WO2002015948A2

WO2002015948A2 - Method of treating and dehydrating bone for implantation

Info

Publication number: WO2002015948A2
Application number: PCT/US2001/026553
Authority: WO
Inventors: Todd M. Boyce; Lawrence A. Shimp
Original assignee: Osteotech, Inc.
Priority date: 2000-08-24
Filing date: 2001-08-24
Publication date: 2002-02-28
Also published as: CA2420113A1; AU2001286755A1; WO2002015948A9; EP1311309A2; WO2002015948A3

Abstract

Monolithic bone intended for implantation is treated in oder to conserve its mechanical strength during dehydration and subsequent packaging and to maintain the strength of the bone during the storage period preceding the rehydration and implantation of the bone. The method of treatment comprises contacting the bone with a mechanical strength-conserving amount of at least one biocompatible mechanical strength-conserving agent, the agent being a liquid organic material which is capable of penetrating and remaining in the bone during its dehydration, packaging and storage, dehydrating the bone containing the mechanical strength-conserving agent and packaging the dehydrated bone.

Description

METHOD OF TREATING AND DEHYDRATING BONE FOR IMPLANTATION AND RESULTING BONE

BACKGROUND OF THE INVENTION

Monolithic bone intended for implantation is treated in order to conserve its

mechanical strength during dehydration and subsequent packaging and to maintain the strength of the bone during the storage period preceding the rehy dration and implantation of the bone. This treatment is useful when combined with a number of methods of dehydrating the bone, e.g. dehydrating under ambient or near ambient conditions,

dehydrating in a vacuum oven, immersion in a graded series of dehydrating agents, etc.,

and serves to reduce the percent shrinkage of dehydrated bone treated in accordance with the invention as compared to untreated lyophilized bone.

The use of preserved bone intended for implantation to replace diseased or

missing parts is common. The successful application of such bone is predicated on sound

knowledge of its biologic properties and its capacity to withstand the stresses to which it

will be subjected. When mineralized bone is used in grafts, it is primarily because of its inherent strength, i.e., its load bearing ability at the recipient site. The biomechanical properties of bone grafts upon implantation are determined by many factors, including the specific site from which the bone is taken; the age, sex, and physical characteristics of the donor; and the method chosen to prepare, preserve, and store the bone prior to implantation. A more detailed explanation of the alteration of the biomechanical properties of bone by the methods chosen for its preservation and storage may be found in Pelker et al., Clin. Orthop. Rel. Res., 174:54-57(1983). However, the needs for

processing (e.g., to preserve the graft for later use and to remove immunogenic cellular materials) can conflict with the need to conserve the mechanical strength of the bone.

During the preparation of bone intended for implantation the porous matrix is typically contacted with one or more treatment fluids to variously clean, defat, sterilize, virally inactivate, disinfect, and/or demineralize the bone or to impregnate the bone with one or more pharmacological agents (antibiotics, bone growth factors, etc.) so the bone can act as a drug delivery system. See U.S. Patent No. 5,846,484 for a detailed explanation of

the treatment of bone intended for implantation. Some treatment processes, such as irradiation and lyophilization, can work against conservation of the mechanical strength of bone and can lessen the bone's weight bearing properties. Processing requirements can also create dimensional changes in the allograft bone. Such changes of dimension can create damage within the tissue, and may also make it difficult for a machined piece to mechanically engage with surgical instruments, other allografts, or the prepared surgical

site. Treatment processes also can have a deleterious effect on such important mechanical properties as toughness. Implants demonstrating improved toughness are

important as the insertion of some allografts can be quite energetic, e.g., the hammering in of cortical rings used in spinal fusion surgery. U.S. Pat. Appln. Serial No. 09/382,331 incorporated herein by reference discloses a method of treating bone intended for lyophilization prior to its storage that conserves the mechanical properties of the bone. It is generally accepted that freezing monolithic bone to temperatures as cold as

-70° C. prior to its packaging and storage results in little if any alteration in its physical properties. However, freezing bone as a preservation technique is costly and can be

logistically difficult, e.g., shipping and storage. Lyophilization (freeze-drying, i.e., freezing, then sublimation of moisture) is commonly performed on bone to permit its

shelf storage for up to several years without spoilage. Lyophilization removes excess moisture from the bone and reduces its antigenicity. According to the American

Association of Tissue Banks ("A.A.T.B"), lyophilized whole bone containing no more than 6% moisture can be stored at ambient temperatures for up to five years after processing. However, adverse changes in the biomechanical properties of the bone have

been found to result from the lyophilization procedure. Lyophilization can result in damage to the bone due to dimensional changes that occur during the freezing and dehydrating operations. The adverse mechanical changes appear to be associated with damage occurring in the bone matrix, specifically, ultrastructural cracks along the collagen fibers. These effects appear to be magnified when lyophilization and gamma irradiation are used together. Studies using rat bones to model the effects of

lyophilization upon the compressive properties of cancellous bone (compression strength

of tail vertebrae) and the bending and torsional properties of the long bones indicate that compressive strength can be reduced by up to 30% with little or no change in stiffness, bending strength can be reduced by as much as 40%, and torsional strength can be reduced by up to 60%. These changes have been found to occur even after the bone has

been rehydrated. A more detailed explanation of the effects of lyophilization on mineralized bone can be found in Kang et al., Yonsei MedJ 36:332-335(1995), and Pelker et al., J. Orthop. Res.1 :405-411(1984). Because freezing and thawing bone is minimally damaging to the bone, whereas lyophilization results in reduction in the

mechamcal strength of the bone, it is the inventors' belief that the mechanical strength- conserving agent is not acting as a cryopreservative (i.e., minimizing crystal growth during freezing) but rather in some new, not entirely understood, manner to diminish the dimensional changes associated with lyophilization. It has been determined that the diminishment of dimensional changes is related to the extent to which the mechanical

strength-conserving agent has penetrated the bone before or during the dehydration

process. Similarly, an improvement in the toughness of the implant has been seen to be

related to the extent to which the mechanical strength-conserving agent has penetrated the bone before or during the dehydration process, providing further evidence of the advantage of the invention herein. Thus, it is desirable to provide a method for treating bone which is to undergo dehydration as a prelude to its packaging and storage that will better conserve the biomechanical properties of the bone, i.e., its mechanical strength and/or dimensions and/or toughness, as compared to untreated lyophilized bone, from the time the bone is harvested through the packaging and storage operations and to time of implantation.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a method for treating

monolithic bone intended for implantation in order to conserve the mechanical strength of the bone during its dehydration and subsequent packaging and storage and to substantially maintain such strength throughout its rehydration and subsequent

implantation. It is a further object of the invention to provide a treatment that reduces the

dimensional change associated with the lyophilization of bone.

It is a further object of the invention to provide a treatment that improves the toughness of the bone graft. It is a further object of the invention to provide a treatment with minimal negative impact to the biological properties of the bone graft.

It is a further object of the invention to provide a treatment that acts as an antimicrobial/preservative agent.

It is a further object of the invention to provide a method for packaging

dehydrated monolithic bone so that the bone may be stored at ambient temperatures for an extended period of time, e.g., up to five years or longer without excessive loss of its

mechanical strength.

It is a further object of the invention to provide a method for the rehydration of dehydrated monolithic bone such that the mechanical strength of the bone at the time of its implantation is optimized.

It is a further object of the invention to provide a method that decreases the time necessary to rehydrate a dehydrated bone intended for implantation.

It is a further object of the invention to provide a method that minimizes the tendency for a partially rehydrated dehydrated graft to fracture due to the insertion forces _t applied by the surgeon.

It is a further object of the invention to provide a dehydrated monolithic bone

implant containing a mechanical strength-conserving agent and, optionally, one or more medically/surgically useful substances, e.g., an osteogenic material such as bone

morphogenic proteins (BMPs).

These and other objects not specifically set forth above will be apparent to those

skilled in the art in view of the objects set forth above and the foregoing specification. In keeping with these and related objectives of the invention, there is provided a method for treating monolithic bone intended for implantation to conserve the mechanical strength of the bone during dehydration and subsequent packaging and to maintain such

strength during the storage of the bone preceding its implantation. The method comprises: contacting the bone with a mechanical strength-conserving amount of at least one

biocompatible mechanical strength-conserving agent, said agent being a liquid organic material or solution, mixture, or suspension thereof, which is capable of penetrating and remaining in the bone during its dehydration, packaging and storage; dehydrating the bone containing the mechanical strength-conserving agent; and,

packaging the dehydrated bone. In another aspect, the invention includes the dehydrated bone obtained by the foregoing method(s) and use of bone obtained by the invention herein.

The expression "monolithic bone" as utilized herein refers to relatively large

pieces of human or animal bone, i.e., pieces of bone, autograft, allograft or xenograft, that are of such size as to be capable of withstanding the sort of mechanical loads to which

functioning bone is characteristically subjected. The monolithic bone of this invention is to be distinguished from particles, filaments, threads, etc. as disclosed in U. S. Patent Nos. 5,073,373, 5,314,476 and 5,507,813, which, due to their relatively small dimensions, are incapable of sustaining significant mechanical loads, either individually

or in the aggregate. It is further to be understood that the expression "monolithic bone" refers to fully mineralized bone, i.e., bone with its full natural level of mineral content, and to such bone that has been demineralized to some minor extent, i.e., to an extent which reduces the original mechanical strength of the bone by no more than about 50 percent. The monolithic bone can be provided as a single integral piece of bone or as a piece of bone permanently assembled from a number of smaller bone elements, e.g., as

disclosed and claimed in U.S. Patent No. 5,899,939 the contents of which are incorporated herein by reference. Although monolithic bone can contain factors which

are osteogenic, monolithic bone can also contain additional materials, e.g., as disclosed in U.S. Patent No. 5,290,558 the contents of which are incorporated herein by reference, which will remain with the bone after its rehydration and will be present at the time of

implantation.

The expression "mechanical strength" as utilized herein is intended to mean any

one of the principal biomechanical properties of bone, specifically including compression strength, flexural modulus, torsional modulus and yield strength, as well as the sum of

these properties, that are characteristic of bone.

The expression "toughness" as utilized herein is intended to refer to any characteristic that qualitatively can be described as the way in which the bone fails, i.e., how the bone undergoes deformation prior to fracture. For example, bone which exhibits improvement in toughness would be more desirable than bone having less toughness.

Quantitatively, "toughness" as utilized herein is a measure of the energy absorbed by the osteoimplant prior to breakage and is expressed in units of Newton-meters (N-m).

The expression "conserving the mechanical strength of the bone" and expressions of like import shall be understood herein, to mean that the monolithic bone treated in accordance with the invention, i.e., dehydrating such bone in the presence of a

mechanical strength-conserving agent, will exhibit a level of mechanical strength which

is at least about 10% greater than that of a comparable specimen of monolithic bone which has been lyophilized in the absence of a mechanical strength-conserving agent.

The expression "dimensional-conserving" and expressions of like import shall be understood herein to mean that the monolithic bone treated in accordance with the

invention, i.e., dehydrating such bone in the presence of a mechanical strength-

conserving agent shall demonstrate at least about 2% less decrease in length dimension as compared to bone that has been lyophilized. That is, bone treated in accordance with the invention herein will demonstrate less shrinkage after dehydration than bone that is lyophilized in the absence of a mechanical strength-conserving agent.

The expression "toughness-enhancing" and expressions of like import shall be

understood herein to mean that the monolithic bone treated in accordance with the invention, i.e., dehydrating such bone in the presence of a mechanical strength- conserving agent shall demonstrate at least greater than about 19 percent increase in toughness as compared to bone that has been lyophilized in the absence of a mechanical strength-conserving agent. That is, bone treated in accordance with the invention herein

will demonstrate improved ability to withstand the forces occurred during implantation a∑ compared to bone that is lyophilized in the absence of a mechanical strength-conserving agent

.The term "biocompatible" and expressions of like import shall be understood to mean the absence of stimulation of an undesired severe, long-lived or escalating biological response to an implant and is distinguished from a mild, transient

inflammation which accompanies implantation of essentially all foreign objects into a living organism and is also associated with the normal healing response. Materials useful to the invention herein shall be considered to be biocompatible if, at the time of

implantation, they are present in a sufficiently small concentration such that the above- defined condition is achieved.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS i*

Figure 1 is a graphical representation of a standard freeze-drying process.

Figure 2 is a graphical representation of an alternative freeze-drying process in which the tissue is subjected to some level of dehydration prior to freezing and sublimation of any remaining moisture.

Figure 3 is a representation of a ramp-shaped implant.

Figure 4 is a graphical representation of the dimension change of bone implant prepared as in Example 5.

Figure 5 is a graphical representation of the treatment effects on dimensional change. DETAILED DESCRIPTION OF THE INVENTION

Bone for implantation is obtained, e.g., aseptically in a morgue or an operating

room from, e.g., a cadaver donor or from a living donor's tissue obtained by surgical excision or amputation. The bone is cleansed, e.g., using 70% ethanol and washed with

water for injection and sonication. The bone may be treated with antibiotics such as polymyxin B sulfate, bacitracin, and/or gentamicin, and may contain trace amounts of

residual antibiotics. Cleansing, cutting, sizing, shaping, container sterilization, filling, lyophilization, and stoppering may be functions are performed under conditions following industry standards for tissue handling. The bone employed in the invention is of monolithic proportions in contrast to "particles," "filaments," "threads," "strips," etc.,

as described in U.S. Patent Nos. 5,073,373, 5,314,476 and 5,507,813. Thus, the bone treated according to the method of the invention is generally a relatively large piece or segment of donor bone and is intended for implantation into a correspondingly relatively large defect or other implantation site. Typically, the bone herein will possess dimensions of length on the order of about 2 mm to about 500 mm and preferably at least

about 5 mm to about 100 mm. Similarly, dimensions of width will be on the order of

about 1 mm to about 600 mm and preferably at least about 1 mm to about 100 mm. Dimensions of thickness will be on the order of about 1 mm to about 30 mm and preferably at least about 1 mm to about 10 mm. Any one of several methods, including but not limited to, cutting, forming and machining can readily obtain such bone.

Prior to dehydration, the prepared bone is contacted with a mechanical strength- conserving amount of a biocompatible mechanical strength-conserving agent. The biocompatible mechanical strength-conserving agent appropriate to the invention is a compound- or solution that is liquid at the temperature at which it is contacted with the bone, more preferably from about 5°C. to about 65°C, and which penetrates the small pores of the bone remaining therein after being dehydrated. The conserving agent is

biocompatible and nontoxic and does not substantially interfere with the normal healing of the graft. A suitable conserving agent will meet these criteria even if mixed with water or other volatile solvent and then subsequently the water or solvent is removed during dehydration leaving the conserving agent behind, i.e., it has a eutectic point significantly

below the freezing point of water and/or a vapor pressure less than that of the volatile

solvent. Suggested classes of conserving agent would include polyhydroxy compound, polyhydroxy ester, fatty alcohol, fatty alcohol ester, fatty acid, fatty acid ester, liquid silicone, mixtures thereof, and the like.

Examples of suitable conserving agent include, but are not limited to: (i) Polyhydroxy compound, for example, glycerol, 1,4-butylene glycol, diethylene

glycol, triethylene glycol, tetraethylene glycol, propylene glycol, dipropylene glycol; polysaccharides and their derivatives, e.g., hyaluronic acid; polyoxyethylene- polyoxypropylene copolymer, e.g., of the type known and commercially available under the trade names Pluronic and Emkalyx; polyoxyethylene-polyoxypropylene block copolymer, e.g., of the type known and commercially available under the trade name

Poloxamer; alkylphenolhydroxypolyoxyethylene, e.g., of the type known and commercially available under the trade name Triton, and the like. (ii) Polyhydroxy ester, for example, monoacetin, triacetin, poly(oxyalkylene) glycol ester, and the like.

(iii) Fatty alcohol, for example primary alcohols, usually straight chain having from 6 to 13 carbon atoms, including caproic alcohol, caprylic alcohol, undecyl alcohol, lauryl alcohol, and tridecanol.

(iv) Fatty alcohol ester, for example, ethyl hexyl palmitate, isodecyl neopentate, octadodecyl benzoate, diethyl hexyl maleate, and the like.

(v) Fatty acid having from 6 to 11 carbon atoms, for example, hexanoic acid, heptanoic acid, octanoic acid, decanoic acid and undecanoic acid.

(vi) Fatty acid ester, for example, polyoxyethylene-sorbitan-fatty acid esters; e.g., mono- and tri-lauryl, palmityl, stearyl, and oleyl esters; e.g., of the type available under the trade name Tween from Imperial Chemical Industries; polyoxyethylene fatty acid esters;

e.g., polyoxyethylene stearic acid esters of the type known and commercially available under the trade name Myrj; propylene glycol mono- and di-fatty acid esters such as propylene glycol dicaprylate; propylene glycol dilaurate, propylene glycol hydroxy

stearate, propylene glycol isostearate, propylene glycol laureate, propylene glycol ricinoleate, propylene glycol stearate, and propylene glycol caprylic-capric acid diester available under the trade name Miglyol; mono-, di-, and mono/di-glycerides, such as the esterification products of caprylic or caproic acid with glycerol; e.g., of the type known and commercially available under the trade name Imwitor; sorbitan fatty acid esters, e.g., of the type known and commercially available under the trade name Span, including sorbitan-monolauryl, -monopalmityl, -monostearyl, -tristearyl, -monooleyl and trioleylesters; monoglycerides, e.g., glycerol mono oleate, glycerol mono palmitate and

glycerol monostearate, for example as known and commercially available under the trade names Myvatex, Myvaplex and Myverol, and acetylated, e.g., mono- and di-acetylated monoglycerides, for example as known and commercially available under the trade name

Myvacet; isobutyl tallowate, n-butylstearate, n-butyl oleate, and n-propyl oleate.

(vii) Liquid silicone, for example, polyalkyl siloxanes such as polymethyl siloxane and poly(dimethyl siloxane) and polyalkyl arylsiloxane.

As stated above, the suitable biocompatible mechanical strength-conserving agent selected from the examples above preferably is capable of penetrating the small pores of

the bone. Therefore, optionally, a solution of a conserving agent can be utilized. This solution can be aqueous or can be one utilizing a polar organic solvent or other volatile

solvent.

The expression "volatile solvent" as utilized herein is intended to refer to any suitable solvent or mixture of solvents having a vapor pressure at relevant temperature,

i.e., the temperature at which dehydration takes place, greater than that of the strength-

conserving agent so that the solvent is readily passed off by evaporation leaving the strength-conserving agent behind. Such volatile solvent(s) will be suitable even if ordinarily considered to be toxic so long as the amount of volatile solvent, if any, present at the time of implantation does not produce a toxic response. Examples of volatile solvents useful in the invention herein would include but not be limited to, water;

alcohols, typically a low molecular weight alcohol such as methanol, ethanol, isopropanol, butanol, isobutanol, ethylbutanol, acetonitrile, pyridine, industrial methylated spirit, etc.; graded series of dehydrating agents in solution with the conserving

agent, e.g.; one solution of 70% ethanol/glycerol followed by two changes of 95%)

ethanol/glycerol and then absolute ethanol; histological solution, e.g., Flex 100™; polar

solvent, e.g., dimethylsulfoxide, small ketones, acetone; chloroform; methylene chloride and ethylene chloride; straight chain hydrocarbons, e.g., hexane, pentane and similar alkanes; low molecular weight alkenes; esters; ether, e.g., ethyl ether, tetrahydrofuran, dioxane, ethylene glycol monoethyl ether, crown ethers, etc.; aldehyde or solutions containing aldehydes, e.g., formaldehyde, formalin, etc., at low temperatures such that cross-linking does not proceed; super critical fluids, e.g., carbon dioxide or hydrogen sulfide at supercritical pressures, mixtures of any of the above liquids, etc. Such volatile solvents when present prior to lyophilization or other method of dehydration, will typically represent between about 20 to about 80, preferably about 30 to about 60 percent by volume of the biocompatible mechanical strength-conserving agent solution.

The biocompatible mechanical strength-conserving agent, neat or solution,

should have a viscosity at 20° C. of no greater than about 1410 cps, preferably the viscosity is between about 2 and about 300 cps. The preferred biocompatible mechanical strength-conserving agent is glycerol, more preferably a 50% aqueous or alcoholic

solution of glycerol, most preferably a series of graded dehydrating alcohols and glycerol. The bone is contacted with a mechanical strength-conserving amount of the mechanical strength-conserving agent in a suitable container, e.g., a 120 ml or 500 ml bottle, optionally with mechanical stirring. Optionally, the conserving agent can be applied by infusing, e.g., employing a pressurized system such as that described in U.S. Patent No. 5,846,484 the contents of which are incorporated herein by reference or by

varying levels of positive pressure. Pretreatment of tissues using the process described in U.S. Pat. No. 5,846,484 can improve the speed at which the strength-conserving agent penetrates the tissue. Optionally, the conserving agent can be contacted with the bone in the presence of a low pressure atmosphere such as that described in U.S. Patent No. 5,513,662 the contents of which are incorporated herein by reference or in the low pressure atmosphere provided by vacuum packaging the bone and strength-conserving agent utilizing a vacuum sealer. Optionally, the conserving agent can be contacted with the bone in the presence of alternating vacuum and positive pressure such as that

provided by the Hypercenter™ XP Enclosed Tissue Processor commercially available from Shandon Lipshaw USA or preferably a Sakura Tissue-TEK® VIP™ vacuum infusion tissue processor commercially available from Sakura Finetek, USA. As one skilled in the art will readily appreciate, the optimal times and levels of alternating vacuum-positive pressure or alternating positive pressure can be determined through

routine experimentation. The tissue processor allows for the simultaneous contacting of the bone with a mechanical strength-conserving agent and dehydrating of the bone when

a graded series of dehydrating agents is used as the volatile solvent for the strength- conserving agent. Such simultaneous contacting/dehydrating may result in an implant having better mechanical properties than one that is lyophilized after being contacted with a strength-conserving agent.

To assist the mechanical strength-conserving agent in penetrating the small pores of the bone, the bone and agent can be advantageously subjected to sonication. It has been determined that contacting the bone with strength-conserving agent in an ultrasonic bath improves the penetration of the agent into the tissue. Sonicating bone is well known

in the art and is described in U.S. Patent 5,797,871 the contents of which are incorporated herein by reference. Of course, it will be understood by one skilled in the art, that the contacting the bone with the strength-conserving agent can be carried out by any combination of one or more of the foregoing.

After the bone has been in contact with the conserving agent for a period of about

5 minutes to about 7 days, preferably at least about one hour, it can optionally be shaped prior to dehydration. Such shaping can be accomplished by cutting, forming, machining or other method of shaping bone. Thus, the bone can be rough cut, processed with strength-conserving agent, further shaped as desired, then subjected to further processing

if necessary. Such shaping of bone intended for implantation is well known in the art and is described in U.S. Pat. No. 6,025,538, the contents of which are incorporated herein by reference. After shaping or other optional processing steps, the bone intended for

implantation is dehydrated following procedures well known in the art. For example, the

bottle containing bone and conserving agent is subjected to, e.g., processes including but not limited to: contacting the bone with a graded series of dehydrating liquids; subjecting the bone to microwave energy such as described in U.S. Pat. No. 4,656,047 the contents of which are incorporated by reference herein; subjecting the bone to heat at ambient or sub-atmospheric pressures, e.g., drying oven at temperatures from about 35°C. to about

85°C, preferably about 40°C. to about 50°C, or vacuum oven at temperatures from about 35°C. to about 85°C, preferably about 40°C. to about 50°C; subjecting the bone to sub-

atmospheric pressure in the presence or absence of a desiccant, e.g., closed container subjected to vacuum optionally containing a desiccant such as anhydrous calcium chloride, anhydrous silica gel or the like; subjecting the bone to ambient temperatures at ambient or sub-atmospheric pressures such as typically found in a laboratory bench-top or conventional fume hood; alternative lyophilization procedures such as starting the lyophilization cycle at a higher temperature to dehydrate the tissue then reducing the

temperature and pressure to freeze the tissue and sublimate any remaining moisture as

described in Balderson, et al., The effects offreeze-drying on the mechanical properties of human cortical bone, 45th Annual Meeting of the Orthopaedic Research Society, : 785, (1999), the contents of which are incorporated by reference herein; or by a combination

of one or more of the foregoing. It will be understood that all references to vacuum herein, unless otherwise specified, refer to vacuum pressures as are usually provide by standard sources of laboratory vacuum, e.g., vacuum pump, air-water venturi device, etc. The monolithic bone treated in accordance with the invention, i.e., dehydrating

such bone in the presence of a mechanical strength-conserving agent, will exhibit a level

of mechanical strength which is at least about 10%, preferably at least about 20%, and more preferably at least about 30% greater than that of a comparable specimen of monolithic bone which has been lyophilized in the absence of a mechanical strength-

conserving agent. In addition, bone treated according to the invention herein demonstrates at least about 2% less decrease in length dimension as compared to bone

that has been lyophilized without being treated according to the invention herein. Further, bone treated according to the invention herein shows at least greater than 19% improvement in overall toughness after dehydrating as compared to bone that has been lyophilized without being treated according to the invention herein. At this point, the bone can optionally be further shaped prior to packaging.

There are a variety of conditions by which dehydrated bone can be rehydrated prior to implantation. Soaking the dehydrated bone in rehydrating solution at normal atmospheric pressure can perform rehydration. Alternatively, the dehydrated bone can be rehydrated in a low atmospheric pressure environment, for example, the rehydration solution can be introduced via hypodermic needle through the sealed rubber stopper. The

strength-conserving agent also acts as a wetting agent decreasing the time necessary to rehydrate the bone at the time of use.

The rehydration solution can be any of a number of suitable agents such as sterile water, normal saline, physiologically buffered saline, dextrose solution, antibiotic solutions, and others of this sort. Optionally, it can contain one or more wetting agents such as any of the Pluronic™ agents or any of a variety of medically/surgically useful

substances such as antiviral agents, particularly those effective against HIV and hepatitis; antimicrobials and/or antibiotics such as erythromycin, bacitracin, neomycin, penicillin B, tetracycline, viomycin, chloromycetin and streptomycin, cetazolin, ampicillin, azactam, tobramycin, clindamycin, gentamicin, etc.; amino acids, peptides, vitamins,

inorganic elements, co-factors for protein synthesis; hormones; endocrine tissue or tissue fragments; synthesizers; enzymes such as collagenase, peptidases, oxidases, etc.; polymer cell scaffolds with parenchymal cells; angiogenic drugs and polymeric carriers containing such drugs; antigenic agents; cytoskeletal agents; bone morphogenic proteins (BMPs),

transfoπning growth factor (TGF-beta), insulin-like growth factor (IGF-1); insulin-like growth factor two (IGF-2); platelet derived growth factor (PDGF), growth hormones such as somatotropin.

The rehydrated dehydrated monolithic bone prepared according to the method herein is intended to be applied at a bone defect site, e.g., one resulting from injury, defect brought about during the course of surgery, infection, malignancy or development malformation. The bone, suitably sized and shaped as required, can be utilized as a graft

or replacement in a wide variety of orthopedic, neurosurgical and oral and maxillofacial surgical procedures such as the repair of simple and compound fractures and nonunions, external and internal fixations, joint reconstruction such as arthrodesis, general arthroplasty, cup arthroplasty of the hip, femoral and humeral head replacement, femoral head surface replacement and total joint replacement, repairs of the vertebral column including spinal fusion and internal fixation, tumor surgery, e.g., deficit filling,

discectomy, laminectomy, excision of spinal cord tumors, anterior cervical and thoracic operations, repair of spinal injuries, scoliosis, lordosis and kyphosis treatments,

intermaxillary fixation of fractures, mentoplasty, temporomandibular joint replacement, alveolar ridge augmentation and reconstruction, onlay bone grafts, implant placement and revision, sinus lifts, etc. Specific bones which can be repaired with the bone-derived implant herein include the ethmoid, frontal, nasal, occipital, parietal, temporal, mandible, maxilla, zygomatic, cervical vertebra, thoracic vertebra, lumbar vertebra, sacrum, rib, sternum, clavicle, scapula, humerus, radius, ulna, carpal bones, metacarpal bones,

phalanges,- ilium, ischium, pubis, femur, tibia, fibula, patella, calcaneus, tarsal and metatarsal bones.

The invention will be more fully understood by way of the following examples which are intended to illustrate but not limit methods in accordance with the present invention. Example 1

Human diaphyseal segments from the humerus were treated for 72 hours in an ultrasonic bath containing 50% (v/v) aqueous glycerol. An M-4 threaded hole was drilled and tapped into the cortex of the diaphyseaj bone. The specimens were then frozen at — 40°C. following this first phase of treatment. Specimens were then treated by one of two processes in a Virtis Unitop 600L lyophilization unit. The first used a standard freeze-

then-dry (FD) process that sublimates the water off from the frozen specimen. The time-

temperature relationship for this process is outlined in figure 1. The second process uses a dry-then-freeze (DF) process that evaporates off much of the liquid prior to freezing and sublimation of the remaining liquid. The time-temperature relationship for this process

is outlined in figure 2. It has been reported by Balderson, et al., The effects of freeze- drying on the mechanical properties of human cortical bone, 45th Annual Meeting of the Orthopaedic Research Society, : 785, (1999), that bone specimens using such a lyophilization procedure as the second process tend to have superior toughness and other mechanical properties. Example 2

. Human cortical bone specimens from the diaphysis of a long bone are manufactured into the shape of a threaded cylindrical dowel. Specimens are then treated

in a stirred 60% propylene glycol/40% ethanol solution, while in an oven at 35° Celsius for at least 24 hours. At the end of 24-48 hours, the specimen is removed from the solution, and is then placed in a vacuum oven at 30° Celsius and standard laboratory ,

vacuum. The specimen remains in the oven for a period of time necessary to evaporate off the remaining solvent, to remove the remaining water from the tissue, and to allow

adherent treatment solution to penetrate. This time is determined by standard assays of solvent content and of moisture content. The samples are then packaged for surgical use.

Example 3

*, _*

Human cortical bone specimens, prepared by cutting on a band saw into strut allografts, are placed into the retort chamber of an automated tissue-processing machine,

such as the Sakura Tissue-Tek® VIP™ vacuum infusion tissue processor (Sakura FineTek, USA, Torrance, CA). Solutions of the following compositions (Table 1) are automatically pumped into the retort chamber for at least 4 hours per solution. This sequence of alcohol/glycerol solutions will concurrently dehydrate the tissue, while at the same time replacing the moisture with glycerol. Solutions are applied using alternating

pressure (0.35 kg/cm² -90 seconds) followed by ambient pressure (30 seconds), then vacuum (50 cm Hg - 90 seconds), then ambient pressure (30 seconds) in a cycle to assist and to speed fluid penetration into the tissues. Following the last solution, specimens are taken out of the retort, and placed into a bell jar with a vacuum attachment. The

remaining volatile alcohol solvent is evaporated off into a hood, until only trace alcohol

remains.

Table 1

Example 4

Human cortical bone specimens, prepared by cutting on a bandsaw into diaphyseal cross-sections, are placed into a closed container with a 50% ethanol/50%

glycerol solution. The specimen is stirred continuously for 24-48 hours, then the container is opened under a hood to allow the volatile ethanol solvent to evaporate at ambient pressure. Specimens are then removed from the container, and placed into a

Virtis Unitop 600L lyophilization unit, using a standard lyophilization procedure, to complete the dehydrating solvent removal steps.

Example 5

Five ramp shaped graft pieces (described in figure 3) were prepared from human

bone tissue that had been treated for viral inactivation using standard techniques. Specimens were individually placed in Kapak™ bags, and partially filled with 50% (v/v) aqueous glycerol solution. The bags were then sealed in a vacuum sealing device so that the air was removed prior to sealing and the implants were each surrounded by the glycerol solution at a reduced pressure. Specimens were allowed to equilibrate in the

solution overnight, and were then removed from the Kapak™ bags. Specimens were then freeze-dried in a Virtis Unitop™ 600L lyophilization unit using standard methods.

Dimensions were measured prior to treatment, and following treatments and with rehydration in physiological saline after 1.5 hours. Figure 4 shows the difference from fresh cut dimensions. Specimens were also visually checked for warpage or deformation

of the ridges. None was found. Specimens were also checked to determine whether they would accept a mating screw into the threaded hole. AH specimens did accept a threaded screw.

Comparative Example 1

Bovine cortical bone specimens, 4mm x 4mm x 40mm (nominal) were prepared

from the same bovine femur. Some specimens were soaked in a 50% aqueous solution of glycerol for three days prior to lyophilization. Other specimens were lyophilized without glycerol. After lyophilization, the specimens were tested in 3 -point bending (30mm span, center loaded) in the MTS servo hydraulic testing system. Loading was conducted at a rate of 25mm/min under displacement control. Specimens were loaded to failure. Data were collected on maximal load, failure load and energy absorbed to break (a measure of how brittle the material is). Factors were compared by the Wilcoxon non-parametric test. The results are given in Table 2 below.

Samples: - Glycerol/Dry n=4 No glycerol/dry n=3 Glycerol/Saline n=2 No glycerol/Saline n=3

Table 2

Break Load (kN) Energy to break (N-m)

Glycerol/Dry 0.277 ± 0.022 0.037 ± 0.0025

Glycerol/Saline 0.205 ± 0.037 0.037 ± 0.0021

No glycerol/Dry 0.234 ± 0.064 0.028 ± 0.0072

No glycerol/Saline 0.152 ± 0.016 0.028 ± 0.0135

Many of the specimens that were exposed to saline showed a number of fine, internal longitudinal cracks that were visible macroscopically. Both glycerol-treated and non-treated specimens displayed this morphology. For all specimens, peak load was equivalent to break load. Glycerol application was a significant factor in determining breaking load (p=0.05), and marginally significant in the energy to breakage (p=0.08).

Saline hydration was significant to the breaking load (p=0.03) but not other parameters.

Glycerol application prior to lyophilization reduces brittleness in the bone

samples. Freeze-drying, composed of a freezing step and a water-removal step, is damaging to bone and has been shown to negatively affect mechanical properties. Yet, the bone literature teaches that freezing itself is not detrimental to bone to any significant degree. Thus, it is believed that the damage protection offered by the strength-conserving

agent does not act by eliminating damage in the freezing, but rather by eliminating damage due to dimensional changes during the dehydrating aspects of freeze-drying. Although the mechanism of the invention is not entirely understood, the inventors believe that this, improvement is achieved by maintaining the liquid environment of the bone to reduce damage during lyophilization. Strength was improved by an average of 34% and energy absorption prior to fracture was improved by up to 32%.

Comparative Example 2

Human bone was treated for viral inactivation using the process described in U.S. Patent No. 5,846,484. From these bones diaphyseal segments, 2 cm in length, were cut

on a band saw. Other specimens were not treated by this 5,846,484 process, but were rather cleaned of adherent soft tissues and processed using standard techniques. To

determine penetration of a treatment solution, penetration was affected by either of two treatment processes: Specimens suspended in a stirred solution; and specimens suspended in an ultrasonic bath. The treatment solution used was 50% (v/v) aqueous glycerol, and also contained 0.5%(w/v) methylene blue dye to allow assessment of penetration. Specimens of each group were removed at 1 hour, 4 hours, 11 hours, 24

hours, and 48 hours. Sectioning the bone transversely to the middle (1cm) point, and

qualitatively describing penetration assessed penetration of the conserving agent. The table below, table 3, summarizes penetration into Haversian Canals (HC) and Matrix (M) regions of the middle ( 1 cm) section.

Table 3

Key: X= Mostly Penetrated; P=Part y Penetrate ; = n mally Penetrated

Pre-treatment of tissues using the process described in patent 5,846,484 improved

the speed at which the tissue was penetrated by the treatment solution. Further, effecting penetration by ultrasonic bath also substantially reduced the penetration time for the

solution, ^**

Comparative Example 3

Human bone was treated for viral inactivation using processes described in US patent 5,846,484. From these treated bones, human diaphyseal bone segments were shaped into a ramped structure (figure 3) using processes described in U.S. Patent Appln.

No. 09/328,242 filed June 8, 1999. One specimen from each of four donors (16 total specimens) received each of four treatments: A.) treated for 3 days in an ultrasonic bath

containing 50% (v/v) aqueous glycerol solution, then freeze-dried using standard methods; B.) treated for 3 days in a container containing 50% (v/v) aqueous glycerol solution stirred continuously (Stir), then freeze-dried using standard methods; C.) freeze- dried only; D.) frozen only (-70°C). Dimensional measurements were taken after initial manufacture, and then again after all treatments and following 1 hour rehydration in

physiological saline. The threaded hole was also tested using a mating screw prior to the

process and at the end of the process. Results in Figure 5 show the difference between final and initial measurements for the overall length (OL) and overall width (OW). Each

of the treated groups showed substantially less dimensional change than the freeze-dried only group, though differences in all groups were greater than that of the specimens that

were frozen and still contained water at the time of rehydration. The threaded hole (shown on the left side of figure 3) was also tested at each stage, and found to accept the screw for the treated specimens (Stir and Ultrasonic) and the frozen specimens at each stage. For the freeze-dried specimens, two of four specimens failed to accept the screw following the freeze-drying step, though one of these did accept a screw after rehydration.

Comparative Example 4

Donor specimens prepared as in Comparative Example 3 where subjected to mechanical testing utilizing a MTS 858 Bionix™ compressive testing instrument at a

loading rate of 25 mm/min for single-cycle compression to 2mm displacement testing to determine the toughness of each treatment group. The results are contained in table 4

below.

Table 4

The results demonstrate that toughness is improved when bone is treated in accordance with the invention herein.

It will be understood that various modifications can be made to the embodiments and examples disclosed herein. Accordingly, the above description should not be construed as limiting but merely as exemplifications of preferred embodiments. Those skilled in the art will envision such various modifications that are within the scope and spirit of the claims appended hereto.

Claims

WHAT IS CLAIMED IS

1. - A method for treating monolithic bone intended for implantation to

conserve the mechanical strength of the bone during dehydration, subsequent packaging and storage of the bone, the method comprising:

contacting the bone with a mechanical strength-conserving amount of at least one biocompatible mechanical strength-conserving agent, said agent being a liquid organic material which is capable of penetrating and remaining in the bone during its dehydration, packaging and storage;

dehydrating the bone containing the mechanical strength-conserving agent; and, packaging the dehydrated bone.

2. The method of Claim 1 further comprising infusing under pressure the

mechanical strength-conserving agent.

3. The method of Claim 1 further comprising sonicating the bone and mechanical strength-conserving agent.

4. The method of Claim 1 further comprising contacting the bone and the mechanical strength-conserving agent in the presence of a low pressure atmosphere.

5. The method of Claim 1 further comprising contacting the bone and the

mechanical strength-conserving agent in the presence of alternating vacuum and positive pressure.

6. The method of Claim 1 further comprising contacting the bone and the mechanical strength-conserving agent in the presence of alternating levels of positive

pressure.

7. The method of Claim 1 wherein the strength-conserving agent is selected from the group consisting of polyhydroxy compound, polyhydroxy ester, fatty alcohol, fatty alcohol ester, fatty acid, fatty acid ester, liquid silicone and mixtures thereof.

8. The method of Claim 7 wherein the polyhydroxy compound is selected

from the group consisting of glycerol, 1,4-butylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, propylene glycol, dipropylene glycol, polysaccharides and their derivatives, hyaluronic acid, polyoxyethylene-polyoxypropylene copolymer, polyoxyethylene-polyoxypropylene block copolymer, and alkylphenolhydroxypolyoxyethylene.

9. The method of Claim 1 wherein the mechanical strength-conserving agent is glycerol.

10. The method of Claim 1 wherein the mechanical strength-conserving agent is in solution with at least one volatile solvent.

11. The method of Claim 10 wherein the volatile solvent is selected from the group consisting of water, methanol, ethanol, isopropanol, butanol, isobutanol, ethylbutanol, acetonitrile, pyridine, industrial methylated spirit, graded series of

dehydrating agents, histological solution, Flex 100™, dimethylsulfoxide, small ketones, acetone, chloroform, methylene chloride, ethylene chloride, straight chain hydrocarbons

of less than 12 carbons, hexane, pentane, low molecular weight alkenes, esters, ethers, ethyl ether, tetrahydrofuran, dioxane, ethylene glycol monoethyl ether, crown ethers, aldehyde, solutions containing aldehydes, formaldehyde, formalin, super critical fluids, liquid carbon dioxide, liquid hydrogen sulfide, and mixtures thereof.

12. The method of Claim 10 further comprising the step of volatile solvent removal.

13. The method of Claim 10 wherein the mechanical strength-conserving agent is an aqueous solution of glycerol.

14. The method of Claim 10 wherein the mechanical strength-conserving agent is an alcoholic solution of glycerol.

15. The method of Claim 14 wherein the alcoholic solution of glycerol further comprises a dehydrating graded series of at least one alcohol,

16. The method of Claim 10 wherein the step of dehydrating is carried out by contacting the bone with a graded series of dehydrating liquids; or, by subjecting the bone to microwave energy; or, by subjecting the bone to heat at ambient or sub-atmospheric pressures; or, subjecting the bone to sub-atmospheric pressure in the presence or absence of a desiccant; or, by a combination of one or more of the foregoing.

17. The method of Claim 12 wherein the step of volatile solvent removal is carried out by subjecting the bone to microwave energy; or, by subjecting the bone to ambient temperatures at ambient or sub-atmospheric pressures; or, by subjecting the bone to heat at ambient or sub-atmospheric pressures; or, subjecting the bone to sub-

atmospheric pressure in the presence or absence of a desiccant; or, by a combination of one or more of the foregoing.

18. A rehydrated strength-conserved shaped bone implant prepared by: contacting bone with a mechanical strength-conserving amount of at least one biocompatible mechanical strength-conserving agent, said agent being a liquid organic material which is capable of penetrating and remaining in the bone during its dehydration, packaging and storage; dehydrating the bone containing the mechanical strength-conserving agent;- -- packaging the dehydrated bone; and,

rehydrating the bone prior to or during implantation.

19. The shaped bone implant of Claim 18 wherein the shaped bone has been shaped before, during, or after its contacting with mechanical strength conserving agent.

20. The shaped bone implant of Claim 18 wherein the shaped bone has at least about 2% less decrease in length dimension as compared to bone that has been

lyophilized without being contacted with a mechanical strength-conserving amount of a mechanical strength-conserving agent.

21. The shaped bone implant of Claim 18 wherein the toughness of the

dehydrated bone is at least greater than 19% as compared to bone that has been dehydrated without being contacted with a mechanical strength-conserving amount of a mechanical strength-conserving agent.

22. The shaped bone implant of Claim 18 wherein the step of rehydrating is

performed by contacting the dehydrated bone with at least one rehydrating liquid selected from the group consisting of sterile water, normal saline, physiologically buffered saline, dextrose solution, antibiotic solutions, wetting agents and medically/surgically useful substance(s).

23. A method of using the bone of Claim 18 for repair of at least one bone selected from the group consisting of: ethmoid, frontal, nasal, occipital, parietal, temporal, mandible, maxilla, zygomatic, cervical vertebra, thoracic vertebra, lumbar vertebra, sacrum, rib, sternum, clavicle, scapula, humerus, radius, ulna, carpal bones,

metacarpal bones, phalanges, ilium, ischium, pubis, femur, tibia, fibula, patella, calcaneus, tarsal and metatarsal bones; the method comprising; exposing a surgical site, inserting the bone of Claim 18 into the surgical site, and, obtaining closure of the surgical site.