone healing.
Calcium phosphate ceramics, particularly hydroxyapatite (HA) and beta-tricalcium phosphate (TCP), have been reported to promote bone ingrowth and are reabsorbed by the host tissue. Moreover, they have been used as carriers for the sustained release of various therapeutic agents, such as growth factors (see, e.g., Uchida et al, J. Orthoptera Res. 10:440 (1992), Matsumoto et al, Biomaterials 25:3807 (2004), Paul et al, J. Biomater. Appl. 17:253 (2003)). Proteins can be favorably adsorbed on the calcium phosphate ceramics because of electrostatic interactions and delivered into a bony defect where they serve as osteoinductive agents.
Injectable calcium phosphate cements are a desirable and less invasive means to treat bony defects caused by trauma or abnormal metabolic bone conditions such as osteoporosis. However, the addition of growth factors to such preparations has been unsuccessful to date. The major issues associated with injectable formulations of calcium phosphate are entrapment of the growth factor resulting in no release and aggregation or denaturation of the growth factor within the setting solution that renders the protein biologically inactive. Decreasing the interaction between the growth factor and the hydroxyapatite surface and increasing the surface area by using porogens or biodegradable polymers increases the release rate and enables controlled release kinetics of growth factors.
Various polymers have also been used in calcium phosphate bone ceramics as supplementary materials to improve tensile strength and hardness, increase fracture toughness, and setting time (U. S. Pat. 20020187104, Li, et al.) Polymer-bioceramic composite have also been reported in fabrication of porous ceramic matrix, where the polymer is molded into the plurality of pores and the porous ceramic matrix is used as drug delivery vehicle (U. S. Pat. 20040002770). The polymer precursors are formed by polymerizing the polymer precursors in the pores.
In the past (U.S. Pat. No. 5,820,632 to Constantz et al., U.S. Pat. No. 5,525,148 to Chow et al), various additives have been included, e.g. calcium chloride, sodium or potassium hydroxide, sugar, sodium bicarbonate and phosphate salts, which may be leached out so as to provide for porosity in the cement. In addition, porosity has also been generated by including citric acid and sodium bicarbonate to produce carbon dioxide during the setting of a calcium phosphate cement. (U.S. Pat. No 20030019396 to Edwards, Brian et al.) The fabrication of non-injectable porous constructs of this manner can significantly alter the release profile of growth factors. For example, triphasic prolonged release profiles have been observed with the use of a polycaprolactone/tricalcium phosphate (PCL-TCP) composite (see Rai et al, Biomaterials, 26:3739 (2005).
U.S. Patent No. 5,866,155 to Laurencin, et al. entitled "Methods for using microsphere polymers in bone replacement matrices and composition produced thereby" teaches methods of preparing flexible matrices composed of a biodegradable, biocompatible polymer and a calcium phosphate based material for use in three dimensional constructs for tissue engineering, and more specifically, bone replacement.
The publication by Liu et al. entitled "Osteoinductive Implants: The Mise-en-scene for Drug-Bearing Biomimetic Coatings" appearing in March 2004 in Vol. 32, pp. 398-406 of Annals of Biomedical Engineering describes titanium metal alloy implants coated with amorphous calcium phosphate and crystalline HAp, and methods of making same. The biocompatible implants described by Liu can exhibit high compressive strength, but are not bioresorbable. Moreover, the requirement for the deposition of multiple calcium phosphate layers, and thus multiple surface treatments, adds layers of complexity and requires additional quality control measures.
U.S. Patent Application Serial No. 2005/0169964 by Zitelli et al. entitled "Antibiotic calcium phosphate coating" describes a method for applying calcium phosphate surface layers containing therapeutic agents such as antibiotics or bone proteins to a metallic prosthesis.
U.S. Patent Application Serial No. 2005/0170070 by Layrolle et al. entitled "Method for applying a bioactive coating on a medical device" describes ceramic coatings containing bioactive agents formed on the surfaces of medical devices made of inorganic, metallic or organic materials, and methods and systems for making same. The coatings are deposited on the implant surface by passing the implant through a stream of a coating solution in a reactor system.
U.S. Patent Application Serial No. 2005/0031704 by Ahn et al. entitled "Tricalcium phosphates, their composites, implants incorporating them, and method for their production" describes bioceramics, particularly tricalcium phosphate bioceramics, composites incorporating these materials, and methods for their production. The surface of a calcium phosphate powder such as TCP or hydroxyapatite may contain therapeutic compositions (e.g., nucleic acids, proteins, or antibiotics) for drug delivery.
U.S. Patent Application Serial No. 2005/0106260 by Constanz et al. entitled "Calcium phosphate cements comprising an osteoclastogenic agent" describes injectable calcium phosphate cement pastes that include osteoclastogenic agents.
U.S. Patent Application Serial No. 20050119761 by Matsumoto et al. entitled "Porous calcium phosphate ceramic and method for producing same" describes sintered calcium phosphate ceramics with macroporosity for use in medical applications. The ceramic is capable of binding polypeptides. U.S. Patent Application Serial No. 20040091544 by Ruff et al. entitled "Coated dibasic calcium phosphate" describes dibasic calcium phosphate coatings as pharmaceutical carriers for sustained release of orally administered peptides.
U.S. Patent Application Serial No. 20020156529 by Lin et al. entitled "Surface- mineralized spinal implants" describes spinal implants with mineralized bioactive surfaces chemically coated on the implant. The coatings are non-hydroxyl containing carbonated calcium phosphate bone mineral nanocrystalline apatite less than about 1 μm in size.
U.S. Patent No. 6,808,561 to Genge et al. entitled "Biocompatible cement containing reactive calcium phosphate nanoparticles and methods for making and using such cement" describes cement powders that contain reactive tricalcium phosphate nanoparticles and methods of making same.
U.S. Patent No. 5,769,897 to Harle entitled "Synthetic bone," discloses a method of manufacturing synthetic bone. Further, it discloses a vacuum vessel for treating, storing and transporting the artificial bone material according to the invention prior to implantation. U.S. Pat. No. 5,037,377 to Alonso entitled "Means for improving biocompatibility of implants, particularly of vascular grafts," discloses a means for improving biocompatibility of implants. A method of applying soluble collagen to vascular grafts is achieved by vacuum impregnation and impregnation under pressure. The grafts are kept in a vessel and which is evacuated to obtain a pressure differential of 120mmHg (0.84atm).
Examples of vacuum impregnation techniques in other industries include U.S. Patent Nos. 6,913,650 to Gilmore et al. entitled "Component impregnation" and 4,620,991 to Young entitled "Apparatus for the impregnation of porous articles" disclose wet and dry- vacuum impregnation methods and machinery for die cast metal parts. Other U.S. Pat. Nos. that disclose vacuum impregnation methods include; 4,479,986; 4,722,295; 4,931,306; 5,416,159.
The aforementioned prior art references are incorporated by reference as though fully set forth herein.
SUMMARY OF THE INVENTION
It is an object of the present disclosure to provide systems and methods for impregnating a porous biomaterial (e.g., a medical implant) with a liquid pharmaceutical carrier medium carrying one or more bioactive compositions. The following embodiments provide means that are in accordance with the preceding objectives.
In an embodiment, a device suitable for perioperatively impregnating a biomaterial with a pharmaceutical agent is provided for. In an embodiment, the device may be operated at a point of care setting. In an embodiment, the device may be operated with a single-hand motion. In an embodiment, a system for impregnating a porous biomaterial with a bioactive composition may include a cassette portion. The cassette portion may include a body; a cavity within the body, the cavity being sized to accept a porous biomaterial; and an opening from the cavity to a surface of the body. In an embodiment, the cassette portion may include a barrel of a syringe.
In an embodiment, the system may include a sealing portion operatively couplable to the cassette portion. The sealing portion may be configured such that, when coupled to the cassette portion, the opening of the cavity may be sealed to form a substantially airtight chamber disposed in the cassette body. In an embodiment, a sealing portion may include a plunger of a syringe.
In an embodiment, the system may further include an actuator operatively coupled to the sealing portion. The actuator may be configured to reversibly apply a vacuum to the chamber. The actuator may include a squeeze trigger. In an embodiment, the system may further include a porous biomaterial configured to reside within the chamber formed by coupling the cassette potion to the sealing portion. The volume of the porous biomaterial may be less than the volume of the chamber.
In an embodiment, the system may further include a pharmaceutical carrier liquid. The pharmaceutical carrier liquid may include a physiologically acceptable liquid. The pharmaceutical carrier liquid may further include a bioactive composition. In an embodiment, the volume of pharmaceutical carrier liquid may be sufficient to occupy at least a portion of the chamber volume not occupied by the porous biomaterial. In an embodiment, the volume of pharmaceutical carrier liquid may be sufficient to allow at least a portion of the surface of a porous biomaterial residing in the chamber to contact the pharmaceutical carrier liquid.
A method of applying a pharmaceutical composition to a porous biomaterial may include providing a system comprising a cassette portion, the cassette portion comprising a body; a cavity within the body, the cavity being sized to accept a porous biomaterial; and an opening from the cavity to a surface of the body; a sealing portion operatively couplable to the cassette portion, the sealing portion being configured such that, when coupled to the cassette portion, the opening of the cavity is sealed to form a substantially airtight chamber disposed in the cassette body; and an actuator operatively coupled to the sealing portion, the actuator being configured to reversibly apply a vacuum to the chamber when engaged; providing a porous biomaterial to the cavity of the system; providing a pharmaceutical carrier liquid comprising a bioactive composition to the cavity of the system; coupling the cassette portion to the sealing portion, thereby sealing the porous biomaterial and the pharmaceutical carrier liquid in the chamber; and applying a vacuum to the chamber by engaging the actuator.
In an embodiment, a vacuum applied to the chamber may be sufficient to achieve a chamber pressure in the range of 0.1 to about 1.0 atm below ambient pressure. In an embodiment, a vacuum may be maintained for up to about lminute. In an embodiment, the step of applying a vacuum to the chamber by engaging the actuator is performed up to about 3 times or more.
BRIEF DESCRIPTION OF THE DRAWINGS
Other objects and advantages of the invention will become apparent upon reading the following detailed description and upon reference to the accompanying drawings in which:
FIGS. IA-B show S.E.M. images (taken at 10,000-fold magnification) of implant surfaces having nanoporous nanocrystalline calcium phosphate material made by soaking the implant in; Fig. IA) Hank's Balanced Salt Solution (with Ca and Mg) for 3 days; and Fig. IB) phosphate buffered saline for 5 days;
FIG. 2 is a graph depicting the enhanced rate of growth factor (BMP) release from hardened CPC materials in the presence of amino acids; and
FIG. 3 is a graph depicting the enhanced rate of growth factor release from hardened CPC materials in the presence of certain polymer and amino acid/polymer combinations. While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will herein be described in detail. It should be understood, however, that the drawing and detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the intention is to cover all modifications, equivalents and alternatives falling within the spirit and scope of the present invention as defined by the appended claims.
DETAILED DESCRIPTION DEFINITIONS
The terms used throughout this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the various embodiments of the invention and how to make and use them. It will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed in greater detail herein. Synonyms for certain terms may be provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term.
As used herein, a material, composition or object that is "bioresorbable," generally refers to a biocompatible material, composition or object that has the ability to be gradually integrated into a host. When used in the context of the subject prosthetic bone implants, the term generally refers to the ability of at least a portion of the prosthetic bone implant to gradually be replaced by natural bone, such replacement typically occurring naturally by the physiological process of bone remodeling. Non-limiting examples of materials that may be considered to be bioresorbable include certain calcium phosphate ceramics (including but not limited to, for example, hydroxyapatite), collagen, tendon, and grafted bone material. As used herein the term "biodegradable" generally refers to a substance or a composition that can be at least partially broken down in the body or by microorganisms into a simpler substance. While not necessarily being mutually exclusive, the term "biodegradable," is generally not used interchangeably with "bioresorbable." Whereas a biodegradable material may be broken down into its constituent materials, such materials, unlike a bioresorbable material, are not integrated into the host and are released locally or systemically.
The term "biodegradable polymer," is generally employed herein to denote a (generally biocompatible, non-toxic) polymeric material that gradually degrades or erodes in vivo to smaller polymer units and/or its substituent monomers. Such degradation may be the result of, for example, enzymatic, chemical and/or physical processes. Exemplary, though non-limiting, biodegradable polymers suitable for the methods and compositions contemplated herein herein include, for example, poly(lactides), polycaprolactic acid, poly(glycolides), poly(lactide-co- glycolides), poly(gamma-glutamic acid)- sulfonate, polyketals, poly(lactic acid)s, poly(glycolic acid)s, polycarbonates, polyesters, polyesteramides, polyanydrides, poly(amino acids) e.g., polypeptides, polyorthoesters, poly(dioxanone)s, poly(alkylene alkylate)s, copolymers or polyethylene glycol and polyorthoester, biodegradable polyurethane, chitosan, or blends or copolymers thereof. The term is also meant to encompass biocompatible polymer systems such as hydrogels. The term is further meant to encompass polymer systems associated with a particular polymer (including but not limited to those systems incorporating, for example, various modifying agents, additives, cross-linkers, and the like). Advantageously, when used as porogens (defined below) in a hardened calcium phosphate cement matrix loaded with one or more bioactive agents, degradation of certain biodegradable polymer (e.g., PLGA which breaks down into lactic acid and glycolic acid) produces an acidic environment, which influences dissolution of the surrounding calcium phosphate material and release kinetics of bioactive agents associated therewith.
As used herein, terms such as "pharmaceutical carrier," "pharmaceutical composition," "pharmaceutical formulation," "pharmaceutical preparation," or the like, generally refer to formulations that are adapted to deliver a prescribed dosage of one or more pharmacologically active compounds to a cell, a group of cells, an organ or tissue, an animal or a human. The determination of an appropriate prescribed dosage of a pharmacologically active compound to include in a pharmaceutical composition in order to achieve a desired biological outcome is within the skill level of an ordinary practitioner of the art. A pharmaceutical composition may be provided as sustained-release or timed-release formulations. Such formulations may release a bolus of a compound from the formulation at a desired time, or may ensure a relatively constant amount of the compound present in the dosage is released over a given period of time. Terms such as "sustained release," "controlled release," or "timed release" and the like are widely used in the pharmaceutical arts and are readily understood by a practitioner of ordinary skill in the art. Pharmaceutical preparations may be prepared as solids, semi-solids, gels, hydrogels, liquids, solutions, suspensions, emulsions, aerosols, powders, or combinations thereof. Included in a pharmaceutical preparation may be one or more carriers, preservatives, flavorings, excipients, coatings, stabilizers, binders, solvents and/or auxiliaries that are, typically, pharmacologically inert. It will be readily appreciated by an ordinary practitioner of the art that, included within the meaning of the term are pharmaceutically acceptable salts of compounds. It will further be appreciated by an ordinary practitioner of the art that the term also encompasses those pharmaceutical compositions that contain an admixture of two or more pharmacologically active compounds, such compounds being administered, for example, as a combination therapy.
As used herein the terms "subject" generally refers to a mammal, and in particular to a human.
Terms such as "in need of treatment," "in need thereof," "who would benefit from such treatment" and the like, when used in the context of administering a pharmaceutical preparation to a subject, generally refers to a judgment made by an appropriate healthcare provider that an individual or animal requires or will benefit from a specified treatment or medical intervention. Such judgments may be made based on a variety of factors that are in the realm of expertise of healthcare providers, but include knowledge that the individual or animal is ill, will be ill, or is at risk of becoming ill, as the result of a condition that may be ameliorated or treated with the specified medical intervention.
The phrases "therapeutically effective amount" and "effective amount" are synonymous unless otherwise indicated, and mean an amount of a compound of the present invention that is sufficient to improve the condition, disease, or disorder being treated. Determination of a therapeutically effective amount, as well as other factors related to effective administration of a compound of the present invention to a patient in need of treatment, including dosage forms, routes of administration, and frequency of dosing, may depend upon the particulars of the condition that is encountered, including the patient and condition being treated, the severity of the condition in a particular patient, the particular compound being employed, the particular route of administration being employed, the frequency of dosing, and the particular formulation being employed. Determination of a therapeutically effective treatment regimen for a patient is within the level of ordinary skill in the medical or veterinarian arts. In clinical use, an effective amount may be the amount that is recommended by the U.S. Food and Drug Administration, or an equivalent foreign agency. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the mammalian host treated and the particular mode of administration.
By "prophylactically effective amount" is meant an amount of a pharmaceutical composition that will substantially prevent, delay or reduce the risk of occurrence of the biological or physiological event in a cell, a tissue, a system, animal or human that is being sought by a researcher, veterinarian, physician or other caregiver. The term "pharmacologically inert," as used herein, generally refers to a compound (e.g., additives, excipients, binders, vehicles, and the like) that is substantially free of any pharmacologic or "drug-like" activity.
As used herein, the term "bone substitute material" generally refers to any biocompatible composition, including but not limited to natural bone graft, particulate or crushed bone, demineralized bone matrix, ceramics, polymer systems, composites, or mixtures thereof, that is suitable for use in medical/dental applications characterized by replacement, augmentation, or filling of a hardened calcified tissue (e.g., bone, teeth, enamel and the like). In the context of certain descriptions set forth herein, the term refers to calcium phosphate-based ceramic materials (such as cements) that form hardened structures having physical and chemical properties approximating those of natural bone mineral.
As used herein, the term calcium phosphate cement (CPC) generally refers to a biocompatible material having particles of at least calcium phosphate ceramic material which, when combined with a suitable setting liquid forms a self-setting composition that hardens to become calcium phosphate-based solid mass. In certain embodiments, CPC particles may have an average diameter in the range of about 0.1 μm to about 500 μm, in the range of about 0.5 μm to about 10 μm, or in the range of about 0.05 μm to about 50 μm. A variety of CPCs are known in the art and detailed descriptions thereof, as well as their method of manufacture may be found, for example, in the disclosures of U.S. Patent Nos. 7,018,460; 6,994,726; 6,972,130; 6,960,249; 6,955,716; 6,953,594; 6,929,692; 6,916,177; 6,908,506; 6,905,516; 6,855,167; 6,849,275; 6,840,995; 6,821,528; 6,808,561; 6,796,378; 6,793,725; 6,777,001; 6,730,324; 6,719,993; 6,719,773; 6,716,216; 6,706,273; 6,706,067; 6,703,038; 6,692,563; 6,670,293; 6,648,960; 6,642,285; 6,641,587; 6,620,236; 6,616,742; 6,613,054; 6,599,516; 6,596,338; 6,593,394; 6,592,513; 6,582,228; 6,558,709; 6,547,866; 6,541,037; 6,537,589; 6,527,810; 6,495,156; 6,417,247; 6,384,197; 6,379,453; 6,375,935; 6,338,752; 6,325,992; 6,296,667; 6,241,734; 6,224,629; 6,214,368; 6,206,957; 6,149,655; 6,139,578; 6,136,029; 6,117,456; 6,083,229; 6,053,970; 6,051,061; 6,027,742; 6,018,095; 6,005,162; 6,002,065; 5,997,624; 5,993,535; 5,980,625; 5,976,234; 5,968,253; 5,964,932; 5,962,028; 5,954,867; 5,952,010; 5,900,254; 5,891,558; 5,885,540; 5,866,155; 5,846,312; 5,820,632; 5,782,971; 5,709,742; 5,697,981; 5,695,729; 5,683,667; 5,683,496; 5,679,294; 5,605,713; 5,571,493; 5,569,442; 5,545,254; 5,542,973; 5,534,244; 5,525,148; 5,522,893; 5,503,164; 5,496,399; 5,460,803; 5,437,857; 5,427,768; 5,338,356; 5,336,264; 5,281,265; 5,262,166; 5,218,035; 5,178,845; 5,152,836; 5,149,368; 5,053,212; and 4,373,217, all of which are expressly incorporated herein by reference. In some embodiments, CPC particles may refer to particles of one or more of alpha- tricalcium phosphate (α-TCP), beta-tricalcium phosphate (β-TCP), tetracalcium phosphate (TTCP), monocalcium phosphate monohydrate (MCPM), monocalcium phosphate anhydrous (MCPA), dicalcium phosphate dihydrate (DCPD), dicalcium phosphate anhydrous (DCPA), octacalcium phosphate (OCP), calcium dihydrogen phosphate, calcium dihydrogen phosphate hydrate, acid calcium pyrophosphate, anhydrous calcium hydrogen phosphate, calcium hydrogen phosphate hydrate, calcium pyrophosphate, calcium triphosphate, calcium phosphate tribasic, calcium polyphosphate, calcium metaphosphate, anhydrous tricalcium phosphate, tricalcium phosphate hydrate, and amorphous calcium phosphate. In some embodiments, the hardened material formed using CPC particles (which may be referred to a "hardened CPC") may be substantially composed of an apatite material, e.g., hydroxyapatite. CPCs may optionally include one or more additives that affect the physico-chemical properties of hardened cements made therewith. Exemplary though non-limiting additives which may find use in certain embodiments include those additives selected from the group consisting of sodium phosphate (Na3PO4), disodium hydrogen phosphate (Na2HPO4), sodium dihydrogen phosphate (NaH2PO4), disodium hydrogen phosphate dodecahydrate (Na2HPO4- 12H2O), disodium hydrogen phosphate heptahydrate (Na2HPO4-7H20), sodium phosphate dodecahydrate (Na3PO4 12H2O), orthophosphoric acid (H3PO4), calcium sulfate (CaSO4), Ca4(PO4)2O, CaHPO4-2H2O, CaHPO4, Ca8H2(PO4)6-5H2O, alpha-Ca3(PO4)2, beta-Ca3(PO4)2, Ca2P2O7, and Ca2H2P2O8, (NH4)3PO4, (NH4)2HPO4, and (NH4)H2PO4. The terms "setting liquid," "setting solution," and the like generally refer to liquid compositions that, when contacted with cement particles, allow cementing reactions to occur between said particles. Exemplary setting liquids suitable for use in the presently disclosed embodiments include, though are not limited to, acidic solutions, basic solutions, solutions having substantially physiological pH (e.g., 6.0-8.5) or substantially pure water. Suitable acidic solutions may include solutions containing nitric acid (HNO3), hydrochloric acid (HCl), phosphoric acid (H3PO4), carbonic acid (H2CO3), sodium dihydrogen phosphate (NaH2PO4), sodium dihydrogen phosphate monohydrate (NaH2PO^H2O), sodium dihydrogen phosphate dihydrate, sodium dihydrogen phosphate dehydrate, potassium dihydrogen phosphate (KH2PO4), ammonium dihydrogen.phosphate (NH4H2PO4), malic acid, acetic acid, lactic acid, citric acid, malonic acid, succinic acid, glutaric acid, tartaric acid, oxalic acid and their mixture. Suitable basic solutions may include solutions containing ammonia, ammonium hydroxide, alkali metal hydroxide, alkali earth hydroxide, disodium hydrogen phosphate (Na2HPO4), disodium hydrogen phosphate dodecahydrate, disodium hydrogen phosphate heptahydrate, sodium phosphate dodecahydrate (Na3PO4* 12H2O), dipotassium hydrogen phosphate (K2HPO4), potassium hydrogen phosphate trihydrate (K2HPθ4 #3H2O), potassium phosphate tribasic (K3PO4), diammonium hydrogen phosphate ((NH4)2HPO4), ammonium phosphate trihydrate ((NH4)3Pθ4 #3H2O), sodium hydrogen carbonate (NaHCO3), sodium carbonate Na2CO3, and their mixture. Examples of solutions having substantially physiological pH include solutions containing phosphate ions, Tris-buffered solution, HEPES -buffered solutions, Hank's solutions, and the like.
The term "injectable self-setting calcium phosphate cement paste" generally refers to a composition resulting contacting calcium phosphate cement particles with a setting solution in a ratio such that the resulting mixtures is sufficiently viscous to allow the injection thereof through a standard medical syringe. Such a mixture may typically be obtained by mixing the CPC particles and the setting solution in a ratio of about 0.1 mg/ml to about 20 mg/ml. Non-limiting examples of self-setting calcium phosphate cement pastes suitable for use herein are described in U.S. patent application No. 20030216777 entitled "Method of enhancing healing of interfacial gap between bone and tendon or ligament," U.S. patent application No. 20040031420 entitled "Calcium phosphate cement, use and preparation thereof," U.S. patent application No. 20040175320 entitled "Tetracalcium phosphate (TTCP) having calcium phosphate whisker on surface and process for preparing the same," U.S. patent application No. 20050069479 entitled "Method of increasing working time of tetracalcium phosphate cement paste," U.S. patent application No. 20050101964 entitled "Spinal fusion procedure using an injectable bone substitute," U.S. patent application Nos. 20050271740, 20050271741, 20050271742 and 20050268819 entitled "Injectable calcium phosphate cements and the preparation and use thereof," U.S. patent No. 6960249 and U.S. patent application Nos. 20050268820 and 20050279252, U.S. patent application No. 20040003757 entitled "Tetracalcium phosphate (TTCP) having calcium phosphate whisker on surface," U.S. patent application No. 20050268821 entitled "Tetracalcium phosphate (TTCP) with surface whiskers and method of making same," U.S. patent application Nos. 20050274282, 20050274286 and 20050274287 entitled "Calcium phosphate cements made from (TTCP) with surface whiskers and process for preparing same," U.S. patent application Nos. 20050274288 20050279256 20050274289 20060011099 20060011100 entitled "Process for affecting the setting and working time of bioresorbable calcium phosphate cements," U.S. patent Nos. 6379453, 6840995 and U.S. patent application No. 20030121450 entitled "Process for producing fast-setting, bioresorbable calcium phosphate cements," U.S. patent No. 6616742 and U.S. patent application No. 20030078317 entitled "Process for preparing a paste from calcium phosphate cement," U.S. patent No. 6648960 entitled "Method of shortening a working and setting time of a calcium phosphate cement (CPC) paste," U.S. patent No. 6994726 and U.S. patent application No. 20050267592 entitled "Dual function prosthetic bone implant and method for preparing the same," U.S. patent application No. 20050184417 entitled "Method for making a porous calcium phosphate article," U.S. patent application No. 20050029701 entitled "Method for making a molded calcium phosphate article," all of which are commonly owed with the present invention, and the entire contents of which are expressly incorporated by reference as though fully set forth herein. As used herein, the term "porogen" generally refers to any particulate non-toxic biocompatible material that may be incorporated into an injectable CPC formulation and that, upon hardening of said CPC formulation to a hardened CPC, is gradually removed from the hardened CPC matrix (by virtue of its bioresorbability, biodegradability, solubility etc.) leaving a void therein. A porogen may have any 3-dimensional shape, e.g., substantially spherical, substantially cuboidal, substantially cylindrical, substantially pyramidal, substantially ovoid, or irregular in shape. Typically, a dimension of a porogen will be in the range of about 0.01 μm to about 1 mm. Porogens suitable for use in the present embodiments include crystalline materials (e.g., salts such), polypeptides, and polymer compositions.
As used herein, the term "microspheres" generally refers to particles that are substantially spherical or ovoid in shape and have an average mid- sectional diameter that is less than about 2 mm, less than about 1 mm, less than about 0.5 mm, less than about 250 μm, less than about 100 μm, less than about 50 μm, less than about 20 μm, less than about 10 μm, less than about 54 μm, less than about 1 μm or less than about 0.5 μm.
As used herein, the term "microfibers" generally refers to fibers that are that have an average mid-sectional diameter that is less than about 2 mm, less than about 1 mm, less than about 0.5 mm, less than about 250 μm, less than about 100 μm, less than about 50 μm, less than about 20 μm, less than about 10 μm, less than about 54 μm, less than about 1 μm or less than about 0.5 μm. As used herein, the term "whisker," when used in the context of a calcium phosphate material, generally refers to thin, needle-like calcium phosphate crystals that form on the surface of calcium phosphate particles after subjecting the particles to specific processes as defined in the following United States patents and published applications, all of which are commonly owned with the present invention and incorporated herein by reference in their entirety: U.S. patent application No. 20040031420 entitled "Calcium phosphate cement, use and preparation thereof U.S. patent application No. 20040175320 entitled "Tetracalcium phosphate (TTCP) having calcium phosphate whisker on surface and process for preparing the same" U.S. patent application No. 20050069479 entitled "Method of increasing working time of tetracalcium phosphate cement paste" U.S. patent application No. 20050101964 entitled "Spinal fusion procedure using an injectable bone substitute" U.S. patent application Nos. 20050271740,
20050271741, 20050271742 and 20050268819 entitled "Injectable calcium phosphate cements and the preparation and use thereof U.S. patent No. 6960249 and U.S. patent application Nos. 20050268820 and 20050279252 U.S. patent application No. 20040003757 entitled "Tetracalcium phosphate (TTCP) having calcium phosphate whisker on surface" U.S. patent application No. 20050268821 entitled "Tetracalcium phosphate (TTCP) with surface whiskers and method of making same" U.S. patent application Nos. 20050274282, 20050274286 and 20050274287 entitled "Calcium phosphate cements made from (TTCP) with surface whiskers and process for preparing same" U.S. patent application Nos. 20050274288 20050279256 20050274289 20060011099 20060011100 entitled "Process for affecting the setting and working time of bioresorbable calcium phosphate cements" U.S. patent Nos. 6379453 6840995 and U.S. patent application No. 20030121450 entitled "Process for producing fast-setting, bioresorbable calcium phosphate cements" U.S. patent No. 6616742 and U.S. patent application No. 20030078317 entitled "Process for preparing a paste from calcium phosphate cement" U.S. patent No. 6648960 entitled "Method of shortening a working and setting time of a calcium phosphate cement (CPC) paste."
As used herein, the term "interconnected porosity" generally refers to pores or cavities in the body or matrix of the subject prosthetic bone implants that are in fluid communication and that form a continuous network of pores capable of conveying liquids or gases, or materials dissolved therein. Typically, the amount of interconnected porosity of the subject implants is related to the bioresorbability thereof.
As used herein, the term "pore throat diameter" generally refers to the size or diameter of the openings between adjacent pores, or between a pore and the implant surface. As used herein, the term "non-dispersible," when used in the context of the presently described calcium phosphate cements, generally refers to a physical property of the cement whereby a paste made by combining the cement powder with a setting liquid resists dispersion in an aqueous environment. The ability of a calcium phosphate cement paste to resist dispersion may be related to the surface structure of its constituent particles. As used herein, the term "nanocrystalline" generally refers to a ceramic material whose polycrystalline grain structure is reduced from the micron range to the nanometer range. The surface of a nanocrystalline ceramic has physico-chemical properties that distinguish its polycrystalline counterpart and may make it more receptive to binding certain molecules and ions. Nanocrystalline calcium phosphate may be formed through the crystallization of amorphous calcium phosphate.
As used herein, the term "nanoporous" generally refers to a porous material (i.e. a calcium phosphate ceramic) whose average pore diameter is in the nanometer range (typically between 1 to 1000 nm).
As used herein, the term "wicking" generally refers to the ability of a porous calcium phosphate article to convey liquid by capillary action.
As used herein, the term "unsintered," when used in the context of the subject prosthetic bone implants, generally refers to a prosthetic bone implant that is made from a hardened calcium phosphate cement and that has not undergone a high temperature sintering step. While sintered calcium phosphate ceramics exhibit relatively high tensile strength and biocompatibility, they typically are less porous, and as a result are generally not bioresorbable. Thus, unsintered calcium phosphate cement articles retain their porosity, and are therefore more bioresorbable than sintered calcium phosphate ceramics. Included within the term "unsintered" are those bioresorbable calcium phosphate articles or cements that have been treated at a temperature up to 75O0C, up to 5000C, up to 2000C, or up to 5O0C. As used herein, the terms "cortical portion" or "cortical," when used in the context of the subject prosthetic bone implants, generally refers to a portion of the prosthetic bone implant that functions in a load-bearing capacity and whose function and structure are substantially similar to that of naturally occurring cortical or compact bone. As used herein, the terms "cancellous portion," or "cancellous" when used in the context of the subject prosthetic bone implants, generally refer to portions of the subject prosthetic bone implants that are more porous than the cortical portions, and whose structure and function of which are substantially similar to that of naturally occurring trabecular or spongy bone. Due to its high degree of porsity, a cancellous portion has a relatively high surface area and can support tissue ingrowth and infiltration of body fluids and cells. A cancellous portion may also increase the wicking profile of a prosthetic bone implant.
As used herein, the term "apatite" generally refers to a group of phosphate minerals, (typically to hydroxyapatite, fluorapatite, and chlorapatite) having the general chemical formula Ca5(PO4)SX, where X is OH, F, or Cl. The term "hydroxyapatite," sometimes referred to as "HA" or "HAp," as used herein generally refers to a form of apatite with the formula Ca5(PO4)S(OH), but is more typically represnted as Ca10(PO4)6(OH)2 to denote that the crystal unit cell comprises two molecules. Hydroxyapatite is the hydroxylated member of the complex apatite group. The hardness of hydroxyapatite may be altered by replacing the OH ion with other anions (e.g., fluoride, chloride or carbonate). Additionally, HAp has a relatively high affinity for peptides, making it an ideal carrier for the delivery and/or sustained release of polypeptides over long periods of time in situ. Materials that are refered to herein as "apatitic," are generally those materials that have apatite as the major phase (i.e., materials that are substantially comprised of apatite). As used herein, the term "porous biomaterial," when used in the context of an implantable medical article (e.g., a material suitable for implantation in a calcified tissue) generally refers to a hardened calcium phosphate cement (CPC) having 2 vol% porosity or greater. The term encompasses those materials, articles and production methods disclosed in U.S. Patent Application Publication Nos. 2005/0029701 by Lin et al. entitled "METHOD OF MAKING A MOLDED CALCIUM PHOSPHATE ARTICLE"; 2005/0184417; 2005/0186354; 2005/0186449; 2005/0184418; and 2005/0186353, by Lin et al., entitled "METHOD FOR MAKING A POROUS CALCIUM PHOSPHATE ARTICLE"; U.S. Patent No. 6,994,726 and U.S. Patent Application Publication Nos. 2005/0267587; 2005/0267593; 2005/0267589; 2005/0267588; 2005/0263927; 2005/0263928; 2005/0263929; 2005/0263931; 2005/0263919; 2005/0263930; 2005/0263920; 2005/0263921 ; 2005/0267604; and 2005/0263922, by Lin et al., entitled "DUAL FUNCTION PROSTHETIC BONE IMPLANT AND METHOD FOR PREPARING SAME." The patent references disclosed immediately above are commonly owned with the present application, and the entire contents thereof are expressly incorporated by reference in their entirety as though fully set forth herein.
As used herein, the term "crystalline" is an art-recognized term that is used to describe a mineral composition having relatively a well-defined crystal structure, with a unique arrangement of atoms within the component crystals. There are at least 7 art-recognized crystals systems. Pure hydroxyapatite typically crystallizes in the hexagonal crystal system, although alternate crystal structures may be realized by altering the composition of the mineral.
As used herein, the term "amorphous," when used in the context of mineral compositions, generally refers to a relatively unstructured, non-crystalline form of a mineral that is capable of acting as a seed and support for the growth of crystals thereon. As used herein, terms such as "bioactive composition," "bioactive agent" or the like generally refer to compositions or agents that are capable of inducing or affecting a biological response action in a biological system, e.g. by inducing or affecting a therapeutic or prophylactic effect, an immune response, tissue growth, cell growth, cell differentiation, cell proliferation, etc. A bioactive composition/agent may be provided by means of a suitable pharmaceutical delivery vehicle. The delivery vehicle would typically be optimized to stably accommodate an effective dosage of one or more compounds having biological activity. The determination of the effective dose of a bioactive compound that should be included in a bioactive composition to achieve a desired biological response is dependent on the particular compound, the magnitude of the desired response, and the physiological context of the composition. The determination of the effective dose range of a particular bioactive compound or composition is within the skill level of an ordinary practitioner of the art. Components of bioactive compositions may include, by way of non-limiting example, growth factors, bone proteins, analgesics, antibiotics, or other pharmacologically active compounds.
As used herein, a composition that is referred to as being "physiologically acceptable" is a composition that is non-toxic, biocompatible and whose physical and chemical features (e.g., pH, osmolarity, temperature, and the like) fall within a range that is substantially unlikely to induce or be the cause of adverse physiological responses (e.g., inflammation, hypersensitivity, toxicity, and the like). A "physiologically acceptable" aqueous solution will typically have a pH in the range of about 6.0 to about 8.5, in the range of about 7.0 to 8.0, or in the range of about 7.2 to about 7.6. Such a solution will typically have an osmolarity in the range of about 200 to about 500 mOsmol/L, about 250 to about 350 mOsmol/L or about 280 to about 310 mOsmol/L. Thus the term "physiological acceptable salts" is generally meant to encompass those salts, as well as aqueous solutions made therefrom, having the chemical and biological properties described above.
The term "excipient" is a term of the pharmaceutical arts that generally refers to a pharmacologically inert substance or composition that serves as a delivery vehicle or carrier medium for a drug or bioactive composition. An excipient may include one or more binders, stabilizers, fillers, lubricants, preservatives and the like. A variety of compositions or agents that serve as excipient are known in the art and include, by way of non-limiting example only, certain polymers, small carbohydrates, amphiphilic molecules. Specific example of agents that may serve as excipient in the presently disclosed embodiments may include, for example amino acids (e.g. glutamic acid), physiological acceptable salts, sodium phosphates, and small polypeptides. Nevertheless, it will readily be appreciated by an ordinary practitioner of the art that various other excipients may be employed during the practice of the invention without departing from the spirit and scope of the embodiments described herein.
As used herein, an "osteoinductive composition" generally refers to a composition that induces and/or supports the formation, development and growth of new bone, and/or the remodeling of existing bone. An osteoinductive composition typically includes one or more osteogenic agents. An "osteogenic agent," as used herein, is an agent that can elicit, facilitate and/or maintain the formation and growth of bone tissue. Many osteogenic agents function, at least in part, by stimulating or otherwise regulating the activity of osteoblast and/or osteoclasts. Exemplary osteogenic agents include certain polypeptide growth factors, such as, osteogenin, Insulin-like Growth Factor (IGF)-I, IGF-II, TGF-βl, TGF-β2, TGF-β3, TGF-β4, TGF-β5, osteoinductive factor (OIF), basic Fibroblast Growth Factor (bFGF), acidic Fibroblast Growth Factor (aFGF), Platelet-Derived Growth Factor (PDGF), vascular endothelial growth factor (VEGF), Growth Hormone (GH), growth and differentiation factors (GDF)-5 through 9, osteogenic protein- 1 (OP-I) and any one of the many known bone morphogenic proteins (BMPs), including but not limited to BMP-I, BMP-2, BMP-2A, BMP-2B, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-8b, BMP-9, BMP-IO, BMP-Il, BMP- 12, BMP-13, BMP-14, BMP-15. An osteoinductive composition may include one or more agents that support the formation, development and growth of new bone, and/or the remodeling thereof. Typical examples of compounds that function in such a supportive manner include, though are not limited to, extracellular matrix-associated bone proteins (e.g., alkaline phosphatase, osteocalcin, bone sialoprotein (BSP) and osteocalcin in secreted phosphoprotein (SPP)-I, type I collagen, fibronectin, osteonectin, thrombospondin, matrix-gla-protein, SPARC, alkaline phosphatase and osteopontin). As used herein, the term "growth factor" generally refers to a factor, typically a polypeptide, which affects some aspect of the growth and/or differentiation of cells, tissues, organs, or organisms.
As used herein, the term "bone morphogenic protein," or "BMP" generally refers to a group of polypeptide growth factors belonging to the TGF- β superfamily. While BMPs are widely expressed in a variety of tissues, many function, at least in part, by affecting the formation, maintenance, structure or remodeling of bone and/or other calcified tissues. Members of the BMP family are potentially useful as therapeutics. For example, BMP-2 has been shown in clinical studies to be of use in the treatment of a variety of bone-related conditions.
As used herein, the term "bone protein" generally refers to a polypeptide factor that supports the growth, remodeling, mineralization or maintenance of calcified tissues. Bone proteins are typically components of extracellular matrix (ECM), or associate with cells and structures that form ECM structures. Exemplary though non-limiting bone proteins that may find use in the embodiments provided for herein may include alkaline phosphatase, osteocalcin, bone sialoprotein (BSP) and osteocalcin in secreted phosphoprotein (SPP)-I, type I collagen, type IV collagen, fibronectin, osteonectin, thrombospondin, matrix-gla-protein, SPARC, alkaline phosphatase and osteopontin. One or more bone proteins may be included in an osteoinductive composition. As used herein, the term "antibiotic" generally refers to a naturally occurring, synthetic or semi- synthetic chemical substance that, when diluted in an aqueous medium, kills or inhibits the growth of microorganisms and can cure or treat infection.
As used herein, the term "analgesic" is used in reference to a pharmacologically active agent or composition that alleviates pain without causing loss of consciousness As used herein, the term "polypeptide" generally refers to a naturally occurring, recombinant or synthetic polymer of amino acids, regardless of length or post-translational modification (e.g., cleavage, phosphorylation, glycosylation, acetylation, methylation, isomerization, reduction, farnesylation, etc...), that are covalently coupled to each other by sequential peptide bonds. Although a "large" polypeptide is typically referred to in the art as a "protein" the terms "polypeptide" and "protein" are often used interchangeably. The term "portion", as used herein in the context of a polypeptide (as in "a portion of a given polypeptide/polynucleotide") generally refers to fragments of that molecule. The fragments may range in size from three amino acid or residues to the entire molecule minus one amino acid. Thus, for example, a polypeptide "comprising at least a portion of the polypeptide sequence" encompasses the polypeptide defined by the sequence, and fragments thereof, including but not limited to the entire polypeptide minus one amino acid. A polypeptide may be made using recombinant means, or may be isolated from its natural source (i.e., partially purified). As used herein, the term "recombinant," when used in reference to a polypeptide, generally refers to a protein, or a fragment thereof, that is made and/or at least partially purified using recombinant DNA technology. Techniques for the production of recombinant proteins are widely known in the art. Briefly, a nucleotide sequence encoding the protein (or portion thereof) that is to be expressed is inserted in-frame into an expression vector (e.g., a viral, bacterial, fungal, yeast, plant, insect or mammalian expression vector) that includes DNA sequences that are required for transcription of the encoding nucleotide sequence and the subsequent translation of the RNA resulting therefrom. The expression vector is then introduced into an appropriate cell system in culture. The recombinant protein produced by cultured cells may be at least partially purified using a variety of techniques. General guidance in techniques used for the production and purification of recombinant proteins may be found, for example, in Sambrook, J. et al. Molecular Cloning: A Laboratory Manual. 2nd, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, which is incorporated herein by reference.
As used herein, the term "at least partially purified" when used in the context of a polypeptide or a composition containing a polypeptide, generally refers to an expressed polypeptide that is at least partially free from other cellular material, or culture medium that was present during the production thereof.
The term "autogenic bone" generally refers to bone material derived from a substantially genetically identical reference source (i.e., the same subject or a genetic clone thereof). Typically, autogenic bone refers to material derived from bone of the same subject in which the material will ultimately be used.
The term "allogenic bone" generally refers to bone material that is derived from a genetically distinct source belonging to the same species as the subject in which the bone material will ultimately be used. The term "xenogenic bone" generally refers to bone material that is derived from a different species as the subject in which the bone material will ultimately be used.
The following embodiments provide for porous, bioresorbable calcium phosphate bone replacement compositions which harden to materials having high compressive strength (> 20 MPa) and which also function as pharmaceutical carriers for bioactive compositions. The presently described embodiments are further directed methods of making same. The bone replacement compositions will typically be made using calcium phosphate cement (CPC) pastes that self -harden to compositions that are substantially apatitic in nature. CALCIUM PHOSPHATE CEMENTS
Calcium phosphate cements suitable for use with the presently described embodiments, including their method of manufacture and use, may include without limitation, those disclosed in U.S. Patent Nos. 6,379,453 and 6,840,995 to Lin et al., entitled "PROCESS FOR PRODUCING FAST SETTING, BIORESORBABLE CALCIUM PHOSPHATE CEMENT"; U.S. Patent Appl. Publ. No. 2004/0031420 by Lin et al., entitled "CALCIUM PHOSPHATE CEMENT, USE AND PREPARATION THEREOF"; U.S. Patent No. 6,960,249 to Lin et al. entitled "TETRACALCIUM PHOSPHATE (TTCP) HAVING CALCIUM PHOSPHATE WHISKER ON SURFACE"; U.S. PATENT APPL. PUBL. NO. 2004-0175320, by Lin et al. entitled "TETRACALCIUM PHOSPHATE (TTCP) HAVING CALCIUM PHOSPHATE WHISKER ON SURFACE AND PROCESS FOR PREPARING THE SAME"; Int'l Patent Appl. Publ. No. WO 2004/094335 by Lin et al entitled "CALCIUM PHOSPHATE CEMENT, USE AND PREPARATION THEREOF"; U.S. Patent Appl. Publ. No. 2005/0076813 by Lin et al. entitled "PROCESS FOR PRODUCING FAST-SETTING BIORESORBABLE CALCIUM PHOSPHATE CEMENT"; U.S. Patent Appl. Publ. No. 2005-0069479 by Lin et al. entitled "METHOD OF INCREASING WORKING TIME OF TETRACALCIUM PHOSPHATE CEMENT PASTE"; U.S. Patent Appl. Publ. No. 2005-0271741by Lin et al. entitled "CALCIUM PHOSPHATE CEMENT, USE AND PREPARATION THEREOF"; U.S. Patent Appl. Publ. No. 2005-0271740 by Lin et al. entitled "CALCIUM PHOSPHATE CEMENT, USE AND PREPARATION THEREOF"; U.S. Patent Appl. Publ. No. 2005-0271742 by Lin et al. entitled "CALCIUM PHOSPHATE CEMENT, USE AND PREPARATION THEREOF"; U.S. Patent Appl. Publ. No. 2005-0268819 by Lin et al. entitled "CALCIUM PHOSPHATE CEMENT, USE AND PREPARATION THEREOF"; U.S. Patent Appl. Publ. No. 2005- 0279252 by Lin et al. entitled "TETRACALCIUM PHOSPHATE (TTCP) HAVING CALCIUM PHOSPHATE WHISKER ON SURFACE"; U.S. Patent Appl. Publ. No. 2005-0268820 by Lin et al. entitled "TETRACALCIUM PHOSPHATE (TTCP) HAVING CALCIUM PHOSPHATE WHISKER ON SURFACE"; U.S. Patent Appl. Publ. No. 2005-0268821 by Lin et al. entitled "TETRACALCIUM PHOSPHATE (TTCP) HAVING CALCIUM PHOSPHATE WHISKER ON SURFACE"; U.S. Patent Appl. Publ. No. 2005-0274287 by Lin et al. entitled "TETRACALCIUM PHOSPHATE (TTCP) HAVING CALCIUM PHOSPHATE WHISKER ON SURFACE AND PROCESS FOR PREPARING THE SAME"; U.S. Patent Appl. Publ. No. 2005-0274286 by Lin et al. entitled "TETRACALCIUM PHOSPHATE (TTCP) HAVING CALCIUM PHOSPHATE WHISKER ON SURFACE AND PROCESS FOR PREPARING THE SAME"; U.S. Patent Appl. Publ. No. 2005-0274282 by Lin et al. entitled
"TETRACALCIUM PHOSPHATE (TTCP) HAVING CALCIUM PHOSPHATE WHISKER ON SURFACE AND PROCESS FOR PREPARING THE SAME"; U.S. Patent Appl. Publ. No. 2005-0274288 by Lin et al. entitled "PROCESS FOR PRODUCING FAST-SETTING BIORESORBABLE CALCIUM PHOSPHATE CEMENT"; U.S. Patent Appl. Publ. No. 2005- 0279256 by Lin et al. entitled "METHOD OF INCREASING WORKING TIME OF
TETRACALCIUM PHOSPHATE CEMENT PASTE"; U.S. Patent Appl. Publ. No. 2006- 0011100 by Lin et al. entitled "PROCESS FOR PRODUCING FAST-SETTING BIORESORBABLE CALCIUM PHOSPHATE CEMENT"; and U.S. Patent Appl. Publ. No. 2006-0011099 by Lin et al. entitled "PROCESS FOR PRODUCING FAST-SETTING BIORESORBABLE CALCIUM PHOSPHATE CEMENT" all of which are commonly owned with the present application and the entire contents of which are hereby incorporated by reference in their entirety as though fully set forth herein. The cements are equally suitable for the production of injectable biomaterials (e.g., injectable CPC formulations), or for the production of hardened CPC implants. In some embodiments, calcium phosphate cements may be formed from acidic calcium phosphates (e.g., calcium phosphates having a calcium to phosphorous ratio of less than 1.33), basic calcium phosphates (e.g., calcium phosphates having a calcium to phosphorous ratio of greater than 1.33) or combinations of acidic and basic calcium phosphates. The presently described CPCs may optionally include one or more bioactive compositions dispersed or dissolved therein, such as are described in detail below.
Particularly suited to the presently described embodiments are CPCs made using calcium phosphate particles having whiskers on the surface of the particles, such as are disclosed in the above-cited references and incorporate by reference herein. Without being bound by any particular mechanism of action, it is believed that the whiskers described in these references increase the surface area of cement particles and allow for improved cementing reactions to occur, resulting in hardened materials having improved compressive strength. Additionally, and by virtue of their ability to form interlocking complexes with the whiskers of adjacent particles, surface whiskers advantageously allow a CPC paste to be non-dispersive in aqueous solutions. Thus, these non-dispersive pastes are well suited to therapeutic applications in which a CPC paste is injected to a site the body of the subject where there exists the possibility that the paste would be washed away by body fluids prior to the hardening thereof. Methods of forming whiskers In an embodiment, whiskers comprising TTCP may be formed on the surface of TTCP particles by soaking the particles in an aqueous phosphate solution having basic pH. Without being bound by any particular theory or mechanism of action, crystalline TTCP that is exposed to alkaline solutions (typically at a pH of about 8.0) for a period of several minutes (e.g. typically bout 5 minutes), may result in the dissolution of a portion of the calcium phosphate material into the aqueous surrounding. The loss of the calcium phosphate material into the aqueous solution may contribute to the formation of TTCP crystals on the surface of TTCP particles (e.g. etching). Typically, the etching seen during formation of the whiskers described above and in the above- cited references follows the grain boundaries of the calcium phosphate crystals.
A portion of the dissolved calcium may react with dissolved phosphate ions in the aqueous surroundings to form amorphous calcium phosphate precipitate. This precipitate may further contribute to the size and shape of calcium phosphate whiskers.
In an embodiment, whiskered TTCP particles may be contacted with a setting solution and heated to result in a hardened apatitic cement suitable for use as an injectable bone filler material, or for use in the manufacture of prosthetic bone implants. Modified calcium phosphate cement compositions suited for use in the presently described embodiments may be chosen according certain chemical and/or physical properties that are advantageous for therapeutic use. It is desirable that the constituent CPCs used herein have the ability to harden into biomaterials having relative high compressive strength. For example, a CPC composition may generally be chosen such that a hardened cement made therefrom has a compressive strength of > 30 MPa, > 50 MPa, or > 100 MPa. In some embodiments, CPC compositions may be chosen such that, when mixed with an appropriate setting solution, a paste having sufficient viscosity so as to allow the paste to be injected through a syringe or other aperture to a site within a body or a mold will be formed. The preceding two parameters are, at least in part, related to the density of whiskers on the surface of constituent calcium phosphate particles, and to the density of particles comprising the paste. The density of surface whiskers will typically be in a range such that the resulting material has the desired characteristics of being non-dispersive and able to withstand high compressive forces, while allowing the paste to remain injectable. Typically, such characteristics may be realized when the density of surface whiskers is > 2.0/μm2 and less than 100/μm2.
In order for CPC materials to be of therapeutic use in a point-of-care setting, a paste made therefrom should have a setting time and working time that is greater than 1 minute and less than 45 minutes. U.S. Patent Application No. 2005/0069479 to Lin et al. entitled "METHOD OF INCREASING WORKING TIME OF TETRACALCIUM PHOSPHATE CEMENT PASTE," which are commonly owned with the present applications and fully incorporated by reference herein, discloses methods to manipulate the setting and working times of various calcium phosphate compositions. For example, 2005/0069479 discloses that heating a TTCP paste to between about 5O0C to 35O0C for at least one minute, results in a CPC paste having a working time and setting time of between about 8 to 45 minutes and about 9.5 minutes to about one hour, respectively. Calcium phosphate prosthetic bone implants
The prosthetic bone implants suitable for use in the presently described embodiments will be any porous biomaterial made from hardened, bioresorbable calcium phosphate cements (CPC), without limitation on the structure, shape, size and/or configuration of the of said implants. In certain embodiments, porous biomaterials suitable for use with the presently disclosed embodiments may include, though are not limited to, those disclosed in U.S. Patent Application Publication Nos. 2005/0029701 by Lin et al. entitled "METHOD OF MAKING A MOLDED CALCIUM PHOSPHATE ARTICLE"; 2005/0184417; 2005/0186354; 2005/0186449; 2005/0184418; and 2005/0186353, by Lin et al., entitled "METHOD FOR
MAKING A POROUS CALCIUM PHOSPHATE ARTICLE"; U.S. Patent No. 6,994,726 and U.S. Patent Application Publication Nos. 2005/0267587; 2005/0267593; 2005/0267589; 2005/0267588; 2005/0263927; 2005/0263928; 2005/0263929; 2005/0263931; 2005/0263919; 2005/0263930; 2005/0263920; 2005/0263921; 2005/0267604; and 2005/0263922, by Lin et al., entitled "DUAL FUNCTION PROSTHETIC BONE IMPLANT AND METHOD FOR
PREPARING SAME," all of which are commonly owned with the present application, and the entire contents thereof are incorporated by reference in their entirety as though fully set forth herein.
In an embodiment, apatite comprising the implant body may be made in the absence of a sintering step. Without being bound by any specific theory or mechanism of action, the lack of a sintering step serves, at least in part, to preserve micro- and nano-sized pores present in the matrix of the calcium phosphate material. The presence of such porosity in the finished CPC article advantageously allows the article to have a wicking profile that encourages the penetration thereof by body fluids as well as infiltration of the implant by cells (e.g. osteoblasts, osteoclasts supportive cells) when compared to implants that are made from conventional sintered CPC.
The hardened CPC will typically be at least partially porous (e.g., as a "porous block" or a "porous biomaterial"), and may accommodate porosity up to about 90-vol%. In general, interconnected porosity of a calcium phosphate implant is directly related to its bioresorbability, and inversely related to its compressive strength. The relationship between porosity, bioresorbability and compressive strength of an implanted may be exploited to configure an implant having both high compressive strength (typically > 50 MPa and up to 170 MPa), and high bioresorbability. In an embodiment, the subject prosthetic bone implants may be adapted to withstand compressive forces equal to or in excess of those typically exerted on naturally occurring bone may be accomplished by coupling hardened calcium phosphate articles having different porosities to each other in configurations that are optimally suited for implantation of the implant in or near a bone of a subject. Typically, a dense CPC block will be less than 40 % by volume and will function in a load bearing capacity, whereas a porous CPC block will be 20- 90% by volume. The porosity of the calcium phosphate matrix may be controlled by altering one or more process and or composition parameters during manufacture of the implant. By way of non-limiting example, the porosity of an implant may be readily controlled by, for example, including a pore forming powder or changing the ratio of a pore forming agent in the CPC. In some embodiments, the porosity of the implant may be constant throughout the calcium phosphate matrix.
By way of non-limiting example, and without intending to limit the scope of the presently described embodiments, prosthetic bone implants particularly suited to presently described embodiments are described in the following U.S. Patent Applications: U.S. Patent Application Serial No. 10/780,728 by Lin et al., entitled "METHOD FOR MAKING A
POROUS CALCIUM PHOSPHATE ARTICLE"; U.S. Patent Application Serial No. 10/852,167 by Lin et al., entitled "DUAL FUNCTION PROSTHETIC BONE IMPLANT AND METHOD FOR PREPARING SAME"; and U.S. Patent Application Serial No. 10/633,511 by Lin et al., entitled "METHOD OF MAKING A MOLDED CALCIUM PHOSPHATE ARTICLE"; all of which are commonly assigned with the present invention and which are hereby incorporated by reference as though fully set forth herein. The unsintered prosthetic bone implants described by Lin are biocompatible, bioresorbable, and can be adapted to withstand compressive forces up to 170 MPa. In some embodiments, an implant may be adapted to have varying porosity throughout the calcium phosphate matrix. The implant may optionally be configured to functionally and structurally mimic the configuration of natural occurring bone, with a denser, load bearing cortical portion, and one or more porous cancellous portions integrally disposed therein. Such a configuration may optimize penetration of body fluids and tissue ingrowth into the implant body. In some embodiments, an implant may have a load bearing cortical portion having at least two opposite surfaces and a cancellous portion integrally disposed in the cortical portion and being exposed through the two opposite sides. Both the cancellous portion and the cortical portion may be formed from hardened calcium phosphate cement. In some embodiments, the cancellous portion may have a porosity that is greater than the porosity of the cortical portion. The porosity of the cancellous portion may be at least about 20% by volume. In some embodiments, the cortical portion may also be formed from a porous calcium phosphate cement. The cortical portion may have a porosity of less than about 40% by volume.
In addition to the implant configurations disclosed in the above-cited references, certain embodiments may be directed to implants having improved bioresorbability properties.
Improved bioresorbability may be realized, at least in part, by including an additional layer of nano- and micro-sized porosity to the surface of the implant. In embodiments, the outer porous layer will be at least 100 μm in thickness. Implants that incorporate such an outer porous layer will exhibit improved wicking profiles, and may allow body fluids, vascularization and cellular infiltration of the implant from the exterior of the implant. Such may be readily achieved by coupling a porous component to the exterior surface of the implant during the manufacture thereof. Alternatively, an exterior porous layer may be formed on the surface of the implant by subjecting the implant surface to a treatment that forms a layer of porous calcium phosphate material on the surface thereof. Such treatments are described in detail below. The bioresorbability of a calcium phosphate implant is related the size and interconnectedness of pores distributed throughout the matrix or body of the implant. Ideally, the pores will be large enough to allow body fluid wicking and osteoblast infiltration. Typically, infiltration of osteoblasts is facilitated when at least a portion of the pores have openings and/or pore throat diameters of approximately 100 μm or larger. Pore throat diameter and pore opening diameter ranges may be from 100-500 μm to about 100-300 μm, respectively. CPC implants having defined porosity
In an embodiment, pore size, pore throat diameter, and the degree of interconnected porosity may be manipulated by incorporating within a CPC paste one or more porogens having defined size and/or shape. While any number of porogens familiar to the skilled practitioner may be used in the present embodiments without departing from the spirit and scope thereof, porogens that are particularly contemplated for use herein include those porogens that may be removed from the hardened CPC material after the setting thereof, and which, when removed, leave a space or void in the hardened CPC matrix. Exemplary porogens that may find use in the presently described embodiments include, though are not limited to, water-soluble materials (e.g., crystalline salts) and/or biodegradable polymers such as are disclosed above and incorporated herein.
Porogens incorporated into a CPC paste may be varied in size and/or shape, and the proportions thereof may be selected such that a desired degree and configuration of interconnected porosity is achieved. In some embodiments, porogens may be substantially spherical or ovoid in shape. Of particular use in certain non-limiting embodiments may be those porogen microspheres having average diameter of less than about 5 mm; less than about 2.5 mm; less than about lmm; less than about 750 μm; less than about 500 μm; less than about 300 μm; less than about 200 μm; less than about 100 μm; less than about 50 μm; less than about 20 μm; less than about 10 μm; less than about 5 μm; less than about 2.5 μm; less than about 1 μm; less than about 0.5 μm; or less than about 0.1 μm. In some embodiments, porogens may be substantially cuboidal in shape. In some embodiments, porogens may be substantially pyramidal in shape. Of particular use in certain non-limiting embodiments may be those porogen microparticles having average particle volume of less than about 0.5 mm3; less than about 0.25 mm3; less than about 0.1 mm3; less than about 0.075 mm3; less than about 0.05 mm3; less than about 0.030 mm3; less than about 0.020 mm3; less than about 0.010 mm3; less than about 0.005 mm3; less than about 0.002 mm3; less than about 0.001 mm3; less than about 0.0005 mm3; less than about 0.00025 mm3; less than about 0.0001 mm3; less than about 0.00005 mm3; or less than about 0.00001 mm3. In some embodiments, porogens may be configured as fibers. Of particular use in certain non-limiting embodiments may be those porogen microfibers having average cross-sectional diameter of less than about 5 mm; less than about 2.5 mm; less than about lmm; less than about 750 μm; less than about 500 μm; less than about 300 μm; less than about 200 μm; less than about 100 μm; less than about 50 μm; less than about 20 μm; less than about 10 μm; less than about 5 μm; less than about 2.5 μm; less than about 1 μm; less than about 0.5 μm; or less than about 0.1 μm.
Moreover, incorporating porogens of varying geometry into the cement paste may improve the interconnected porosity of an implant. In an embodiment, the relative degree of interconnected porosity of an implant may be manipulated by varying the ratio of porogen having different particle size and geometry (e.g. combination of spherical and cuboidal particles). Certain embodiments may incorporate microfiber porogens therein. In the non- limiting embodiment where the porogen includes mixtures of one or more salts, KCl, whose crystal structure is substantially spherical in shape may be used to incorporate microspherical pores in the matrix of a hardened CPC material, and NaCl, whose crystal structure is substantially cuboidal in shape may be used to incorporate microcuboidal pores in the matrix of a hardened CPC material. Of course, it will be readily appreciate by an ordinary practitioner of the art that various mixtures of such porogens may be employed to result in a hardened CPC matrix having pores of defined density, interconnectedness and geometry without departing from the spirit and scope of the present invention. Moreover, porogens may be formulated so as to incorporate one or more therapeutically active compositions therein.
Generally, spherical salt particles will have less adverse effect on the mechanical strength of the implant, but do not allow maximum interconnected porosity. The lack of interconnected communication between adjacent pores in the implant body may be remedied by including non- spherical salt crystals therein. The degree of interconnected porosity may be further manipulated by varying the ratio of spherical to non-spherical salt crystals. In an embodiment, the ratio of spherical to non-spherical salt crystals comprising the pore forming powder will be from about 9:1 to about 1:4, or from 3:4 to about 1:4. In an embodiment, the ratio of spherical to non- spherical salt crystals comprising the pore forming powder will be about 1:1.
In an embodiment, interconnected porosity and pore size may be influenced by the average particle size of constituent particles comprising the hardened cement. Typically, CPC particles having an average diameter between about 0.1 μm to about 500 μm are used to form the implants. When forming CaP implants having interconnected porosity, the ratio of porogen to CPC powder (dry weight ratio) will not exceed about 1:1. Using higher ratios may adversely affect the compressive strength of the resulting implant.
In a further non-limiting embodiment, microcavities and or internal voids may be created in the body of the subject implants by suspending one or more porogens therein. The density of porogen particles is such that they do not substantially touch adjacent particles. The particles may function as drug reservoirs when the drug is loaded therein. Advantageously, the microcavities formed in this manner may serve as reservoirs for bioactive compositions, thus increasing the elution time and or effective treatment time of a pharmaceutical agent. The porogens may be removed from the hardened calcium phosphate implant by soaking the implant in an aqueous solution, as set forth in above-cited references. The porogens may alternatively be removed by allowing the degradation, dissolution or resorption thereof after the biocomposite material is in place in a calcified tissue. Bioactive Compositions
In some embodiments, incorporating one or more bioactive agents into a prosthetic implant may enhance the biocompatibility and/or bioresorbability of the implant. Constituents of the bioactive composition may be selected to impart certain advantageous therapeutic or physiological properties on the implant. In an embodiment, bioactive agents may include one or more osteoinductive compounds.
The local inclusion of one or more osteoinductive compounds with the implant in situ may accelerate healing, vascularization, tissue and cellular infiltration of the implant. Suitable osteoinductive compounds include osteogenic compounds. Numerous osteogenic compounds are known to practitioners of ordinary skill in the art including any one of a number of polypeptide growth factors known for their ability to induce the formation or remodeling of bone. By way of non-limiting example, osteogenic compounds suitable for inclusion in the presently described embodiments include, but are not limited to, osteogenin, Insulin-like Growth Factor (IGF)-I, IGF-II, GDF-5 through GDF-9, Transforming Growth Factor (TGF)-βl, TGF- β2 , TGF- β3 , TGF- β4, TGF- β5, osteoinductive factor (OIF), basic Fibroblast Growth Factor (bFGF), acidic Fibroblast Growth Factor (aFGF), Platelet-Derived Growth Factor (PDGF), vascular endothelial growth factor (VEGF), Growth Hormone (GH), and osteogenic protein- 1 (OP-I). In certain embodiments, growth factors belonging to the Bone Morphogenic Protein (BMP) family of growth factors, which include, but are not limited to, BMP-I, BMP-2A, BMP- 2B, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-8b, BMP-9, BMP-IO, BMP-Il, BMP- 12, BMP- 13, BMP- 14, BMP- 15, or combinations thereof, may be especially suited for inclusion in the subject implants.
In some embodiments, bioactive agents may include one or more compounds that support the formation, development and growth of new bone, and/or the remodeling thereof. Typical examples of compounds that function in such a supportive capacity include, though are not limited to, bone matrix proteins (e.g., alkaline phosphatase, osteocalcin, bone sialoprotein (BSP) and osteocalcin in secreted phosphoprotein (SPP)-I, type I collagen, type IV collagen, fibronectin, osteonectin, thrombospondin, matrix-gla-protein, SPARC, alkaline phosphatase and osteopontin). In an embodiment, a peptide or peptide fragment may contain the amino acid sequence Arg-Gly-Asp, which has been shown to bind to and enhance the recruitment of osteoblasts.
Bioactive agents may, in some embodiments, further include pharmacologically active compounds that do not act locally to stimulate bone growth and healing, but that may nonetheless be therapeutically advantageous in certain applications, such as, for example, antibiotic and or analgesic agents. Exemplary analgesic agents suitable for use herein include, but are not limited to, norepinephrine, bupivacaine, ropivacaine, 2-chloroprocaine, lidocaine, mepivacaine, ropivacaine, mepivacaine, benzocaine, tetracaine, dibucaine, cocaine, prilocaine, dibucaine, procaine, chloroprocaine, prilocaine, mepivacaine, etidocaine, tetracaine, xylocaine, morphine, fentanyl, alphaxalone and active analogs, 5-alpha-pregnane-3 alpha-21-diol-20-one (tetrahydro-deoxycorticosterone or THDOC), allotetrahydrocortisone, dehydroepiandrosterone, benzodiapenes, nifedipine, nitrendipine, verapamil, aminopyridine, benzamil, diazoxide, 5,5 diphenylhydantoin, minoxidil, tetrethylammonium, valproic acid, aminopyrine, phenazone, dipyrone, apazone, phenylbutazone, clonidine, taxol, colchicines, vincristine, vinblastine, levorphanol, racemorphan, levallorphan, dextromethorphan, cyclorphan, butorphanol, codeine, heterocodeine, morphinone, dihydromorphine, dihydrocodeine, dihydromorphinone, dihydrocodeinone, 6-desoxymorphine, heroin, oxymorphone, oxycodone, 6-methylene- dihydromorphine, hydrocodone, hydromorphone, metopon, apomorphine, normorphine, N-(2- phenylethyl)-normorphine, etorphine, buprenorphine, phenazocine, pentazocine and cyclazocine, meperidine, diphenoxylate, ketobemidone, anileridine, piminodine, fentanil, ethoheptazine, alphaprodine, betaprodine, l-methyl-4-phenyl-l,2,5,6-tetrahydropyridine (MPTP), loperamide, sufentanil, alfentanil, remifentanil, lofentanil, 6,7-benzomorphans, ketazocine, aryl-acetamides, U-50,488, spiradoline (U-62,066), enadoline (CI-977), asimadoline, EMD-61753, naltrexone, naltrindole. Exemplary though non-limiting antibiotic agents include, but are not limited to, tylosin tartrate, tylosin, oxytetracycline, tilmicosin phosphate, ceftiofur hydrochloride, ceftiofur sodium, sulfadimethoxine cefamandole, tobramycin, penicillin, cefoxitin, oxacillin, vancomycin, cephalosporin C, cephalexin, cefaclor, cefamandole, ciprofloxacin, bisphosphonates, isoniazid, ethambutol, pyrazinamide, streptomycin, clofazimine, rifabutin, fluoroquinolones, ofloxacin, sparfloxacin, rifampin, azithromycin, clarithromycin, dapsone, tetracycline, erythromycin, ciprofloxacin, doxycycline, ampicillin, amphotericine B, ketoconazole, fluconazole, pyrimethamine, sulfadiazine, clindamycin, lincomycin, pentamidine, atovaquone, paromomycin, diclarazaril, acyclovir, trifluorouridine, foscarnet, penicillin, gentamicin, ganciclovir, iatroconazole, miconazole, Zn-pyrithione.
The amount of a pharmacologically active agent to include in the subject bioactive coating compositions may typically vary with the identity of the agent, the physiological context in which the agent is being employed, and the magnitude of the desired response. Typical dosages of pharmacologically active agents that will be loaded onto the calcium phosphate carrier may be in the range of 2 ng/m3 to 1 mg/m3, according to the volume of pharmaceutical carrier used to deliver the bioactive agent. General guidance in determining effective dose ranges for pharmacologically active compounds may be found, for example, in the publications of the International Conference on Harmonisation and in REMINGTON'S PHARMACEUTICAL SCIENCES, chapters 27 and 28, pp. 484-528 (Mack Publishing Company 1990), which is incorporated by reference as though fully set forth herein.
Bioactive agents may be coupled to the implant by way of a pharmaceutically acceptable carrier. Desirable characteristics for pharmaceutical carriers employed in the presently described embodiments include at least one of i) biocompatibility; ii) bioresorbability; iii) ability of the carrier to stably store the bioactive agents and/or allow its sustained release to surrounding tissues and cells. Such characteristics may be realized using a thin (10-50 μm in thickness) crystalline hydroxyapatite layer formed on the surface of the implant. METHODS OF INCORPORATING BIOACTIVE COMPOSITIONS INTO CPCs
In accordance with an objective of the present disclosure, set forth below are methods by which a bioactive composition may be coupled, added or incorporated to a calcium phosphate- based pharmaceutical carrier (e.g., CPC paste or hardened CPC material). Co-precipitating a bioactive composition with hydroxyapatite
In a first set of embodiments, a method is provided whereby a layer of crystalline calcium phosphate is formed on the surface a calcium phosphate prosthetic bone implant by co- precipitating apatite and one or more bioactive agents from a physiologically acceptable aqueous calcium phosphate solution. The co-precipitated bioactive agents will be stably integrated and dispersed within the matrix of said crystalline calcium phosphate surface layer. When implanted into recipient bone, bioactive agents are gradually released from the crystalline calcium phosphate layers of the subject implants in a sustained manner. Thus, it is an object of the presently described embodiments to provide an improved prosthetic bone implant comprising unsintered calcium phosphate, that is bioresorbable, biocompatible, and acts as a carrier for therapeutically effective bioactive agents, and methods of making said improved implants.
Calcium phosphate layers produced using current art-recognized techniques are typically composed of large, partially molten HAp particles. HAp produced synthetically under these conditions is prone to delamination and is poorly degraded in situ. The calcium phosphate layers of the present embodiments, in addition to being bioresorbable and biocompatible, are produced under physiological conditions and thus have the additional advantage of being able to integrally accommodate bioactive molecules, such as osteogenic agents, that typically cannot withstand harsh processing treatments (e.g., elevated temperature pressure, osmotic conditions and pH). The bioactive molecules may be co-precipitated with the inorganic mineral components that will form the crystalline calcium phosphate. As a consequence, the bioactive agents are incorporated into the crystal structure of the precipitated mineral coating, rather than being merely deposited upon the surface of the implant and or the coating. In forming an integral part of the calcium phosphate coatings, the elution profile of the integrated bioactive agent is more constant and sustained rather than being a single burst (as when superficially adsorbed). The reduced elution rate advantageously prolongs the osteoinductive and healing potential of therapeutics agents acting locally at the implantation site. In an embodiment, the crystalline coating may involve the nucleation and growth of HAp crystals on the surface of a calcium phosphate prosthetic bone implant. Unlike similar treatments in prior art coating procedures, the subject implants, being substantially composed of HAp, do not require a pre-treatment process to deposit a nucleating layer on the implant surface, although such a pre-treatment step may be performed if desired. In an embodiment, formation of the crystalline coating may include contacting the implant with a coating composition that includes a source of calcium and a source of phosphate. Contacting the implant with the coating composition may include fully or partially immersing the implant in the coating composition. Typically, this step will be carried out at a temperature that is within physiologic range (e.g., between about 2O0C to about 450C, between about 250C to about 370C, or at about 370C). The implant will be contacted with the coating composition for a period of time sufficient to allow the precipitation of crystalline calcium phosphate on the surface of the implant. Typically, a layer crystalline calcium phosphate mineral that is at least 0.5 to about 100 μm thick, between 20 to about 50μm thick, or about 40 μm thick, will be allowed to form on the surface of the implant. Layers of such thickness will typically be achieved in less than 100 hours at 370C, or more typically, in less than about 48 hours at 370C. The thickness of a calcium phosphate mineral layer may be monitored using techniques widely familiar to practitioners, such as densitometry, reflectometry, scanning electron microscopy, spectroscopy, or the like. The coating composition will contain amounts of calcium and phosphate that are sufficient to precipitate crystalline HAp at physiological temperature and pH. The concentration of calcium ions in the coating composition may range from 0.5 to 10 mM, or from 0.5 to 5 mM. The concentration of phosphate ions in the coating composition may range from 0.5 to 6 mM, or from 0.5 to 3 mM. Sodium chloride, or any suitable salt may be added to maintain the ionic strength of the coating composition. Typically the ionic strength of the solution should be between 100 mM to 200 mM sodium chloride, and more typically 150 mM.
The size of HAp crystals may be controlled by varying the amount of crystal growth inhibitors in the coating composition (e.g., magnesium and carbonate), with crystal size being inversely proportion to the concentration of crystal growth inhibitors present in the solution. In order to form HAp crystals, the concentration of magnesium should be less than 7.5 mM, more typically less than 2.5 mM, and most typically less than 0.5 mM. Similarly, HAp crystals ideally form when the concentration of carbonate ions is less than 25 mM, more typically less than 10 mM, and most typically less than 5 mM. Typically, precipitation of HAp crystals will occur at a substantially physiological pH
(from 6-8, or about 7.4). An appropriate buffer, like tris (amino-ethane) or HEPES (N- [2- hydroxyethyl] piperazine-N'-[4-ethanesulfonic acid]) is preferably used to maintain the desired pH. Suitable buffers to maintain a desired pH are known from the art. The relationship between temperature, pH and calcium phosphate solubility per se is known in the art. The skilled practitioner will be able to derive suitable conditions from the guidelines described above.
Information and further guidance on solubility calculations for various calcium phosphates may also be found in "G. Vereecke & J. Lemaitre: Calculation of the solubility diagrams in the system Ca(OH)2-H3PO4-KOH-HNO3-CO2-H2O, J. Crystal growth 104 (1990) 820-832.
Generally, the bioactive agents that are to be co-precipitated with HAp crystals will be solubilized in the coating composition. Typically the concentration of the one or more bioactive agents in the solution will be in a concentration range of 0.1 mg/1 to 10 g/1, in the range of 0.1- 1000 mg/1, in the range of 0.1-500 mg/1, or in the range of 0.1-20 mg/1. Depending upon the desired type of crystals to be grown, the skilled professional may choose to use particular concentrations, pH ranges and temperatures to form the crystals. Most preferably calcium and phosphate are among the inorganic ions used to incorporate bioactive agents into an implant. A coating composition for depositing crystalline HAp on the surface of a calcium phosphate implant will typically be buffered at a pH in the range of 6 to 8.
The pH of the coating composition may depend upon the isoelectric point (pi) of a bioactive agent that is to be incorporated into the coating. Co-precipitation of bioactive agent with inorganic crystals is related to electrostatic interactions. For chargeable compounds, and in particular for amphoteric compounds, the efficiency of incorporation depends on the pi of the bioactive agent and pH of the coating composition. The pi of a compound can be measured by isoelectric focusing using polyacrylamide gel electrophoresis. In some embodiments, the bioactive agent is charged at the pH at which the bioactive agent is incorporated into the implant, because this positively affects the amount of bioactive agent that is incorporated.
For the purpose of non-limiting illustration, BMP-2 has a IEP of 9.2. Accordingly the protein has a positive charge below 9.2 and negative charge above 9.2. At a pH of 7.4 for the coating composition, the protein is positively charged and thereby interacts with anions (such as phosphate) in solution. The interaction of the protein with the anions, enhances co-precipitation thereof with HAp crystals growing on the implant surface. For instance, a concentration of BMP-2 in a coating composition of 5 mg/L may lead to an incorporation of 5 μg/mg of coating at pH 7.4. BMP-7, however, has an IEP of 7.7. At a pH of 7.4, the efficiency for incorporation is low due to insufficient difference between IEP and coating pH. Under the same conditions, the incorporation of BMP-7 is only 0.25 μg/mg coating at pH 7.4 for 5 mg/1 of BMP-7 in coating solution. In order to increase efficiency of incorporation, a lower pH for coating solution may be selected (e.g. 6.7). Ideally, the difference between pH and pi for each bioactive agent in the composition should be at least about 1 pH unit for optimal co-precipitation of bioactive agents with the growing inorganic layer. For basic amphoteric compounds (pL>7.0) co-precipitation is preferably performed at a pH below pi, for acidic amphoteric compound (pI<7.0) co- precipitation is preferably performed at a pH higher than pi. For compounds with a pi of 7.0 a pH close to 6 or close to 8 is preferred. In case several compounds with different pi's are to be incorporated, it is preferred to choose a pH where all bioactive agents are charged, if possible. If this is not possible, more than one co-precipitation procedure may be performed, with each procedure incorporating bioactive compositions using conditions are close to ideal as possible, resulting in an implant with more than one crystalline coating. This may be advantageous in some cases, since in vivo, HAp crystals typically degrade from the outside in. Thus, therapeutic agent such as osteogenic compounds and analgesic compounds may be precipitated on an outer layer of the implant, while therapeutic agents such a bone proteins or antibiotics may be deposited first.
The pH of the calcium phosphate solution typically has less influence on the incorporation rate of uncharged bioactive agents. In general physiological pH, around 7.4 is suitable for this purpose.
In an embodiment, including one or more bioactive agents, in particular one or more osteoinductive agents, in the coating may stimulate cell activity and cell differentiation near an implant. Accordingly, the subject coated implants may regenerate or repair bone tissue more efficiently and more rapidly than implants which do not contain bioactive agents. The release of bioactive agent(s) is related to the rate of coating degradation. After implantation, the mineral coating is remodeled or degraded by osteoclastic activity, leading to a gradual release of the bioactive agent(s), around the implanted medical device. Thus an optimal concentration of bioactive agent(s) can be maintained around the medical device, and burst-release of bioactive agent(s), which may lead to unwanted side effects and premature cessation of therapeutic activity of the implant may be avoided.
In vitro, the degradation of the coating and release of the bioactive agent(s) may be monitored by measuring the calcium and or bioactive agent(s) release under physiological conditions as a function of time. Methods to monitor levels of these compounds are known in the art and include monitoring via a calcium-ion selective electrode, chromatography or enzyme- linked immunosorbant assay to measure the elution profiles of polypeptide factors. Ideally, a growth factor incorporated into a crystalline calcium phosphate layer as described herein will have an elution profile at physiological pH (about 7.4) that roughly corresponds to the dissolution rate of the calcium phosphate matrix in which it is incorporated. Optionally, it may be desirable, under certain situations to "pre-coat" the surface of the prosthetic bone implant with an initial layer (e.g., an amorphous mineral) of inorganic compounds, such as with an initial layer comprising calcium and phosphate. The amorphous layer may be obtained by contacting the implant surface with an aqueous calcium phosphate pre- coat solution under high nucleation conditions to obtain a thin and amorphous calcium phosphate layer. The optional amorphous layer may act as a seed to enhance the ability of more highly structured crystalline HAp to be precipitated on the implant surface. In some applications, including the optional amorphous layer may improve the stability and the activity of the crystalline HAp coating and the bioactive agent(s) incorporated therein. The implant may be pre-coated for a period of time sufficient to deposit an amorphous layer of calcium phosphate material at least 1 μm in thickness (typically, between 12-24 hours).
The composition of the inorganic components of the pre-coat solution may be chemically similar to that found in body fluids. The concentration of calcium ions in the pre-coat solution may range from 0.5 to 20 mM, or from 8 to 12.5 mM. The concentration of phosphate in the pre-coat solution may range from 0.5 to 10 mM, or from 2.5 to 5 mM. The concentrations of calcium and phosphate may have to be adjusted to maintain a desired pH. The solubility of calcium phosphate is inversely proportional to pH, that is, as pH increases the solubility of calcium phosphate decreases. For example, at 370C, and at a pH of 6.7, calcium phosphate is more soluble than at physiological pH (about 7.4). Concentrations of calcium and phosphate, in some embodiments, will be between 4 mM to 15 mM for calcium and 2 mM to 20 mM for phosphate.
Furthermore, the presence of magnesium ions is thought to inhibit the deposition of crystalline calcium phosphate mineral coatings. Particularly, the presence of magnesium has been found to inhibit or reduce the crystal growth of the coating during deposition from the calcium phosphate solution, resulting in an amorphous calcium phosphate layer that may act as a seed to enhance formation of crystalline HAp subsequently precipitated thereon. Optimum control of crystal growth leads to a uniform, strong and wear resistant coating. Magnesium and carbonate ions may be present in the pre-coat solution at concentrations below 10 and 25 mM, respectively. The quantity of magnesium and carbonate, both inhibitors of crystal growth may be adjusted for optimal formation and attachment of the optional amorphous pre-coat layer. In embodiments where apatite crystals are to be formed it is desirable to produce apatite crystals of submicrometer dimensions (<1 microns), which may result in a mechanically stronger coating. In an embodiment, increasing the magnesium and carbonate ion concentration may decrease the average crystal size.
Formation of nanoporous nanocrystalline HAp
In an alternate embodiment, bioactive compositions may be coupled to prosthetic bone implants by first forming a layer of nanoporous HAp nanocrystals on the surface of at least a portion of the implant. Nanoporous HAp nanocrystals may also be formed on the surface of a calcium phosphate implant surfaces using any art-recognized technique. In some embodiments, the nanocrystalline HAp surface will be highly porous and have a surface area in the range of about 25 m2/g to about 150 m2/g. The surface area of the nanocrystalline HAp coating the subject implants will be directly proportional to amount of bioactive composition that can be coupled to the implant. Advantageously, the surface area of the nanocrystalline HAp coating is inversely related to the elution rate of the bioactive composition when implanted in a subject.
FIG. 1 shows SEM images (at 10,000 fold magnification) of the surface of calcium phosphate implants having a surface layer of nanoporous HAp nanocrystals according to some embodiments. Individual particles of calcium phosphate are cemented to each other, and a layer of nanoporous HAp nanocrystals is formed thereon.
In one non-limiting embodiment, a layer of nanoporous HAp nanocrystals that is well suited for prolonged retention and slow elution of bioactive agents (such as drugs, growth factors or other agents having biological activity) may be formed on the surface of a CaP implant by contacting the portion of the implant that is to be coated with an aqueous solution containing a source of phosphate ions. Optionally, the solution may contain a source of calcium ions. The implant will be soaked in the solution for a period of time that is sufficient to form nanocrystalline HAp on the implant surface. In an embodiment, the implant may be soaked for up to 8 days. After soaking, the implant may be rinsed with the solution, with water, or with an appropriate physiological buffer. Optionally, the implant may be dried and stored under sterile conditions for use in a point-of-care setting.
In an embodiment, the nanoporous nanocrystalline HAp layer will have a surface area of between about 25 m2/g to about 150 m2/g, or between about 60 m2/g to about 100 m2/g. The increased surface area of the prosthetic bone implants significantly increases the drug binding capacity of the implant (i.e. results in a greater amount of bioactive composition to be coupled thereto).
In an embodiment, the physical and chemical properties of surface nanoporous HAp nanocrystals may be by altered by including one or more additives in the aqueous solution. The additives may include, for example, inhibitors of crystal formation, such as magnesium and/or carbonate ions (as described extensively above). By controlling the amount of such additives in an aqueous solution, the morphology of nanoporous HAp nanocrystals may be regulated.
In an embodiment, the physical and chemical properties of surface nanoporous HAp nanocrystals may also be determined by the amount of time that the implant is left in contact with the aqueous solution. Typically, the implant will be contacted with the aqueous solution for a period of time ranging from between 1 to 8 days. The amount of time that the implant is to be contacted with the aqueous solution is dependent on factors such as the chemical composition of the solution, and the surface area that is desired. FIG. 1 demonstrates the dependence of the surface area nanocrystalline HAp on chemical composition and incubation time. FIGs. IA and IB each show an SEM image of 10,000-fold magnification of the surface of a hardened calcium phosphate cement that has undergone the indicated treatment. The image depicted in FIG. IA corresponds to a CaP sample that has been immersed in Hank's Balanced Salt Solution (HBSS, with calcium and magnesium) for 3 days. The image depicted in FIG. IB corresponds to a CaP sample that has been immersed in Phosphate Buffered Saline (PBS) for 5 days. Nanophase nanocrystalline HAp is formed under both sets of conditions.
Alternatively, deposition of nanophase HAp nanocrystals on the surface of the subject implants may be performed using techniques such as ion- spray or sol-gel surface chemistry techniques. Formation of nanophase HAp nanocrystals typically occurs under physiologically unfavorable conditions and may be performed in the absence of bioactive agents whose stabilities are intolerant to such conditions. In these cases, the implant and nanophase HAp coating may be prepared and packaged under ascetic conditions. Bioactive agents may be loaded onto the surface thereof in a point-of-care setting by immersing the coated prosthetic bone implant in a sterile, physiologically buffered aqueous solution containing the dissolved bioactive composition. After loading onto the implant, the implant is delivered to its desired site in the body. Due to its high surface area and affinity for polypeptides, in particular BMPs, the elution rate of bioactive agents from the nanophase HAp is similar to the dissolution rate of the HAp crystals.
Bioactive compositions may be loaded onto the CaP subject bone implant by soaking the implant in an aqueous composition including a bioactive agent. This soaking step may be performed in addition to co-precipitating a drug onto the surface of an implant as described above. Alternatively, loading a bioactive agent onto the implant surface by performing a soaking step may be suited to situations where the implant was manufactured under conditions that would destabilize, degrade, or otherwise adversely affect the function of the drug. The soaking step may be performed without limitation with regard to strength, composition, pH or temperature of the soaking solution. Charging the implant by performing a soaking step may be suited to situation where activation of the drug must be performed under conditions that are adverse to the precipitation and/or formation of crystalline HAp on implant surfaces. Addition of bioactive compositions to pharmaceutical carriers Methods for the use of injectable CPC cements as pharmaceutical carriers may include the use thereof for delivery of bioactive compositions with controlled release profile (e.g., multimodal, exhibiting an initial burst followed by gradual release or slow release profile over longer time). Certain structures of the cement may be capable of being substantially or completely reabsorbed by the host tissue. In one embodiment, the release can range from 2% to 40% of the initial load within 24 hrs.
A variety of strategies may be employed to realize controlled release kinetics of bioactive composition from the subject carriers. In an embodiment, bioactive compositions that modulate the affinity of a component thereof (e.g., one or more growth factors) for a CPC material may be included in one or more components used to in fabrication of a CPC paste.
In an embodiment, formation of pores and or interconnected porosity in accordance with the procedures set forth above and incorporated herein may facilitate controlled release of a bioactive composition from a CPC matrix during use. The pores may be made prior to injection or created slowly as a result of the dissolution, degradation or resorption of one or more of the porogens, in conjunction with the dissolution, degradation or resorption of the hardened CPC material in the presence of said porogen.
In an embodiment, controlled release of a bioactive composition from a CPC matrix may be facilitated by a combination of the two preceding embodiments to produce a heterogeneous injectable construct with biphasic desorption profile, release by passive dissolution of the soluble phase and long-term release by active resorption of the cement.
Features of such embodiments may include the addition of an excipient for bone growth factor (such as BMPs and TGF-beta) delivery using injectable calcium phosphate cement as a carrier to treat defects in various calcified tissue (such as bone). In an embodiment, an excipient may include a mixture of one or more of sodium phosphate, amino acids, bone growth factors, polymers, sugars and carbohydrates.
Any bioactive agent that facilitates or stimulates new bone growth may be delivered to a calcified tissue in accordance with the embodiments described herein to produce an excipient combined with injectable calcium phosphate cement as a carrier. In certain specific embodiments, certain osteoinductive agents may be included in an injectable CPC bone substitute material. Osteoinductive agents that may find use according to such embodiments may include one or more bone morphogenetic proteins e.g. BMP-2, BMP-7, BMP-9, GDF-5, GDF-6, and GDF-7, one or more transforming growth factors (e.g., TGF-beta), one or more IGFs (e.g., IGF-I, and IGF-2), or various portions and/or combinations thereof. In an embodiment, autologous bone marrow (e.g., derived from the subject who will be receiving it) or bone-derived TGF-beta, insulin-like growth factors, platelet-derived growth factor and BMP2, or any of the bioactive agents disclosed herein, may be combined with the injectable bone substitute materials.
In some embodiments, various excipients (e.g., amino acids and/or polypeptides) that function, at least in part, by decreasing the binding affinity between polypeptide growth factors (e.g., BMPs) and a calcium phosphate matrix may be included in the subject bone substitute compositions. Turning to FIG. 2, the effect of various excipients on growth factor release kinetics from CPC biomaterials is shown. Hardened CPC articles were manufactured in accordance with the present disclosure and the growth factor BMP was equally loaded on the articles. A portion of the hardened articles contained no additional excipient (control; shown in FIG. 2 as closed diamonds), a portion further contained the amino acid glutamate (GIu; shown in FIG. 2 as closed squares); a portion further contained the various amounts of the amino acid glycine (GIy; shown in FIG. 2 as closed triangles (having an amino acid:CPC solid component ratio of 0.5:1) and as light X (having an amino acid:CPC solid component ratio of 1:1), respectively) and a portion further contained the amino acid alanine (Ala; shown in FIG. 2 as darker X). The CPC articles were immersed in a physiological solution and the amount of BMP that was released therefrom into the surrounding solution at the indicated times was determined. It was determined that Ala and GIy increased the release profile of BMP from the CPC implants. After an incubation time of 24 hours, the growth factor/CPC composite had a compressive strength of 64.7 MPa.
In some embodiments, allowing the leaching out of various water-soluble porogens (e.g., sodium chloride and potassium chloride salts) may generate pores. Hardened CPC particles made therefrom may typically have compressive strengths of about 5.5 MPa or more after incubation thereof in a Hank's solution for about 24 hours. The cumulative release after 24 hrs is up to 46%.
In some embodiments, the biodegradable polymers may be mixed with cement powder or setting solution and act as porogen. Without being bound by any particular theory or mechanism of action, it is believed that the degradation of such polymers in situ results in an at least partially acidic micro-environment (e.g., pH < about 7). Acidic conditions in the vicinity of the degrading porogen may enhance localized dissolution of the calcium phosphate mineral matrix, resulting in a porous bioceramic matrix that releases the growth factors from the cement over the time. Polymers such as polylactic acid, polylactic acid-polyethylene glycol block copolymer and their derivatives are used to fabricate microspheres or microfibers and combined with calcium phosphate cement to deliver growth factors. Optionally, the biodegradable polymers may be provided to the subject bone replacement material in combination with one or more additional excipients, such as, for example, molecules that decrease the binding affinity of apatite fro peptide growth factors (e.g., certain amino acids such as alanine). Turning now to FIG. 3, the effect of various excipients on growth factor release kinetics from CPC biomaterials is shown. Hardened CPC articles were manufactured in accordance with the present disclosure and the growth factor BMP was equally loaded on the articles. A portion of the hardened articles contained no biodegradable polymer porogen (control; shown in FIG. 3 as closed triangles), a portion contained the biodegradable polymer poly lactic acid (PLA; shown in FIG. 3 as closed diamonds) as a porogen; a portion contained the biodegradable polymer polyethylene glycol in combination with the amino acid alanine (Ala+PEG; shown in FIG. 3 as light crosses; and a portion contained the biodegradable polymer polyethylene glycol in combination with PEO (PLA-PEO; shown in FIG. 3 as closed squares). The CPC articles were immersed in a physiological solution and the amount of BMP that was released therefrom into the surrounding solution at the indicated times was determined. It was determined that release profile changed significantly when compared to control especially in combination with molecules that decrease the binding of the growth factor to the apatite cement. Furthermore, In data not shown, the release rate of such implants was increased up to 46% within the first 24 hours by the addition of porogens specifically NaCl and KCl in a 1:1 ratio.
In an embodiment, a bone substitute material suitable for use as a pharmaceutical carrier medium may include an injectable paste having calcium phosphate cement (CPC) particles, an excipient comprising a physiologically acceptable aqueous phosphate solution and an osteoinductive composition. In some embodiments, a bone substitute material paste may further include one or more porogens mixed therein. Exemplary porogens may include one or more biodegradable and/or bioresorbable compositions such as salts, polymers, biopolymers, and the like. Porogens may be provided in combination with CPC particles and/or mixed with the physiologically acceptable setting solution. In an embodiment, porogens may include certain biodegradable polymers, the degradation of which results in an acidic (e.g., pH < about 7.0) local micro-environment that encourages localized dissolution of hardened CPC matrix, in addition to creating voids (i.e., pores) in the space occupied by the biodegradable/bioresorbable polymer composition or other porogen. The inclusion of such biodegradable/bioresorbable porogens may advantageously result in a porous bioceramic matrix that locally releases the bioactive composition from the hardened cement over the time, thereby delivering a therapeutic dose of said bioactive composition.
In an embodiment, a bone substitute material paste may further include one or more porogens in a ratio such that the hardened CPC may develop a predetermined porosity (i.e., >90 vol%, >70 vol.%, >50 vol.%, >30 vol.%, >15 vol.%, >5 vol.%, or > 1 vol.%). Porogens may include crystals of physiologically acceptable salts, biodegradable polymers, or combinations thereof. Biodegradable polymers suitable for applications described herein may include, though are not limited to, natural or synthetic polypeptides, polylactic acid, chitosan, polylactic acid- polyethylene glycol block copolymer and their derivatives. In some embodiments, suitable biodegradable polymers may include polyesters, poly(L-lactic acid), poly(D,L-lactic acid), poly(glycolic acid), polycaprolactone, block copolymers and copolymers thereof. In an embodiment, at least a portion of the porogen may be formulated as microspheres. In an embodiment, at least a portion of the porogen may be formulated as microfibers. In an embodiment, the dry weight ratio of porogen to CPC particles in bone substitute material paste may be up to about 1:1.
In one set of non-limiting embodiments, the presently disclosed bone substitute materials may be formulated to act as a pharmaceutical carrier for one or more bioactive agents. The bioactive agent may, in turn be formulated to include an osteoinductive composition, a growth factor composition, an antibiotic composition, and analgesic composition, or combinations thereof. In an embodiment, a bioactive composition may include at least one growth factor from the TGF-β superfamily of growth factors, at least one growth factor from the BMP family of growth factors, at least one growth factor from the GDF family of growth factors, at least one growth factor from the IGF family of growth factors, or their combination. In an embodiment, a bioactive composition may include BMP-2, BM-4, BMP-12, or their combination. In certain embodiments, a bioactive composition may include at least a portion of one or more polypeptides, including but not limited to at least a portion of a polypeptide growth factor, at least a portion of one or more TGF-β superfamily growth factors, at least a portion of one or more BMP growth factors, or various combinations thereof. In an embodiment, a polypeptide for use in an osteoinductive composition as described herein may be at least partially purified. Source materials for at least partial purification of the polypeptides as described herein may include natural source material (e.g., natural bone, bone marrow, cultured cells), or recombinant material (e.g., protein whose expression is facilitated and or enhanced by way of a suitable viral, bacterial, yeast, insect, plant or mammalian protein expression system including a suitable expression vector). In some embodiments, one or more of the bone growth factors may be derived from autogenic bone, allogenic bone, xenogenic bone, or from recombinant sources.
In an embodiment, CPC particles and a physiologically acceptable aqueous setting solution comprising an excipient may be provided in a ratio sufficient to form an injectable paste. The excipient may include one or more of amino acids, physiological acceptable salts, sodium phosphates, and polypeptides.
In an embodiment, the injectable calcium phosphate cement bone substitute compositions may harden to form a calcium phosphate material. In embodiments in which porogens were included in the paste, the hardened material may be soaked in a physiologically acceptable aqueous solution to promote the dissolution/degradation of at least a portion of the porogen embedded throughout the hardened matrix, thereby creating a network of pores therein. Alternatively, the hardened material may be implanted into a site in the body (e.g., a tooth or a bone). Over time, body fluids in contact with the hardened calcium phosphate material/porogen composite may penetrate the matrix thereof and allow dissolution/degradation of the porogen, thereby creating a network of pores therein.
In an embodiment, the injectable calcium phosphate cement bone substitute compositions may further include physiologically acceptable excipient.
In an embodiment, the injectable calcium phosphate cement bone substitute compositions may be a paste that is made by a process of providing a solid phase that includes calcium phosphate cement (CPC) particles in combination with at least one porogen; and contacting said solid phase with a liquid phase; and further with an osteoinductive composition. In an alternate embodiment, the osteoinductive composition may be included as a component of the solid phase. In a further alternate embodiment, the osteoinductive composition may be included as a component of the liquid phase. In yet a further embodiment, the osteoinductive composition may be included as a component of both the solid phase and the liquid phase. The ratio of the solid phase to liquid phase of the composition may be sufficient to form a paste that can readily be injected using syringe to a site on a bone or to a mold in order to make an implantable structure. In an embodiment, the ratio of solid phase to liquid phase may be in the range of about 0.1 mg/ml to about 20 mg/ml.
In an embodiment, the injectable calcium phosphate cement bone substitute compositions may be delivered to a bone defect or to a mold and form a hardened bioresorbable calcium phosphate material. In an embodiment, certain structures of the hardened CPC may be substantially reabsorbed by or released around the site of its application. In certain embodiments, porogens within the hardened calcium phosphate matrix may gradually be removed from the hardened material, leaving pores and/or an interconnected network of porosity dispersed throughout the matrix. In some embodiments, bioactive compositions incorporated in the hardened calcium phosphate material and/or in the porogens may be gradually released to surrounding tissue. In some embodiments, any bioactive agent that facilitates or stimulates new bone growth
(e.g., an osteogenic agent) may be delivered to a bone or to an implant using a suitable excipient combined with injectable calcium phosphate cement as a carrier. In certain embodiments, bioactive compositions may include osteoinductive compositions such as bone morphogenetic proteins e.g. BMP-2, BMP-7, BMP-9, growth differentiation factors (GDF)-5, GDF-6, and GDF- 7, platelet-derived growth factors (PDGFs), transforming growth factors (e.g., TGF-β), insulin- like growth factors (IGF)-I, and IGF-2, autogenic or allogenic bone or bone marrow, bone- derived TGF-β, IGF, and BMP2. In some embodiments, amino acids or polypeptides, may be employed to decrease the binding affinity between polypeptide factors and the hardened calcium phosphate cement matrix. In an embodiment, cumulative release of components of a bioactive composition from the hardened CPC material approximately 24 hrs after the introduction thereof to a bone defect may be up to about 2.5%. In an embodiment, composite (e.g., growth factor/CPC) bone substitute materials may have a compressive strength of up to about 150 MPa after incubation in a physiologic solution.
In some embodiments, the porosity in the hardened CPC matrix may be realized, at least in part, by the dissolution, degradation, or resorption of porogens found therein. The hardened CPC bone substitute materials may be adapted to have a compressive strength up to about 150 MPa after incubation of said materials in a physiologically acceptable aqueous solution. The cumulative release profile of therapeutic compositions therefrom after 24 hrs may be up to about
46%.
Devices suitable for the addition of pharmaceutical agents to porous biomaterials
In some embodiments, bioactive agents may be loaded onto a porous biomaterial using systems and/or devices that allow an operator to control the amount of a bioactive agent that is delivered to a porous CPC matrix. Of course, it will be readily appreciated by an ordinary practitioner of the art that the use of one or more mechanical means for the loading of pharmaceutical agents to the subject bone substitute compositions as described below in no way precludes the use of additional methods for the loading of the same or of different agents to the CPC matrix. On the contrary, it will be evident to the skilled practitioner that any number of methods presently disclosed that are suitable for the addition of bioactive agents to CPC articles may be employed concurrently or sequentially to incorporate various bioactive agents thereto, without departing from the spirit or scope of the presently described embodiments.
In an embodiment, a device is provided for that allows for the perioperative addition of a bioactive composition to a porous biomaterial. Advantageously, addition of the bioactive composition to a porous biomaterial in accordance with the presently described embodiments may optionally be performed in advance of a surgical procedure, or may alternatively be performed immediately prior to implantation of an implant in a subject (e.g., at a point-of-care setting).
In an embodiment, a device suitable for the perioperative addition of a bioactive composition to a porous biomaterial may include a cassette portion operatively coupled to an actuator. The cassette portion may include a body having a cavity with an opening to the surface of the body. No limitations are place on the size, dimensions or configuration of the cassette portion, or of the cavity formed therein, except that the cavity is capable of accepting the porous implant that is to be impregnated, and that the volume of the cavity is larger than the volume of the porous implant. The cassette portion may be made from any suitable rigid, semi-rigid, pliable or resilient material that is able to withstand a pressure differential of up to about 1 atm between the cavity and the outside surface of the cassette, such as, for example, metals, metal alloys, metal polymer composites, polymers materials, and/or thermoplastic materials. In some embodiments, the cassette portion may be injection molded from a suitable biocompatible thermoplastic material. In an embodiment, the cassette portion may further include a sealing portion. The sealing portion may be coupled to the cassette portion or may be removable from the cassette portion. In an embodiment, the sealing portion may be configured such that the shape thereof is substantially complementary to at least a portion of the cavity or of the opening connecting the cavity to the surface of the body of the cassette portion. In an embodiment, the sealing portion may be configured such that, when the sealing portion is in operation, a substantially airtight seal may be formed between the sealing portion and the cavity, thereby producing a sealed chamber. In an embodiment, the sealing portion may be operatively coupled to an actuator. No limitation is placed on the means by which the sealing portion and the actuator may be coupled, except that the coupling thereof will generally not interfere with the placement and/or retention of a porous biomaterial article and/or the liquid in which it is suspended in the cavity of the cassette portion, nor with the ability of the sealing portion to form an at least partially airtight seal with the cavity of the cassette portion.
An actuator may include, without limitation, any system or device that enables an operator apply a vacuum to the porous biomaterial and liquid carrier medium residing within the cavity of the cassette portion. In an embodiment, an actuator may include a means (e.g., a squeeze trigger, a plunger, a piston, or the like) for the reversible operation thereof. The actuator may, according to some embodiments, be operable using a single hand motion. For example, in certain embodiments, an actuator may include a squeeze trigger operatively coupled to the sealing portion.
During use, a porous biomaterial article onto which a bioactive composition is to be loaded may be provided to the cavity of the cassette portion. The dimensions of the porous biomaterial article will generally be smaller than the dimensions of the chamber formed when the sealing portion is operatively coupled to the cassette portion, such that when the porous biomaterial article is placed in the cavity and the cavity is sealed by the sealing portion to form the chamber, at least a portion of the chamber volume will not be occupied by the porous biomaterial article. A liquid carrier composition may be provided to cavity. The liquid carrier composition may include any physiologically acceptable solution that stably carries bioactive agents dispersed therein, in combination with one or more bioactive agents.
A sufficient volume of said liquid carrier composition will be provided such that the porous article is at least partially suspended therein, and further such that at least a portion of the surface of the porous article is in contact with said liquid carrier composition. The volume of liquid carrier composition provided thereto will typically not exceed the volume of the chamber that is not occupied by the porous biomaterial article when the device is in use. The order in which the article and the liquid carrier composition are provided to the cavity is arbitrary.
The device may then be sealed by coupling the cassette portion to the sealing portion thereby sealing the porous article suspended in the liquid carrier composition within the chamber. The actuator may then be engaged such that a vacuum is achieved within the chamber. In some embodiments, the pressure within the chamber may be < about 0.1 atm, < about 0.2 atm, < about 0.5 atm, or less than about 1 atm. In an embodiment, the vacuum may be maintained for at least about 5 seconds, at least about 10 seconds, at least about 20 seconds, at least about 30 seconds, or at least about 1 minute, after which time, the actuator is released, and the pressure of the chamber return to ambient pressure. Without being bound to any specific mechanism, the vacuum created within the chamber displaces air trapped inside the porous network of the biomaterial. As the chamber is returned to ambient pressure (typically 1 atm) the air that was displaced from the pores attempts to refill the pores, however, since the pharmaceutical carrier liquid is surrounding the biomaterial it is driven into pores that were once occupied by air. The preceding steps may be repeated any number of times until a desired volume of the liquid carrier composition occupies the porous network of the biomaterial. Typically, such may be achieved after about three wet-vacuum cycles. Thus, a device that functions in accordance with the preceding description may allow for deep impregnation of pharmaceutical agents to porous implants that would otherwise not have been driven to the core of the biomaterial under normal atmospheric loading.
EXAMPLES
The following will serve to illustrate, by way of one or more examples, systems and methods for inhibiting, reducing or otherwise disrupting prolactin signaling in pain neurons according to some embodiments. The examples below are non-limiting and are intended to be merely representative of various aspects and features of certain embodiments. Although methods and materials similar or equivalent to those described herein may be used in the application or testing of the present embodiments, suitable methods and materials are described below. EXAMPLE 1
Formation of a hardened calcium phosphate cement article
Porous calcium phosphate cement coupons were made by the following procedure.
An injectable paste of calcium phosphate cement was prepared by mixing 0.6 g of whiskered
TTCP powder (made according to the procedures set forth in U.S. Patent Appl. Publ. No. 2004/0003757) with concentrated (NfLO2HPO4 solution in water at a liquid to solid ratio of 0.3 for 1 min. The paste was then thoroughly mixed with a mixture (1:1) of NaCl and KCl salt particles (pore forming powder). The amount of salt mixed with the paste was equal to the dry weight of the salt used to make the paste. The resulting paste mixture was filled into a cylindrical stainless steel mould having a diameter of 12 mm and compressed with a gradually increased pressure up to about 45 MPa and the cement was allowed to harden. The hardened material was immersed in distilled water at 370C for 48 hours and dried in air for 24 hours.
Formation of a nanocrystalline HAp layer in implant surfaces
EXAMPLE 2
The dried material made in Example 1 was immersed in Hank's balanced salt solution IX, HyQ ®HBSS cell culture reagents without Phenol Red, 0.1 μm sterile filtered; HyClone,
(Logan, Utah) for 3 days, rinsed with distilled water and air dried for 24 hours.
EXAMPLE 3
The dried material made in Example 1 was immersed in phosphate buffered saline (PBS) for 5 days, rinsed with distilled water and then dried in air for 24 hours. The hardened CPC discs made in Examples 2 and 3 were gold coated and the surface morphology of nanocrystalline HAp was examined using scanning electron microscopy.
Representative images are shown in Fig. IA (samples incubated in HBSS for 3 days), and Fig.
IB (sample incubated in PBS for 5 days). As shown in Figure 1, a nanocrystalline nanoporous mineral layer was formed after the surface modification with either HBSS or PBS
In this patent, certain U.S. patents, U.S. patent applications, and other materials (e.g., articles) have been incorporated by reference. The text of such U.S. patents, U.S. patent applications, and other materials is, however, only incorporated by reference to the extent that no conflict exists between such text and the other statements and drawings set forth herein. In the event of such conflict, then any such conflicting text in such incorporated by reference U.S. patents, U.S. patent applications, and other materials is specifically not incorporated by reference in this patent.
Further modifications and alternative embodiments of various aspects of the invention may be apparent to those skilled in the art in view of this description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as the presently preferred embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description to the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims. In addition, it is to be understood that features described herein independently may, in certain embodiments, be combined.

Claims

WHAT IS CLAIMED IS:
1. A system for impregnating a porous biomaterial with a bioactive composition comprising: a cassette portion, the cassette portion comprising a body; a cavity within the body, the cavity being sized to accept a porous biomaterial; and an opening from the cavity to a surface of the body; a sealing portion operatively couplable to the cassette portion, the sealing portion being configured such that, when coupled to the cassette portion, the opening of the cavity is sealed to form a substantially airtight chamber disposed in the cassette body; and an actuator operatively coupled to the sealing portion, the actuator being configured to reversibly apply a vacuum to the chamber.
2. The system according to claim 1, further comprising a porous biomaterial configured to reside within the chamber formed by coupling the cassette potion to the sealing portion, wherein the volume of the porous biomaterial is less than the volume of the chamber.
3. The system according to claim 2, wherein the porous biomaterial resides in the cavity.
4. The system according to any one of claims 1-3, further comprising a pharmaceutical carrier liquid.
5. The system according to claim 4, wherein the pharmaceutical carrier liquid comprises a physiologically acceptable liquid.
6. The system according to any one of claims 4-5, wherein the pharmaceutical carrier liquid comprises a bioactive composition.
7. The system according to any one of claims 4-6, wherein the volume of pharmaceutical carrier liquid is sufficient to occupy at least a portion of the chamber volume not occupied by the porous biomaterial.
8. The system according to any one of claims 4-7, wherein the volume of pharmaceutical carrier liquid is sufficient to allow at least a portion of the surface of a porous biomaterial residing in the chamber to contact the pharmaceutical carrier liquid.
9. The system according to any one of claims 1-8, wherein the actuator comprises a squeeze trigger.
10. The system according to any one of claims 1-9, wherein the cassette portion comprises the sealed barrel of a syringe.
11. The system according to any one of claims 1-10, wherein the sealing portion comprises the plunger of a syringe.
12. The system according to any one of claims 1-11, wherein the actuator comprises a squeeze trigger coupled to the plunger of a syringe.
13. A method of applying a pharmaceutical composition to a porous biomaterial comprising: providing a system comprising a cassette portion, the cassette portion comprising a body; a cavity within the body, the cavity being sized to accept a porous biomaterial; and an opening from the cavity to a surface of the body; a sealing portion operatively couplable to the cassette portion, the sealing portion being configured such that, when coupled to the cassette portion, the opening of the cavity is sealed to form a substantially airtight chamber disposed in the cassette body; and an actuator operatively coupled to the sealing portion, the actuator being configured to reversibly apply a vacuum to the chamber when engaged; providing a porous biomaterial to the cavity of the system; providing a pharmaceutical carrier liquid comprising a bioactive composition to the cavity of the system; coupling the cassette portion to the sealing portion, thereby sealing the porous biomaterial and the pharmaceutical carrier liquid in the chamber; and applying a vacuum to the chamber by engaging the actuator.
14. The method according to claim 13, wherein the vacuum applied to the chamber is sufficient to achieve a chamber pressure in the range of 0.1 to about 1.0 atm below ambient pressure.
15. The method according to any one of claims 13-14, wherein the vacuum is maintained for up to about I minute.
16. The method according to any one of claims 13-15, wherein the step of applying a vacuum to the chamber by engaging the actuator is performed up to about 3 times.
17. A system for impregnating a porous biomaterial with a bioactive composition.
18. A method of applying a pharmaceutical composition to a porous biomaterial.
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