WO2010138627A2

WO2010138627A2 - A method of enhancing bioactivity of implant materials

Info

Publication number: WO2010138627A2
Application number: PCT/US2010/036248
Authority: WO
Inventors: Takahiro Ogawa
Original assignee: The Regents Of The University Of California
Priority date: 2009-05-29
Filing date: 2010-05-26
Publication date: 2010-12-02
Also published as: WO2010138627A3

Abstract

Provided herein is methods of enhancing the bioavailability of an implant material, an implant formed by the method thereof, and a method of using the implant.

Description

A METHOD OF ENHANCING BIOACTIVITY OF IMPLANT MATERIALS

CROSS REFERENCE

This application claims the benefit of U.S. Provisional Application No. 61/182,456 filed on May 29, 2009, the teaching of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention generally relates to a method for enhancing bioactivity of implant materials. BACKGROUND

Reconstruction and repair following femoral neck fracture, degenerative changes of knee and hip joints and missing teeth are quite common procedure and have considerable medical and societal impact. We experience 300,000 incidences of hip fracture alone in the United States, and annual expenditures for treating the osteoporotic fractures are estimated at $13.8 billion. Titanium is a proven biocompatible material, and the use of titanium implants as an endosseous anchor has become essential in such treatments. Successful implant anchorage depends upon the magnitude of bone directly contacting the titanium surface without soft/connective tissue intervention, which is referred to bone-titanium integration or osseointegration. Despite the growing needs for titanium implants, a percentage of unsuccessful implants, for instance, ranging 5%-40% in orthopedic implants, limited application due to unfavorable host site anatomy, and protracted healing time of implants particularly in dental implants, are the immediate challenges. Furthermore, the implant placement, facing often times the impaired bone regenerative potential, such as osteoporotic and aged metabolic properties, increase the level of difficulty to achieve the biological requirements of bone- titanium integration. Therefore, technologies to enhance the bioactivity of titanium surfaces are desired.

The present inventor recently reported a novel phenomenon of biological aging of implant materials, such as titanium and chromium-cobalt, i.e., freshly prepared titanium surfaces are highly bioactive and the bioactivity degrades with time during their storage since processing. For example, the storage for 4 weeks reduces the level of titanium bioactivity to half. This discovery drew an immediate attention in implant therapeutics market. Clinically, metal implant products, regardless of dental or orthopedic use, have been considered biologically stable and to exert invariable clinical performance. There is no regulation or expiration of manufacture, distribution, and storage in these products, except for the expiration of sterilization, which is normally 5 years.

It is practically unlikely for implant products to be delivered to the users within one month after manufacturing. Therefore, there is a need for enhanced bioactivity of implants prior to use thereof. The embodiments described below address the above identified issues and needs.

SUMMARY OF THE INVENTION

Provided herein is a method for increasing the bioactivity of medical implants. In some embodiments, the method comprises irradiating an implant with a high energy radiation for a sufficient period of time to cause the implant to form a surface comprising positive charges.

In some embodiments, the high energy radiation is gamma ray. In some embodiments, the implant is in a package when the implant is subjected to irradiating.

The implant material can be any implant material. In some embodiments, the implant material can be metallic material, non-metallic material, or combination thereof. In some embodiments, the implant material is a metallic material, examples of which include titanium, gold, platinum, tantalum, niobium, nickel, iron, chromium, cobalt, zirconium, magnesium, magnesium, aluminum, palladium, an alloy formed thereof, e.g., stainless steel, or combinations thereof.

In some embodiments, the implants can be, e.g., tooth implants, jaw bone implant, repairing and stabilizing screws, pins and plates for bone, spinal implants, femoral implants, neck implants, knee implants, wrist implants, joint implants such as an artificial hip joint, maxillofacial implants such as ear and nose implants, limb prostheses for conditions resulting from injury and disease, and combinations thereof.

In some embodiments, optionally in combination with any other embodiments, the implant is a titanium implant. In some embodiments, optionally in combination with any other embodiments, the implant is a zirconium implant or chromium-cobalt alloy implant.

In some embodiments, optionally in combination with any other embodiments, the implant material is a non-metallic material, examples of which include polymeric material and a bone cement material. Examples of bone cement materials can comprise a material such as polyacrylates, polyesters, bioglass, ceramics, calcium-based materials, calcium phosphate-based materials, and combinations thereof. In some embodiments, the bone cement material comprises poly(methyl methacrylate) (PMMA) or methyl methacrylate (MMA). In some embodiments, optionally in combination with any other embodiments, the implant is a non-metallic implant selected from the group consisting of tooth implants, jaw bone implant, repairing and stabilizing screws, pins and plates for bone, spinal implants, femoral implants, neck implants, knee implants, wrist implants, joint implants, an artificial hip joint, maxillofacial implants, ear and nose implants, limb prostheses for conditions resulting from injury and disease, and combinations thereof.

In some embodiments, optionally in combination with any other embodiments, the method further comprises treating at least a portion of the surface of the implant by physical treatment or chemical treatment prior to irradiating the implant with the high energy radiation. Such physical treatment can be, e.g., machining, sand-blasting, or metallic or nonmetallic deposition. Such chemical treatment can be, e.g., acid-etching, sand-blasting, oxidation, or alkaline treatment.

In some embodiments, it is provided an implant treated with a method according to any of the above embodiments.

In some embodiments, it is provided a method of treating a medical disorder. The method comprises implanting in a mammal an implant material according to any of the above described embodiments.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows the amount of albumin adsorbed to 4-week-old titanium surfaces with and without high energy irradiation (gamma ray treatment). The albumin was incubated on the titanium disks for 2 hours. Data are mean ± SD (n=3). Figure 2 shows the number of bone- forming cells (osteoblasts) attached to 4-week- old titanium surfaces with and without high energy irradiation (gamma ray treatment). The cells were incubated on these titanium disks for 2 and 24 hours. Data are mean ± SD (n=3).

Figure 3 shows the number of bone- forming cells (osteoblasts) proliferated on the 4-week-old titanium surfaces with and without high energy irradiation (gamma ray treatment) for 2 days of culture. Data are mean ± SD (n=3).

Figure 4 shows the initial spread and cytoskeletal arrangement, and establishment of focal adhesion of osteoblasts 3 h after seeding onto 4-week-old titanium surfaces with and without high energy irradiation (gamma ray treatment). Representative confocal microscopic images of cells stained with rhodamine phalloidin for actin filaments (red) and anti-vinculin (green), along with cytomorphometric evaluations, are presented. Data are mean ± SD (n=10).

Figure 5 shows the alkaline phosphatase activity, an early stage marker of cell differentiation (functional development), in osteoblasts cultured on 4-week-old titanium surfaces with and without high energy irradiation (gamma ray treatment). Data are mean ± SD (n=3).

Figure 6 shows mineralizing capability, a late stage marker of cell differentiation (functional development), in osteoblasts cultured on 4-week-old titanium surfaces with and without high energy irradiation (gamma ray treatment). The mineralizing capability was evaluated as the amount of calcium deposition in the cultures. Data are mean ± SD (n=3).

Figure 7 shows the change of hydrophilicity of 4-week-old titanium surfaces with and without high energy irradiation (gamma ray treatment). Data are mean ± SD (n=3).

DETAILED DESCRIPTION

As used herein, the terms "implant" and "implant material" can be used interchangeably. As used herein, the terms "increase" and "enhance" can be used interchangeably. As used herein, the terms "bioactivity" and "bioavailability" can be used interchangeably.

Provided herein is a method for increasing the bioactivity of medical implants. The technology can be used to restore the age-related bioactivity degradation of implants and to enhance the bioactivity of freshly prepared implants. The methods comprise irradiating an implant with a high energy radiation, such as gamma ray, for a sufficient period of time prior to use of the implant. Because gamma ray permeates most materials and can be applied to an implant surface without unpacking the products, this technology will provide a significant technical advantage in its clinical and commercial application.

As used herein, the term "prior to use" generally refers to a period within days, e.g., within a week, within 6 days, within 5 days, within 4 days, within 3 days, within 2 days, or within 1 day of the use of an implant material. In some embodiments, the term "prior to use" can refer to a period within hours, e.g., within 18 hours, within 12 hours, within 8 hours, within 4 hours, within 2 hours, or within 1 hour of the use of an implant material.

The length of such prior to use period is an important factor since performance or properties of a freshly generated surface of invention described herein can be time sensitive.

In some embodiments, the methods comprise irradiating an implant material with a high energy radiation for a sufficient period of time prior to use of the implant material to cause the implant material to form a surface comprising positive charges.

In some embodiments, the high energy radiation is gamma ray. In some embodiments, the gamma ray dose is in a range of from about 0 kGy to about 1000 kGy. In some embodiments, the gamma ray dose is about 5 KGy, 10 kGy, 20 kGy, 3OkGy, 50 KGy, 100 kGy, 150 kGy, 200 kGy, 300 kGy, 500 kGy, 750 kGy, or 1000 kGy. In some embodiments, the implant is in a package when the implant is subjected to irradiating.

The implant material can be any implant material. In some embodiments, the implant material is a metallic material, non-metallic material, or combination thereof.

In some embodiments, the implant material is a metallic material, examples of which include titanium, gold, platinum, tantalum, niobium, nickel, iron, chromium, cobalt, zirconium, magnesium, magnesium, aluminum, palladium, an alloy formed thereof, e.g., stainless steel, or combinations thereof.

In some embodiments, the implant can be, e.g., tooth implants, jaw bone implant, repairing and stabilizing screws, pins and plates for bone, spinal implants, femoral implants, neck implants, knee implants, wrist implants, joint implants such as an artificial hip joint, maxillofacial implants such as ear and nose implants, limb prostheses for conditions resulting from injury and disease, and combinations thereof.

In some embodiments, optionally in combination with any of the above embodiments, the implant material is non-metallic, examples of which can be a polymeric implant material or can be a bone cement material. Examples of bone cement materials can comprise a material such as polyacrylates, polyesters, bioglass, ceramics, calcium-based materials, calcium phosphate-based materials, and combinations thereof. In some embodiments, the bone cement material comprises poly(methyl methacrylate) (PMMA) or methyl methacrylate (MMA).

In some embodiments, optionally in combination with any other embodiments, the implant is a non-metallic implant such as tooth implants, jaw bone implant, repairing and stabilizing screws, pins and plates for bone, spinal implants, femoral implants, neck implants, knee implants, wrist implants, joint implants, an artificial hip joint, maxillofacial implants, ear and nose implants, limb prostheses for conditions resulting from injury and disease, and combinations thereof. In some embodiments, optionally in combination with any other embodiments, the method further comprises treating at least a portion of the surface of the implant material by physical treatment or chemical treatment prior to irradiating the implant material with the high energy radiation. Such physical treatment can be, e.g., machining, sand-blasting, or metallic or nonmetallic deposition, and such chemical treatment can be, e.g., acid-etching, sand-blasting, oxidation, or alkaline treatment.

In some embodiments, it is provided a method of treating a medical disorder. The method comprises implanting in a mammal an implant according to any of the above described embodiments. In some embodiments, the bioactivity of the implant is tissue- implant integration or bone-implant capacity.

As used herein, wherever and whenever applicable, the term "tissue-implant integration capability" refers to the ability of an implant material to be integrated into the tissue of a biological body. The term "bone-implant integration capacity" refers to the ability of an implant material to be integrated into the bone of a biological body.

The tissue integration capability of an implant can be generally measured by several factors, one of which is wettability of the implant surface, which reflects the hydrophilicity or oleophilicty (hydrophobicity), and hemophilicity of an implant surface. Hydrophilicity and oleophilicity are relative terms and can be measured by, e.g., water contact angle

(Oshida Y, et al., J Mater Science 3:306-312 (1992)), and area of water spread (Gifu-kosen on line text, http://www.gifu-nct.ac.jp/elec/tokoro/fft/contact-angle.html). For purposes of the present invention, the hydrophilicity or oleophilicity can be measured by contact angle or area of water spread of an implant surface described herein relative to the ones of the control implant surfaces. Relative to the implant surfaces not treated with the process described herein, an implant material treated with the process described herein has a substantially lower contact angle or a substantially higher area of water spread.

The tissue integration capability of an implant can also be measured by protein affinity, cell affinity, ability to increase cell spread, ability to increase osteoblast proliferation, ability to increase osteoblast differentiation, ability to increase osteoblast mineralization, or combination thereof.

In some embodiments, the bioactivity of the implant material can be protein affinity, cell affinity, ability to increase cell spread, ability to increase osteoblast proliferation, ability to increase osteoblast differentiation, ability to increase osteoblast mineralization, or combination thereof. The protein can be bovine serum albumin, fraction V, or bovine plasma fibronectin. The cell can be human mesenchymal stem or osteoblast cell.

It is to be noted that while gamma ray has been used to sterilize an implant material, such sterilization is insufficient to enhance the bioactivity of the implant material. Besides, any benefit of such gamma ray sterilization would disappear since such sterilization is performed in manufacture of an implant material, which is generally days or months, if not years, prior to use of the implant material in a patient. Implant Materials

The implant materials described herein with enhanced tissue integration capabilities include any implant materials currently available in medicine or to be introduced in the future. The implant materials can be metallic material, non-metallic implant material, or combination thereof.

In some embodiments, the implants are metallic such as titanium implants, e.g., titanium implants for replacing missing teeth (dental implants) or fixing diseased, fractured or transplanted bone. Other exemplary metallic implants include, but are not limited to, titanium alloy implants, chromium-cobalt alloy implants, platinum and platinum alloy implants, nickel and nickel alloy implants, stainless steel implants, zirconium, chromium- cobalt alloy, gold or gold alloy implants, and aluminum or aluminum alloy implants.

The implants described herein include titanium implants and non-titanium implants. Titanium implants include tooth or bone replacements made of titanium or an alloy that includes titanium. Titanium bone replacements include, e.g., knee joint and hip joint prostheses, femoral neck replacement, spine replacement and repair, neck bone replacement and repair, jaw bone repair, fixation and augmentation, transplanted bone fixation, and other limb prostheses. None-titanium implant materials include tooth or bone implants made of gold, platinum, tantalum, niobium, nickel, iron, chromium, cobalt, magnesium, magnesium, aluminum, palladium, zirconium, chromium-cobalt alloy, alloy formed thereof, e.g., stainless steel, or combinations thereof. In some embodiments, the metallic implant can specifically exclude any of the aforementioned metals.

Non-metallic implants include, for example, ceramic implants, calcium phosphate, or polymeric implants. Useful polymeric implants can be any biocompatible implants, e.g., bio-degradable polymeric implants. Exemplary polymeric implants include, e.g., poly- lactic-co-glycolic acid (PLGA), polyacrylate such as polymethacrylates and polyacrylates, and poly- lactic acid (PLA) implants. Representative ceramic implants include, e.g., bioglass and silicon dioxide implants. Calcium phosphate implants include, e.g., hydroxyapatite and tricalciumphosphate (TCP). In some embodiments, the implant material described herein can specifically exclude any of the aforementioned materials. In some embodiments, the implant comprises a bone-cement material. The bone cement material can be any bone cement material known in the art. Some representative bone cement materials include, but are not limited to, polyacrylate or polymethacrylate based materials such as poly(methyl methacrylate) (PMMA)/methyl methacrylate (MMA), polyester based materials such as PLA or PLGA, bioglass, ceramics, calcium phosphate- based materials, calcium-based materials, and combinations thereof. In some embodiments, the implant material described herein can specifically exclude any of the aforementioned materials.

The implants described herein can be porous or non-porous implants. Porous implants can impart better tissue integration while non-porous implants can impart better mechanical strength.

The implants described herein can be made by a process that includes applying a high energy radiation to the implant. The implants provided herein can be used to treat, prevent, ameliorate, or reduce symptoms of a medical condition such as missing teeth, a need for orthodontic anchorage or bone related medical conditions such as femoral neck fracture, neck bone fracture, wrist fracture, spine fracture/disorder or spinal disk displacement, fracture or degenerative changes of joints such as knee joint arthritis, bone and other tissue defect or recession caused by a body condition or disorder such as cancer, injury, systemic metabolism, infection and aging, limb amputation resulting from injuries and diseases, and combinations thereof.

The implants provided herein can be subjected to various established surface treatments to increase surface area or surface roughness for better tissue integration or tissue attachment. Representative surface treatments include, but are not limited to, physical treatments and chemical treatments. Physical treatments include, e.g., machined process, sandblasting process, metallic deposition, non-metallic deposition (e.g., apatite deposition), or combinations thereof. Chemical treatment includes, e.g., etching using a chemical agent such as an acid, base (e.g., alkaline treatment), oxidation, and combinations thereof. For example, a metallic implant can form different surface topographies by a machined process or an acid-etching process. In some embodiments, the high energy radiation treated implant surface has a higher hydrophilicity than the untreated surface. The treated implant can have a contact angle of the implant surface and water lower than that of the untreated surface. In some embodiments, the treated implant can have a contact angle lower than 10°, 20°, 30°, 40°, or 50°.

In some embodiments, the high energy radiation treated implant surface can have higher protein affinity than the untreated implant surface by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%. The affinity can be measured by adsorption of proteins, such as bovine serum albumin, fraction V, and bovine plasma fibronectin. The albumin adsorption of the treated implant surface can be higher than the untreated surface by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%.

The high energy radiation treated implant surface can have higher cell affinity than the untreated implant surface by at least 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, and 200%. The affinity to cell can be measured by attachment of cells such as human mesenchymal stem cell and osteoblastic cell. The cell attachment of the treated implant surface can be higher than the untreated surface by at least 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, and 200%. The high energy radiation treated surface can be capable of any of the following: increasing adsorption of proteins, increasing attachment of cells such as osteoblasts, facilitating osteoblast spread, and increasing cell functions such as osteoblast proliferation, osteoblast differentiation, osteoblast mineralization, and combination thereof.

The treated implant surface can cause osteoblasts attached thereto to have higher mineralization capacity than the untreated surface by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, and 300%.

The treated surface can cause osteoblasts attached thereto to have higher differentiation capacity than the untreated surface by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, and 300%. The high energy radiation treated implant surface causes tissue-implant integration and/or bone-implant integration.

High Energy Radiation

The implant materials with enhanced tissue integration capabilities provided herein can be formed by treating the implant materials with a high energy radiation for a period of time. The length of the radiation period depends on the type of implants. For a metallic implant (e.g., a titanium implant), the period of radiation generally ranges from about 1 minute to about 1 month, e.g., from about 1 minute to about 1 hour, from about 1 hour to about 5 hours, from about 5 hours to about 24 hours, from about 1 day to about 5 days, from about 5 days to about 10 days, or from about 10 days to about 1 month. For non- implant materials, e.g., a biocompatible, biodurable polymeric implant, the period of radiation generally ranges about 1 minute to about 1 month, e.g., from about 1 minute to about 1 hour, from about 1 hour to about 5 hours, from about 5 hours to about 24 hours, from about 1 day to about 5 days, from about 5 days to about 10 days, or from about 10 days to about 1 month. The term "high energy radiation" includes radiation by gamma ray, light or a magnetic wave. In some embodiments, the term "high energy" refers to a radiation having a wavelength at or below about 400 nm, e.g., about 350 nm, about 300 nm, about 250 nm, about 200 nm, about 150 nm, about 100 nm, about 50 nm, or about 10 nm. In some embodiments, the radiation can have a wavelength at or below about 5 nm, about 1 nm, about 0.5 nm, about 0.1 nm, about 0.05, about 0.01, about 0.005 or about 0.001 nm.

The radiation having a wavelength from about 400 nm to 10 nm is generally referred to as ultraviolet light UV, the radiation having a wavelength from about 10 nm to 0.1 nm is generally referred to as x-rays, and the radiation having a wavelength from about 0.1 nm to about 0.001 nm is generally referred to as gamma-rays. In some embodiments, the high energy radiation is gamma ray. In some embodiments, the gamma ray dose is in a range of 0 kGy to about 1000 kGy. In some embodiments, the gamma ray dose is about 5 KGy, 10 kGy, 20 kGy, 3OkGy, 50 KGy, 100 kGy, 150 kGy, 200 kGy, 300 kGy, 500 kGy, 750 kGy, or 1000 kGy.

In anther aspect of the present invention, it is provided a facility or device for radiating implant materials. In one embodiment, the facility or device includes a chamber for placing implant materials, a source of high energy radiation and a switch to switch on or turn off the radiation. The facility or device may further include a timer. In some embodiments, the facility or device can further include a mechanism to cause the implant materials or the high energy radiation source to turn or spin for full radiation of the implants. Alternatively, the chamber for placing implant materials can have a reflective surface so that the radiation can be directed to the implant materials from different angles, e.g., 360 ° angle. In some embodiments, the facility or device may include a preservation mechanism of the enhanced bone-integration capability, e.g., multiple irradiation of light, radio-lucent implant packaging, packing and shipping.

Medical Uses

The implant materials provided herein can be used for treating, preventing, ameliorating, correcting, or reducing the symptoms of a medical condition by implanting the implant materials in a mammalian subject. The mammalian subject can be a human being or a veterinary animal such as a dog, a cat, a horse, a cow, a bull, or a monkey.

Representative medical conditions that can be treated or prevented using the implants provided herein include, but are not limited to, missing teeth or bone related medical conditions such as femoral neck fracture, missing teeth, a need for orthodontic anchorage or bone related medical conditions such as femoral neck fracture, neck bone fracture, wrist fracture, spine fracture/disorder or spinal disk displacement, fracture or degenerative changes of joints such as knee joint arthritis, bone and other tissue defect or recession caused by a disorder or body condition such as, e.g., cancer, injury, systemic metabolism, infection or aging, and combinations thereof.

EXAMPLES

The embodiments of the present invention will be illustrated by the following set forth examples. All parameters and data are not to be construed to unduly limit the scope of the embodiments of the invention. Gamma-enhanced protein adsorption capacity of titanium

Materials and methods Titanium sample

Disks (20 mm in diameter and 1.5 mm in thickness) made of commercially pure titanium (Grade 2) were used. The surfaces of the disks were freshly prepared by acid- etching the disks with 67% H₂SO₄ at 120⁰C for 75 seconds, and then stored in an ambient dark for 4 weeks. A half number of the disks were treated with gamma ray (0-1000 kGy). Hydrophilic status of titanium surfaces was examined by the contact angle of 1 μl H₂O droplet measured by a contact angle meter (CA-X, Kyowa Interface Science, Tokyo, Japan). Electrostatic status was examined by a coulomb meter. Protein adsorption assay

Bovine serum albumin, fraction V (Pierce Biotechnology, Inc., Rockford, IL), was used as model protein. Three hundred μl of protein solution (1 mg/ml protein/saline) was pippetted onto, and spread over a titanium disk. After 2 hours of incubation at 37°C, nonadherent protein removed as well as the initial whole solution were mixed with microbicinchoninic acid (Pierce Biotechnology) at 37°C for 60 minutes. The amount of protein was quantified using a microplate reader at 562 nm.

Bone-forming cell (osteoblast) cell culture

Bone marrow cells isolated from the femur of 8-week-old male Sprague-Dawley rats were placed into alpha-modified Eagle's medium supplemented with 15% fetal bovine serum, 50mg/ml ascorbic acid, 10^"8M dexamethasone, lOmM Na-^-glycerophosphate and Antibiotic-antimycotic solution containing 10000 units/ml Penicillin G sodium, 10000 mg/ml Streptomycin sulfate and 25 mg/ml Amphotericin B. Cells were incubated in a humidified atmosphere of 95% air, 5% CO₂ at 37°C. At 80% confluency, the cells were detached using 0.25% Trypsin- ImM EDTA-4Na and seeded onto titanium disks at a density of 3 x 10⁴ cells/cm². The culture medium was renewed every three days.

Cell attachment and density assays

Initial attachment of cells was evaluated by measuring the quantity of the cells attached to titanium substrates after 2 hours and 24 hours of incubation. In addition, the propagated cells were quantified as cell density at culture day of 2. These quantifications were performed using WST-I based colorimetry (WST-I, Roche Applied Science, Mannnheim, Germany). The culture well was incubated at 37°C for 4 hours with 100 μl tetrazolium salt (WST-I) reagent. The amount of formazan product was measured using an ELISA reader at 420 nm. Morphology and morphometry of cells

Confocal laser scanning microscopy was performed to examine the morphology and cytoskeletal arrangement of osteoblasts. After 3 hour of culture, the cells were fixed in 10% formalin, and stained using a fluorescent dye, rhodamine phalloidin (actin filament red color, Molecular Probes, OR). The cultures were also immunochemically stained with mouse anti-paxillin monoclonal antibody (Abeam, Cambridge, MA), followed by the adding of FITC-conjugated anti-mouse secondary antibody (Abeam, Cambridge, MA). The cell area, perimeter, and Feret's diameter were quantitatively assessed using an image analyzer (ImageJ, NIH, Bethesda, ML).

Alkaline phosphatase (ALP) activity

Cultures were rinsed with ddH2O and added with 250 μl p-Nitrophenylphosphate (LabAssay ATP, Wako Pure Chemicals, Richmond, VA), and then incubated at 37⁰C for 15 minutes. The ALP activity was evaluated as the amount of nitrophenol released through the enzymatic reaction and measured at 405 nm wavelength using ELISA reader.

Mineralization assay

The mineralization capability of cultured osteoblasts was examined by a calcium colorimetry-based assay. Cultures were washed with PBS and incubated overnight in 1 ml of 0.5 M HCl solution with gentle shaking. The solution was mixed with o-cresolphthalein complexone in alkaline medium (Calcium Binding and Buffer Reagent, Sigma, St Louis, MO) to produce a red calcium-cresolphthalein complexone complex. Color intensity was measured by an ELISA reader at 575 nm absorbance. Statistical Analysis

ANOVA was used to examine differences in variables between titanium disks with and without gamma ray treatment; <0.05 was considered statistically significant.

Results

Gamma -enhanced protein adsorption capacity of titanium Four-week old titanium disks (titanium disks stored in ambient dark for 4 weeks) were compared for their protein adsorption capacity with or without gamma ray (high energy: HE) pre-treatment. Albumin, a model protein, was incubated on the titanium disks for 2 hours and the amount of albumin adsorbed onto the titanium disks was quantified. Gamma treatment increased albumin adsorption by 80% (p<0.05; Fig. 1). Enhanced cell attractiveness of titanium by gamma treatment

Bone forming cells (osteoblasts) were cultured in the liquid medium on titanium disks (untreated and gamma-treated 4-week-old disks). After a 2-hour incubation, the number of the cells that attached to titanium surfaces was approximately twofold greater for gamma-treated (HE) titanium surfaces compared with untreated surfaces (Fig. 2). The gamma- induced advantage was present even after 24 hours of incubation. Pre-treatment of titanium disks with gamma ray doubled their cell attractiveness. The propagated cells on gamma-treated titanium surfaces was greater in number after 2 days of culture, indicating that the cells proliferated more on the surfaces compared with untreated ones (p<0.05; Fig. 3).

Enhanced cell affinity of titanium by gamma treatment

The bone-forming cells (osteoblasts) were cultured in the liquid medium on titanium disks. Three hours after seeding, the shape and skeletal arrangement of the cells were examined under confocal laser microscopy. Cells were clearly larger and the cellular processes stretched to a greater extent on gamma-treated titanium surfaces than on untreated surfaces (Fig. 4). Cell skeleton (highlighted in red) and focal points of adhesion to titanium (highlighted in green) were more established on the gamma-treated (high energy: HE) titanium surfaces. Morphometric evaluations for the area, perimeter, and Feret's diameter of the cells showed greater values of these parameters for gamma-treated titanium surfaces (p<0.01; histograms in Fig. 4).

Enhanced cell function on gamma-treated titanium

At day 7, 60% greater ALP activity was obtained in the osteoblast culture on gamma-treated titanium surfaces compared with untreated control surfaces (p<0.05; Fig. 5). At days 21 of culture, the area of mineral deposition was also approximately 2 times greater on gamma-treated titanium surfaces (p<0.05; Fig. 6). These indicate that function of osteoblasts, including the speed and degree of maturation and mineralization, was enhanced on gamma-treated surfaces.

Surface chemistry of gamma-treated surfaces

Four-week old titanium disks with and without gamma treatment were examined for their wettability to water and electrostatic potential. Gamma-treated titanium surfaces showed hydrophilicity (contact angle of water drop <30°), whereas the surfaces were hydrophobic before gamma treatment (Fig. 7). The gamma-treated titanium surfaces were electropositively charged (> 0 nano C), whereas the surfaces without gamma treatment were electronegative. REFERENCES

[1] Ray NF, Chan JK, Thamer M, Melton LJ, 3rd. Medical expenditures for the treatment of osteoporotic fractures in the United States in 1995: report from the National Osteoporosis Foundation. J Bone Miner Res 1997; 12:24.

[2] Hudson JI, Kenzora JE, Hebel JR, Gardner JF, Scherlis L, Epstein RS, Magaziner JS. Eight- year outcome associated with clinical options in the management of femoral neck fractures. Clin Orthop Relat Res 1998:59.

[3] Lu-Yao GL, Keller RB, Littenberg B, Wennberg JE. Outcomes after displaced fractures of the femoral neck. A meta-analysis of one hundred and six published reports. J Bone Joint Surg Am 1994;76: 15.

[4] Tidermark J, Ponzer S, Svensson O, Soderqvist A, Tornkvist H. Internal fixation compared with total hip replacement for displaced femoral neck fractures in the elderly. A randomised, controlled trial. J Bone Joint Surg Br 2003;85:380.

[5] Ravikumar KJ, Marsh G. Internal fixation versus hemiarthroplasty versus total hip arthroplasty for displaced subcapital fractures of femur— 13 year results of a prospective randomised study. Injury 2000;31:793.

[6] van Steenberghe D, Jacobs R, Desnyder M, Maffei G, Quirynen M. The relative impact of local and endogenous patient-related factors on implant failure up to the abutment stage. Clin Oral Implants Res 2002;13:617.

[7] Ozawa S, Ogawa T, Iida K, Sukotjo C, Hasegawa H, Nishimura RD, Nishimura I. Ovariectomy hinders the early stage of bone- implant integration: histomorphometric, biomechanical, and molecular analyses. Bone 2002;30: 137.

[8] Nevins ML, Karimbux NY, Weber HP, Giannobile WV, Fiorellini JP. Wound healing around endosseous implants in experimental diabetes. Int J Oral Maxillofac Implants 1998;13:620.

[9] Zhang H, Lewis CG, Aronow MS, Gronowicz GA. The effects of patient age on human osteoblasts' response to Ti-6A1-4V implants in vitro. J Orthop Res 2004;22:30.

[10] Takeshita F, Murai K, Ayukawa Y, Suetsugu T. Effects of aging on titanium implants inserted into the tibiae of female rats using light microscopy, SEM, and image processing. J Biomed Mater Res 1997;34: 1. [11] Yamazaki M, Shirota T, Tokugawa Y, Motohashi M, Ohno K, Michi K, Yamaguchi A. Bone reactions to titanium screw implants in ovariectomized animals. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1999;87:411.

[12] Hori N, Att W, Ueno T, Sato N, Yamada M, Saruwatari L, Suzuki T, Ogawa T. Age-dependent degradation of protein adsorption capacity of Ti. J Dent Res; in press.

[13] Suzuki T, Hori N, Att W, Kubo K, Iwasa F, Ueno T, Maeda H, Ogawa T. UV Treatment Overcomes Time-Related Degrading Bioactivity of Titanium Tissue Eng;doi: 10.1089/ten.TEA.2008.0568.

[ 14] Att W, Hori N, Iwasa F, Yamada M, Ueno T, Ogawa T. The effect of UV- photofunctionalization on the time-related bioactivity of titanium and chromium-cobalt alloys. Biomaterials 10.1016/j.biomaterials.2009.04.048.

Claims

CLAIMSWhat is claimed is:

1. A method for enhancing bioactivity of an implant, comprising: irradiating the implant with a high energy radiation for a sufficient period of time prior to use of the implant to cause the implant to form a surface comprising positive charges, wherein the high energy radiation is gamma ray.

2. The method of claim 1 , wherein the gamma ray has a wavelength of 0.1 nm to about 0.01 nm.

3. The method of claim 1 , wherein the gamma ray dose is in a range of from about 0 kGy to about 1000 kGy.

4. The method of claim 6, wherein the gamma ray dose is about 5 KGy, 10 kGy, 20 kGy, 3OkGy, 50 KGy, 100 kGy, 150 kGy, 200 kGy, 300 kGy, 500 kGy. 750 kGy, or 1000 kGy.

5. The method of claim 1, wherein the implant is in a package when the implant is subjected to irradiating.

6. The method of claim 1, wherein the implant is a metallic implant.

7. The method of claim 1, wherein the implant material comprises titanium, gold, platinum, tantalum, niobium, nickel, iron, chromium, cobalt, zirconium, magnesium, magnesium, aluminum, palladium, an alloy formed thereof, or combinations thereof.

8. The method of claim 1, wherein the implant material comprises titanium.

9. The method of claim 1, wherein the implant is selected from the group consisting of tooth implants, jaw bone implants, repairing and stabilizing screws, pins and plates for bone, spinal implants, femoral implants, neck implants, knee implants, wrist implants, joint implants, maxillofacial implants, limb prostheses for conditions resulting from injury and disease, and combinations thereof.

10. The method of claim 9, wherein the joint implant is an artificial hip joint implant, the maxillofacial implant is an ear implant or a nose implant.

11. The method of claim 1, wherein the implant is a non-metallic implant.

12. The method of claim 11, wherein the implant comprises a polymer.

13. The method of claim 11, wherein the implant comprises a bone cement material.

14. The method of claim 13, wherein the bone cement material comprises a material selected from the group consisting of polyacrylates, polyesters, bioglass, ceramics, calcium-based materials, calcium phosphate-based materials, and combinations thereof.

15. The method of claim 13, wherein the bone cement material comprises poly(methyl methacrylate) (PMMA) or methyl methacrylate (MMA).

16. The method of claim 11 , wherein the implant is a non-metallic implant selected from the group consisting of tooth implants, jaw bone implant, repairing and stabilizing screws, pins and plates for bone, spinal implants, femoral implants, neck implants, knee implants, wrist implants, joint implants, an artificial hip joint, maxillofacial implants, ear and nose implants, limb prostheses for conditions resulting from injury and disease, and combinations thereof.

17. The method of any of claims 1 , further comprising treating at least a portion of the surface of the implant material by physical treatment or chemical treatment prior to irradiating the implant material with the high energy radiation.

18. The method of claim 17, wherein the physical treatment is machining, sandblasting, or metallic or nonmetallic deposition, and wherein the chemical treatment is acid- etching, sand-blasting, oxidation, or alkaline treatment.

19. The method of claim 1, wherein the irradiation is conducted in a duration of about 1 minute to about 1 hour, about 1 hour to about 5 hours, about 5 hours to about 24 hours, about 1 day to 5 days, about 5 days to about 10 days, about 10 days to about 1 month.

20. The method of claim 1, wherein the irradiation is conducted within a period of time prior to implanting the implant, wherein the period of time is about 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 18 hours, 12 hours, 8 hours, 4, hours, 2 hours, or 1 hour.

21. The method of claim 1 , wherein the bioactivity is tissue-implant integration or bone-implant integration capacity.

22. The method of claim 1, wherein method causes the bioactivity of the implant to increase, wherein the bioactivity is one of the following or combination thereof: protein affinity, cell affinity osteoblast proliferation on the implant surface, osteoblast differentiation on the implant surface; and osteoblast mineralization on the implant surface.

23. The method of claim 22, wherein the protein is bovine serum albumin, fraction V, or bovine plasma fibronectin.

24. The method of claim 22, wherein the method causes the protein affinity to increase by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%.

25. The method of claim 22, wherein the cell is human mesenchymal stem cell and osteoblastic cell.

26. The method of claim 22, wherein the method causes the cell affinity to increase by at least 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, and 200%.

27. The method of claim 22, wherein the method causes the osteoblast differentiation or osteoblast mineralization to increase by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, and 300%.

28. An implant treated with a method according to any of claims 1-27.

29. A method, comprising implanting in a mammal the implant of claim 28.