WO2005030137A2 - Improving biomechanical performance of irradiated biological material - Google Patents

Abstract

Compositions, and methods of making, at least one irradiated, sterilized and biomechanically strengthened biological material are described. The biological materials are treated with at least one substance that has a high affinity for free radicals. The biological materials have a molecular or supramolecular level of porosity which allows for the penetration of molecules of the high affinity free radical scavenging substance into the biological materials.

Description

TITLE

METHOD FOR IMPROVING THE BIOMECHANICAL PERFORMANCE OF IRRADIATED BIOLOGICAL MATERIALS AND THE MATERIALS MADE THEREBY

TECHNICAL FIELD This invention relates in general to the use of biological materials and, in particular, to the elimination of contaminants from such biological materials.

BACKGROUND OF THE INVENTION Increasingly, many types of biological materials are being used for human, veterinary, diagnostic and/or experimental purposes. In order to prepare such biological materials for use, it is necessary to ensure that such materials do not contain unwanted and potentially dangerous biological contaminants or pathogens. There are many different types of biological materials, such as organic tissues . In particular, bone tissue used as transplants can be in the form of allograft or xenograft. An allograft is a graft of tissue obtained from a donor of the same species as, but with a different genetic make-up from the recipient, such as a bone tissue transplant between two humans whereas a xenograft is a graft of tissue obtained from a donor of different species, such as animals. Typically, the bone tissue donor for the transplant allograft is a human cadaver. In 1985 there were about 10,000 allograft surgeries. By 1996 that number had risen to about 145,000 allograft surgeries. Many companies associated with allografts, including such companies as Osteotech, Regeneration Technologies, Center Pulse, Bone Bank and such non-profit organizations as the Musculoskeletal Transplant Foundation, have been established in the last several decades in proportion to the increase in demand. Currently, an estimated 450,000 bone allografts are transplanted each year in the U.S. for repair of fractures and damage caused by illness and injury. Typical use is in replacing bone lost in tumor removal and in reconstructing skeletal defects. Tissue transplants using allografts are preferred because of biocompatibility and suitability for anatomical matching to repair defects. Regardless of their expediency, risk of infection and disease transmission through allografts is a concern and terminal sterilization is often essential. Gamma radiation has been widely used for sterilization of bone allografts due to its efficacy against viral and bacterial disease transmission. However, gamma radiation sterilization impairs the material properties of bone which is a major clinical concern since bone grafts are used in load bearing applications. In assessing the mechanical properties of bone irradiated with gamma particles, it is observed that pre-yield (elastic) behavior of cortical bone tissue is unaffected while post-yield (plastic) properties suffer a significant reduction as a result of radiation sterilization. Degradation in post-yield properties results in the loss of tissue ductility and gamma radiation sterilized bone tissue becomes brittle. This behavior is hypothetically explained by the dependency of pre-yield properties of cortical bone tissue on the mineral phase and the post-yield properties on the collagen. Therefore, it is largely believed that the collagen phase is more vulnerable to gamma radiation than the mineral phase of cortical bone tissue. Besides gamma radiation induced embrittlement of bone, age and disease related alterations in the collagen biochemistry have also been documented to cause bone brittleness. Thus, it is widely accepted that the integrity of bone's collagen profoundly affects the mechanical strength and fracture resistance of bone tissue. The effect of gamma radiation on the biochemistry of collagen has also been extensively investigated for collagen extracted from tendons. It is shown that gamma radiation leads to scission of the peptide backbone, and reduces the concentration of intermolecular crosslinks in tendon collagen. Similar to tendon, destruction of the peptide backbone of bone's collagen and reduction in intermolecular crosslink density have also been reported for human femoral cortical bone secondary to gamma radiation sterilization. The majority of damage to biomolecules are induced by damaging species (i.e., free radicals) resulting from the radiolysis of water molecules during gamma radiation. In this regard, collagen is a primary target for radiation based free radical attack due to significant amount of water bound to its structure. Free radicals resulting from the radiolysis of water react with target molecules within a lifetime on the order of 0.01 to 1 nanoseconds and render irrecoverable changes in the target molecule's chemical structure. Particularly, the hydroxyl (OH«) free radical induces the greater portion of the in vivo and in vitro damage to biological systems during gamma radiation.

Supporting the existence of a water based free radical attack, bone irradiated at -78°C was less brittle and had less collagen damage than when irradiated at room temperature. Referring now to the drawings, a donor femur 10 is illustrated in Fig. 1. The donor femur 10 has been obtained from a human cadaver (not shown). The donor femur 10 is cut along cut lines 12 and 14 to form a bone section 16. The bone section 16, as shown in Fig. 2 is cut along cut lines 18 and 20 to form a bone tissue fragment (bone allograft) 106, as best seen in Fig. 3, for transplantation into the body of a human recipient. The bone allograft 106 or the bone section 16 may also alternatively be machined, for example, into dowels, pins, screws or other geometric forms. Also, pieces as large as the complete bone section 16 are commonly used without further reduction. One example of the need for bone tissue transplants involves situations where patients lose significant amount of bone tissue as a result of trauma or due to tumor removal, necessitating transplantation of bone tissue obtained from cadavers (i.e. allografts). Other examples include the reconstruction of skeletal defects, augmentation of fracture healing, fusion of joints, joint reconstruction, trauma surgery, and tumor surgery. In these situations patients lose significant amount of bone tissue which is unmatched by their own bone supply, necessitating transplantation of bone tissue obtained from cadavers (i.e. allografts). One of the problems associated with bone allograft transplants is the risk of infection and disease transmission to the transplant recipient through the allograft. To reduce the risk of infection and disease, terminal sterilization of the bone tissue of the bone allograft is often performed before transplantation into the recipient. One known method for sterilization of allografts is gamma irradiation. Gamma radiation has been widely used for sterilization of bone allografts due to its efficacy for minimizing viral and bacterial infection, and disease transmission. However, gamma radiation sterilization impairs the mechanical properties of bone, apparently, by damaging the collagen structure. The collagen structure is the source of the toughness of bone tissue because it provides ductility to the bone tissue. While the gamma radiation is effective in sterilization, it has the effect of impairing the mechanical properties of the allograft. This is of particular concern in situations when bone grafts are used in load bearing applications during their function in the body. For example, gamma radiation sterilization requires 25,000 gamma rays (Gy) to sterilize HIV, human immunodeficiency virus, in liquid cultures and up to 30,000 to 40,000 Gy may be necessary for sterilization of HIV in bone. As discussed above, there is some concern about the effects of gamma radiation sterilization on the biomechanical properties of bone allografts. One proposed procedure in order to avoid damage to the biomechanical properties of bone allografts is sterilization in an argon environment. However, sterilization in an argon environment requires expensive equipment and involves complicated procedures. Another proposed procedure is to perform sterilization at sub-zero temperatures. For example, it has been found that bone irradiated at -78 Celsius was less brittle and had less collagen damage than bone irradiated at room temperature. However, sterilization at sub-zero temperatures generally requires the use of dry ice and appropriate containment. Referring again to the drawings, a known process for the gamma irradiation sterilization of a bone allograft is illustrated schematically in Figs. 4 and 5. A gamma radiation source 102, for example, cobalt-60, emits gamma rays (gamma radiation) 104. The bone allograft 106 is exposed to the gamma radiation 104. The bone allograft 106 is composed of bone tissue 108. The bone tissue 108 is composed of compliant collagen phases 110 and stiff mineral phases 112. The collagen phases 110 provide the ductility of the bone tissue 108, whereas the mineral phases 112 provide the stiffness of the bone tissue 108. The gamma radiation 104 sterilizes the bone allograft 106. The gamma radiation 104 also ionizes water (not shown), which, for example, may be contained in the bone allograft 106 by its porous nature. Typically, a significant amount of water is bound to the structure of the bone tissue 108. The ionization of water creates free radicals 114. It is known that the gamma radiation 104 impairs the ductility of the bone tissue 108, whereas the stiffness is not affected. Therefore, it has been postulated that the free radicals 114 attack and randomly break down the collagen phases 110 into a cleaved state with breaks 116, as illustrated in Fig. 5. The mineral phases 112 are believed to be left intact. Various objects and advantages of this invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiment, when read in light of the accompanying drawings.

SUMMARY OF INVENTION This invention relates to a method for improving the biomechanical performance of sterilized biological material, and, in particular, of irradiated sterilized biological material. The invention also relates to sterilized biological material having improved mechanical integrity. The presence of free radical scavengers during the sterilization process suppresses the formation of free radicals and/or it suppresses the access of free radicals for the target molecule. Suppression of the free radical activity by free radical scavengers results in sterilized biological material that is mechanically superior to that sterilized in the absence of the free radical scavengers. According to one aspect, the present invention relates to a composition comprising at least one irradiated, sterilized and biomechanically strengthened biological material having a molecular or supramolecular level of porosity which allows for the penetration of molecules, wherein the biological material has been treated with at least one substance that has a high affinity for free radicals. In certain embodiments, the biological material comprises at least one of bone tissue, demineralized bone tissue, cartilage tissue, skin tissue, collagen tissue, ligament and/or tendon tissue harvested from donors or in processed form. Also, in certain embodiments, the biological material comprises at least one mineral phase and at least one collagen phase. In certain embodiments, where the biological material comprises bone, the biological material has a desired ductility value that is substantially improved when compared with a collagen phase of a tissue exposed to the gamma radiation in the absence of the substance having the high affinity for free radicals. In certain embodiments, the molecules have a preferred molecular weight that allows penetration into the biological material. For example, in certain embodiments, the molecular substances have a molecular weight less than about300 Da. According to another aspect, the substance comprises at least one antioxidant, including where the substance comprises at least one free radical scavenger having a high solubility and high diffusibility into the biological material. According to another aspect, the present invention relates to a composition comprising at least one irradiated, sterilized and biomechanically strengthened biological material having a molecular or supramolecular level of porosity which allows for the penetration of substances into the biological material to a desired depth; at least one substance that has a high affinity for free radicals; and at least one aqueous solvent, wherein the aqueous solvent and the free radical affinity substance are present in a combined amount effective to prevent biomechanical degradation of the biological material. According to yet another aspect, the present invention relates to a method for improving the biomechanical performance of at least one sterilized biological material, such as tissue, and for at least minimizing and/or reversing the extent of damage to the biological material with an associated recovery in the mechanical strength of such sterilized biological material. In certain embodiments, the method includes providing at least one tissue containing collagen, the tissue having a molecular or supramolecular level of porosity which allows for the penetration of molecules with a molecular weight less than about 300 Da; treating the tissue with at least one substance that has a high affinity for free radicals; and exposing the tissue to radiation. According to yet another aspect, the present invention relates to a method for improving the biomechanical performance of at least one sterilized biological material and for minimizing and/or reversing the extent of damage to the biological material with an associated recovery in the mechanical strength of such sterilized biological material. The method includes administering at least one free radical scavenger, via an aqueous solution to the biological material; administering at least one free radical scavenger substance, via an aqueous solution, to the biological material; immersing the biological material in the aqueous free radical scavenger solution under vacuum; optionally, storing the immersed at least one biological material under cold conditions for a suitable length of time; and, exposing the biological material to an effective dosage of radiation. In another aspect, the present invention relates to a method for improving the biomechanical performance of at least one irradiated biological material and for minimizing damage to the at least one irradiated biological material with an associated recovery in the mechanical strength of such at least one irradiated biological material. In certain embodiments the method includes the steps of: optionally, treating at least one biological material with at least one nucleic acid targeted radiosensitizer; administering at least one free radical scavenger substance, via an aqueous solution, to the biological material; optionally, adjusting the aqueous free radical scavenger solution to a pH of about 7.0; optionally, adding at least one proteolytic inhibitor to the solution; immersing the biological material in the aqueous free radical scavenger solution under vacuum; optionally, storing the immersed at least one biological material under cold conditions for a suitable length of time; optionally, preserving the biological material by cryopreservation to limit formation of ice crystals and to freeze the biological material rapidly; and, exposing the at least one biological material to an effective dosage of radiation. According to yet another aspect, the present invention relates to a method for prophylaxis or treatment of a condition or disease or malfunction of at least one biological material in a mammal comprising introducing into a mammal in need thereof one or more tissues sterilized according to the methods described herein. Various objects and advantages of this invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiment, when read in light of the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is an illustration of a prior art donor femur. Fig. 2 is an illustration of a prior art bone section cut from the donor femur depicted in Fig. 1. Fig. 3 is an illustration of a prior art bone allograft cut from the bone section depicted in Fig. 2. Fig. 4 is a schematic illustration of a prior art process for the gamma irradiation sterilization of the bone allograft depicted in Fig. 3. Fig. 5 is a schematic illustration of the prior art bone allograft of Fig. 4 with breaks in the collagen phase. Fig 6 is a schematic illustration of a process for the gamma irradiation sterilization of the bone allograft of Fig. 3 in accordance with this invention. Fig. 7 is a schematic illustration of the bone allograft of Fig. 6 with free radicals suppressed by free radical scavengers. Fig. 8 is an illustration of a recipient femur including the bone allograft depicted in Fig. 7. Fig. 9 is a Table I which shows the mechanical properties of treatment groups where: §: PSTER. Pτmo and P_ST-_TH designate the level of significance for the main effect of gamma radiation, main effect of thiourea treatment and the interaction of main effects, respectively, (These values are the outcome of generalized MANOVA test); a, b: 'a' and 'b' designate significant difference between the groups I (irradiated) and 1.51 (thiourea-treated irradiated) at the levels of p < 0.1 and p < 0.05 as determined by a Mann Whitney U-test; c: The marginal mean represents the average value of a given property after pooling the data along a row (i.e. thiourea treatment) or column (i.e. radiation); and * : Percent reduction in a given mechanical property with respect to the control group C. The mechanical property is subtracted from that of the treatment group C and divided with the value of the treatment group C to calculate the percent reduction. Figs. 10a, 10b and lc are graphs showing: Energy (Joules) v. Elastic Energy (Fig. 10a), Energy (Joules)/ Post Yield Energy (Fig. 10b), and Energy (Joules)/Fracture Energy (Fig. 10c) of treatment groups The error bars represent standard deviation of the mean. Fig. 11 is a graph showing typical stress strain curves obtained during the monotonic tensile testing of treatment groups, showing fracture points. Tensile curves for thiourea treated control specimens were similar to that of controls. Fig. 12 shows SEM images of failed osteons. The failure pattern of the treatment group 1.5C did not differ from that of controls. Fibrillar extensions on the fracture surfaces are seen.

Fig. 13 shows the effects of gamma radiation and thiourea treatment on the integrity of collagen molecules. Figs. 14a and 14b are schematic illustrations of a proposed mechanism of failure for collagen fibrils of normal bone (Fig. 14a) and irradiated bone (Fig. 14b). Fig. 15 is a photograph of the tensile testing fixture for monotonic and cyclic loading of cortical bone specimens. Final specimen dimensions are 2 x 1 x 16 mm at the gage region. Load and displacement are recorded by a load cell and a strain gage extensometer, respectively. Figs. 16a-16d are schematic illustrations of various steps showing how free radical scavenger (FRS) protect the integrity of the collagen phase in the organic tissue that is sterilized, and, thus, its mechanical integrity.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Unless defined otherwise, all technical and scientific terms used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the relevant art. As used herein, the singular forms "a," "an," and "the" include the plural reference unless the context clearly dictates otherwise. As used herein, the term "sterilize" is intended to mean a reduction in the level of at least one active biological contaminant or pathogen found in the biological material being treated according to the present invention. As used herein, the term "radiation" includes radiation of sufficient energy to sterilize at least some component of the irradiated biological material. In certain embodiments of the present invention different types of radiation are useful. It should be understood, that the particular type of radiation used depends, at least in part on the biological material being irradiated. Radiation, therefore, includes electromagnetic radiation which originating in a varying electromagnetic field, such as radio waves, visible (both mono and polychromatic) and invisible light, infrared, ultraviolet radiation, x-radiation, and gamma rays and mixtures thereof; sound and pressure waves, and in certain embodiments, streams of subatomic particles such as neutrons, electrons, and/or protons. As used herein, the term "biological material" includes both natural and man- made materials, including tissues. Further, the term "tissue" includes at least one substance derived or obtained from at least one multi-cellular living organism that performs one or more functions in the organism or a recipient thereof. Examples of tissues include bone, collagen, tendons, elastin, fibronectin, fibrin, and the like. As used herein, the term "tissue" also includes naturally occurring tissues, such as tissues removed from a living organism and used as such, or processed tissues, such as tissue processed so as to be less antigenic, for example allogenic tissue intended for transplantation. The term "tissue" also includes natural, artificial, synthetic, semi- synthetic or semi-artificial materials that are useful in the replacement of at least some function(s) of a natural tissue when implanted into a patient According to certain aspects of the present invention, terminal sterilization of bone allografts by gamma radiation is often essential prior to the clinical use to minimize the risk of infection and disease transmission. While gamma radiation has superior efficacy to other sterilization methods it also impairs the material properties of bone allografts which may result in the premature clinical failure of the allograft. According to one aspect of the present invention, gamma-radiation-induced biochemical damage to bone's collagen is reduced by scavenging for the free radicals generated during the ionizing radiation. According to the methods of the present invention, the one or more biological material to be sterilized are irradiated with the radiation for a time effective for the sterilization of the one or more biological materials. Combined with irradiation rate, the appropriate irradiation time results in the appropriate dose of irradiation being applied to the one or more biological materials. Suitable irradiation times may vary depending upon the particular form and rate of radiation involved and/or the nature and characteristics of the particular one or more biological materials being irradiated. Suitable irradiation times can be determined empirically by one skilled in the art. According to the methods of the present invention, the one or more biological materials to be sterilized are irradiated with radiation up to a total dose effective for the sterilization of the one or more biological materials, while not causing significant degradation in the biomechanical strength of those one or more biological materials. Suitable total doses of radiation may vary depending upon certain features of the methods of the present invention being employed, such as the nature and characteristics of the particular one or more biological materials being irradiated, the particular form of radiation involved, and/or the particular biological contaminants or pathogens being inactivated. According to another aspect, the present invention provides a lessening in the extent of biochemical degradation of biological materials, such as collagen, that is also accompanied by alleviation in the extent of biomechanical impairment secondary to gamma radiation sterilization According to another aspect of the present invention, biological materials irradiated in the presence of a free radical scavenger demonstrate intact α-chains. Thus, the present invention also provides a method for reversing the extent of damage to biological materials, such as collagen, with an associated recovery in the mechanical strength of such sterilized biological material. The present invention, therefore, also improves the functional life-time of the allograft component following transplantation. During gamma radiation sterilization highly reactive radical species, such as hydroxyl radicals, are formed due to ionization of water molecules. These free radicals have been speculated to impair the integrity of collagen molecules. In particular, the improvement in the fracture resistance of irradiated specimens of bone with the free radical scavenger thiourea treatment was substantial. The improvement in the fracture resistance of irradiated tissue occurs through the recovery of post-yield deformability of bone. When the mechanical, biochemical and fractographic observations are considered, the following picture emerges on the failure of gamma radiation sterilized bone tissue: if the interface between the matrix and the fiber is weak, then the interface fails and the crack is bound to travel around the fiber; in turn, the crack path in the bone becomes more tortuous and crack bridging comes into play, increasing the fracture toughness essential to drive the crack growth. As shown in the examples below, in the control specimens the collagen fibrils were intact; therefore the crack path was likely deflected along the length of collagen fibrils, demonstrating fibrillar extensions on the fracture surfaces of control specimens. Therefore, the failure was resisted at the supramolecular level and loads were transmitted to the microstructural level as evidenced by the tortuous crack path involving lamellar extrusions on the SEM images of control specimens. As demonstrated by SDS-PAGE analyses, the radiation cleaved the backbone of collagen molecules which rendered the collagen fibrils weaker, probably to a level weaker then the strength of the interface between the fibrils and their environment. Therefore, the crack path was not deflected and the failure occurred transversely across the collagen fibrils without the involvement of the microscale as evident by the dull appearance of the fracture surfaces of irradiated specimens. The nature of loads experienced by allografts are cyclic in nature; therefore, fatigue loading is expected to better represent physiological loading. The examples of the present invention demonstrate a five-fold increase in the fatigue life of bone tissue which has been subjected to gamma radiation in the presence of a free radical scavenger. This improvement in the biomechanical performance of irradiated bone tissue has important clinical repercussions from the perspective of increased functional life-time of the allograft component following transplantation. Long-term survival of allografts provide additional valuable time for the host structures to recover their strength. Consequently, clinical complications related with premature failure of allografts is alleviated or prevented. The present invention is also especially useful for bone banking and bone graft technology as well. The use of free radical scavengers to reduce the biomechanical and biochemical impairment of tissue allows for biomechanically more stable grafts and it allows for the application of higher doses of irradiation to be used. The examples given below describe in detail the method for performing the present invention. It will be recognized that variations of this method may include different free radical scavengers dependent upon the target tissue. Thiourea was chosen as the free radical scavenger in the following examples; however, in other embodiments, the method outlined below is applicable to different free radical scavengers as well as antioxidants and are also considered to be separate inventions within the contemplated spirit and scope of this invention. As such, the following examples merely illustrate the nature of the invention

Example I: Treatment of Bone Tissue with Free Radical Scavenger Thiourea Referring to the drawings, the process for the gamma irradiation sterilization of a bone allograft of the invention, and the resulting sterilized bone allograft product, is illustrated schematically in Figs. 6 and 7. Preferably, the bone allograft 106 is kept wet before the process in an aqueous saline solution supplemented with calcium chloride, and when stored, the bone allograft 106 is kept frozen at -40°C. Gamma radiation induces damage to collagen by direct (i.e. photon-electron collision) and indirect (i.e. formation of highly reactive free radicals generated by ionization of water) mechanisms. The majority of the damage to the mechanical properties of the allograft is caused by free radicals formed from the radiolysis of water molecules during gamma radiation. It is highly desirable to reduce the damage caused by the free radicals. Free radical scavengers, preferably thiourea, 118 are administered via an aqueous solution, preferably at a concentration of 0.5 to 1.5 M (moles of solute/liters of solution), to the bone allograft 106. Thiourea is a preferred free radical scavenger 118 because thiourea has a high solubility and high diffusibility into the bone tissue, i.e., thiourea tends to permeate the tissue. In one embodiment of the invention, the aqueous free radical scavenger solution is adjusted to a pH of about 7.0, for example, with the addition of a phosphate buffer. Also, in another embodiment of the invention, proteolytic inhibitors, such as, for example, PMSF (Phenylmethanesulfonyl Fluoride) 0.1 mmol/liter, and sodium azide 0.2 mmol/liter are added. In certain embodiments, at least one or more suitable disinfecting and bioburden-reducing amounts of at least one antibiotic agent, antiviral agent and/or antimycotic agent are also added. The bone allograft 106 is preferably immersed in the aqueous free radical scavenger solution in a glass jar, placed under vacuum, and kept 4° C for a suitable time. Other temperature ranges and other treatment durations could be attained by adjusting the vacuum level. In an alternative embodiment of the invention, the bone allograft is immersed for 14 days with the aqueous free radical scavenger solution being resupplemented every 3 days, i.e. the molarity of the solution is returned to its original molarity, by the addition of further free radical scavengers. Alternatively, the solution may be replaced every 3 days with a new solution with the original molarity. The bone allograft 106 is then removed and placed under the gamma radiation source 102. Optionally, the bone allograft 106 may be rinsed with distilled water between removal from the solution and placement under the radiation source 102. Referring now to the schematic illustration in Fig. 4, the gamma radiation source 102 emits gamma radiation 104. The bone allograft 106 is exposed to the gamma radiation 104. In certain embodiments, the dose of gamma radiation 104 that the bone allograft is exposed to is within the range from about 25 to about 35 kGy. The gamma radiation 104 sterilizes the bone allograft 106. The gamma radiation 104 also ionizes water, which may be contained in the bone allograft 106 by its porous nature. The ionization of water creates the free radicals 114. The free radical scavengers 118 interfere with or combine with the free radicals 114 and, as illustrated in Fig. 7, the free radical scavengers 118 neutralize the free radicals 114. Thus, the collagen integrity is less amenable to impairment from free radical damage, and the ductility of the bone tissue 108 is impaired minimally. Preferably, the collagen phases 110 are affected minimally the free radicals 114, as illustrated in Fig. 7. Thus, any significant impairment that occurs would be due to directly induced damage of the gamma radiation, for example, by photon-electron collisions. The bone allograft 106 may then be implanted in a recipient femur 210 as shown in Fig. 8. The use of the chemical agents with a high affinity for free radicals (i.e., substances to which free radicals are more preferentially attracted relative to the attraction of the free radicals to collagen), for example, free radical scavengers, can block selective damage to collagen by free radicals. Free radical scavengers have a greater affinity with free radicals than the affinity between collagen and the free radicals. Thus, the free radicals react with the free radical scavengers before the free radicals react with the collagen. The free radical scavengers interfere with and neutralize the free radicals before the free radicals impair the collagen. Therefore, the integrity of the collagen molecules, the ductility of the collagen phase, and thus, the mechanical strength of allograft, is substantially maintained. When any substance with a high affinity for free radicals is present in the tissue during gamma irradiation, it is expected that trace amounts (any amount greater than zero) of that substance will be present in the tissue after the gamma irradiation process. The trace amounts may only be present in the range millimoles or micromoles per cubic centimeter of tissue. The trace amounts can be detected, for example, by a mass spectrometer, electron spin resonance, infrared spectrometer, Raman spectrometer or a nuclear magnetic resonance device.

Example II - Biomechanical Testing of Biological Materials Preparation of Tensile Test Specimens Femurs from three male cadavers (ages 31, 31, 38) were obtained from the Musculoskeletal Transplant Foundation (Jessup, PA, USA). The diaphyses of femurs were sectioned into two segments each approximately 55 mm long using a hacksaw. The first segment was taken immediately distal to the minor trochanter and the second was taken distally from the first segment. A low-speed metallurgical saw with a diamond coated blade (SouthBay Tech, CA, USA) was used to cut the rings into the four anatomical quadrants. One millimeter thick wafers were sectioned from the quadrants within the circumferential-longitudinal plane (parallel to osteonal orientation) using the low-speed metallurgical saw. Wafers were then cut into beams with final dimensions of 40mm x 5mm x 1mm. Coupon shaped tensile test specimens were machined from the beams by reducing the width of the mid gage region using a table-top milling machine (Sherline, CA, USA) and a 0.5" diameter end-mill. The gage region had the final dimensions of 2mm x 1mm x 16mm. Specimens were kept wet with calcium supplemented saline solution by using an air-pressure driven nozzle spray during the milling process. Four treatment groups were included in the study: controls (C), thiourea treated controls (1.5C), irradiated (I) and thiourea treated- irradiated (1.51). Specimens were randomly assigned to each of the four treatment groups. Fifteen monotonic and five fatigue specimens were assigned to each treatment group, reaching a total of 80 specimens. Specimens were kept wet at all times and they were stored at -40 °C. Thiourea Treatment and Sterilization Free radical scavengers are substances that inhibit free radical damage to a target molecule by direct chemical reaction with the radical or by minimizing the formation of the radical. In this example, thiourea (CH₄N₂S, 76.12 Da) was selected for scavenging of free radicals. The supramolecular level of porosity of bone tissue allows for the penetration of molecules with a molecular weight less than 300 Da. The lack of charged groups in thiourea further facilitates its diffusion in bone. Also, thiourea has low toxicity and is useful for the treatment of various diseases. Specimens which received thiourea treatment (1.5C and 1.51) were placed in polyethylene containers in groups of 20 and soaked in 40 ml of 1.5 [M] thiourea solution supplemented with calcium and protease inhibitors to minimize leaching of mineral and bacterial degradation. This concentration was determined based on pilot tests in which tensile specimens machined from bovine bone were treated at concentrations of 0.1 [M], 0.5 [M] and 1.5 [M]. Since none of these concentrations altered the native mechanical properties of bovine bone the maximum concentration of 1.5 [M] was selected. The other factor which determined the maximum concentration was the solubility of thiourea in aqueous environment such that it was not possible to obtain concentrations greater than 1.5 [M]. Solutions were replaced once in every 3 days and the entire treatment lasted for 14 days at 4 °C for further minimization of bacterial degradation. The control and irradiated treatment groups were kept under similar conditions with the only exception of thiourea being absent in the solutions in which they are maintained. Specimens were individually wrapped in gauze pads dipped in calcium supplemented saline solution and placed in polyethylene bags. Treatment groups which received gamma radiation were placed in polystyrene coolers filled with dry ice and mailed to Steris Corporation (Steris Corporation, Columbus, OH, USA) for gamma radiation sterilization. Control samples were placed in same type of polystyrene coolers filled with dry ice and placed on the lab bench until the irradiated specimens were sent back to our facilities. Samples were irradiated at an average dose of 36.4 kGy as measured by perspex dosimeters on the site of irradiation. The standard dose range for sterilization of bone grafts is 25 kGy to 35 kGy; thus, the level of radiation in this study was slightly greater than the higher end of the standard range. Monotonic Tensile Tests The specimen was locked into the grips of an electromagnetic mechanical testing machine (ELF 3200, Enduratec, Minnetonka, MN, USA) and the clip-on strain gage extensometer (Epsilon, Jackson, WY, USA) was attached to the gage region using orthodontic heavy gauge rubber bands (3M, 3/16" Heavy, MN, USA).

Mechanical tests were performed under strain control at the rate of 1.5%/s. The load was measured by a 400 N load cell (Honeywell-Sensotec, Columbus, OH, USA). Load-strain data were acquired at the rate of 400 Hz. Tensile specimens were kept wet by drips of calcium supplemented saline solution at ambient temperature. Stress was calculated by dividing the measured load with the cross-sectional area at the reduced gage region. Stress/strain curves were constructed from the raw data and mechanical properties were calculated using custom-written software (Matlab, The Mathworks, Natick, MA, USA). The yield point (limit for elastic deformability) was determined by 0.2% offset-method. The elastic deformation capacity (elastic energy) was calculated from the area within the elastic region whose endpoint was defined by the yield limit. The energy required to fracture the specimen (fracture energy) was calculated from the area under the entire stress-strain curve. The post-yield deformation capacity (post-yield energy) was calculated as the difference between the fracture energy and the elastic energy. Tensile Fatigue Life Fatigue tests were conducted under load-control using sinusoidal waveform at 2 Hz. The initial load level was arranged such that the resulting strain acting on the specimen was 0.2% which corresponded to about 25% of the average yield strain obtained from monotonic tests of control specimens. This level of strain is less than the reported threshold strain of 0.25% above which cortical bone fails at a much faster rate in tension. Therefore, the fatigue loading was within the high-cycle range and the initial strain was physiologically relevant. A loading ratio of R = 0.1 was used such that the minimum load was set at 10% of the maximum load value. Specimens were kept wet by continuous drip of calcium supplemented saline solution at ambient temperature during the entire loading. If the test specimen did not break within two days (i.e. by 300,000 cycles) the test was interrupted. Scanning Electron Microscopy Two monotonic and two fatigue specimens were selected from each treatment group and their fracture surfaces were sputter coated with gold and qualitatively investigated via scanning electron microscopy (JEOL, Akishima, Japan). SDS-PAGE Analysis of Collagen α-chains The grip regions of 4 randomly picked monotonic tensile specimens were cut off and pooled for each treatment group. Tissue was frozen in liquid nitrogen and pulverized manually using a stainless-steel mortar and pestle. The powder was defatted and dehydrated in ethanol for 30 min, lyophilized overnight (Labconco, Kansas City, MO, USA) and demineralized in 0.5 [M] EDTA adjusted to pH 7.2 for 3 days, at 4 °C. The EDTA solution was centrifuged and the precipitate was placed in distilled water and dialyzed (1000 Da MW cut-off) against ultrapure distilled water for three days to clear the mineral ions from the solution. Dialyzed samples were centrifuged and the precipitates were solubilized in 0.5 [M] acetic acid solution with pepsin (10:1 weight ratio of bone/enzyme) for 48 hours at 4 °C. Solubilized collagen in the supernatant was precipitated by addition of NaCl to attain a concentration of 2.0 [M] for 24 hours and the precipitates were recovered by centrifuging at 35,000 g for 1 hour. The pellet was redissolved in 0.5 [M] acetic acid, dialyzed against 0.2 [M] acetic acid solution and lyophilized to obtain soluble collagen. Salt precipitated collagen and collagen standards (Sigma-Aldrich, St. Louis, MO, USA) were run on %5 SDS-PAGE slabs [20] at a concentration of 5 mg/ml, the gel was stained with Coomassie blue and destained. The locations of bands were verified by molecular weight markers (Sigma-Aldrich, St. Louis MO, USA). Four separate gels were run to confirm the consistency of the emerging gel-profiles. Statistical Analyses Generalized multivariate analysis of variance (MANOVA) was performed to determine the significances of the main effect of gamma radiation sterilization (P_STER)_. the main effect of thiourea treatment (P_THIO) ^and the interaction effects of the two factors (P_ST-_TH) on mechanical properties. If MANOVA of main or interaction effects denoted significance then the difference between any two treatment groups was tested by a Mann- Whitney U test (p). A difference at the level of p <0.05 was reported as significant and a difference at the level of 0.05 < p < 0.1 is reported as borderline significant. Results The elastic, the post-yield and the overall deformation energies suffered significant reductions following gamma radiation sterilization at a dose of 36.4 kGy (Fig. 9 - Table 1, and Figure 11). The post-yield and overall deformation energies of bone tissue were dramatically reduced by 70% and 87%, respectively, whereas the elastic energy experienced a relatively modest reduction of 26%. The fatigue life of irradiated treatment group also suffered a significant reduction of 87% secondary to gamma radiation sterilization. Thiourea treatment alone did not alter the native mechanical properties of bone tissue such that none of the variables of thiourea treated controls differed from those of the control specimens (Mann Whitney U-test p > 0.1) (Fig. 9 - Table 1). Generalized MANOVA tests revealed that the main effect of thiourea treatment was insignificant for post-yield and overall energies; however, the interaction effects demonstrated borderline significance at the level of P_ST-_TH ⁼ 0.07 (Fig. 9 - Table 1). These observations indicate that the effect thiourea varies with gamma radiation, i.e., thiourea does not have an effect in the absence of irradiation whereas following irradiation the effect of thiourea on post-yield and fracture energies becomes observable. The effect of thiourea treatment was such that the post-yield and fracture energy values of the treatment group 1.51 (thiourea treated-irradiated) were 1.9-fold and 3.3-fold greater than those of the treatment group I (irradiated), respectively (p < 0.05, Mann Whitney U-test) (Fig. 9 - Table 1 and Figure 11). On the other hand, the post-yield and fracture energy values of the treatment group 1.51 (thiourea treated- irradiated) were significantly less than those of controls (p < 0.05, Mann Whitney U- test). Therefore, thiourea treatment has a noteworthy radioprotective effect on the post-yield and fracture energies of irradiated specimens; however, the extent of this radioprotective effect was not enough to improve the mechanical properties of sterilized specimens to the level of unirradiated controls. General MANOVA analyses of the elastic energy resulted in the main effect of gamma radiation only whereas the main effect of thiourea treatment as well as the interaction effect were insignificant. Therefore, the modest reduction in the elastic deformability of bone tissue secondary to gamma radiation sterilization was not reversed by the thiourea treatment. Generalized MANOVA analyses of fatigue life indicated that the main effect of radiation, the main effect of thiourea treatment and the interaction effect were significant. The radioprotective effect of thiourea on the fatigue life of gamma radiation sterilized cortical bone was substantial such that the fatigue life of the treatment group 1.51 was 4.7 times greater than the fatigue life obtained from the treatment group I (p < 0.05, Mann Whitney U-test). Yet, the mean fatigue life of the treatment group 1.51 was significantly less than that of the treatment group C, indicating that the fatigue life was not improved to the level of controls. The elastic, fracture and post-yield energies were impaired following gamma radiation sterilization (P_STER<0.05) (Figs. 10a, 01b and 10c). Scavenger treatment affected all mechanical properties at the level of P_THIo < 0.1. The effect was such that: a) (C vs. 1.5C) mechanical properties of control group and the scavenger treated group did not differ indicating that scavenger treatment does not alter native mechanical properties of bone, b) (I vs. 1.51) fracture and post-yield energies of scavenger treated-irradiated group were about 100% and 200% greater (p<0.05) than those of irradiated specimens, respectively, indicating that free radical scavenger treatment improves the plastic and overall deformability of irradiated specimens, c) (C vs. 1.51) post-yield and fracture energies of scavenger treated-irradiated group converged to those of controls such that post-yield and fracture energies of 1.51 were lower but not different (p>0.05) from those of controls, d) elastic deformation capacity of both irradiated groups (I and 1.51) were significantly smaller than that of unirradiated controls (C and 1.5C), and e) the elastic energy of scavenger treated-irradiated group was not significantly different from that of the irradiated group suggesting that the impairment in the elastic deformation capacity was not ameliorated by scavenger treatment. Osteons on fracture surfaces of control and scavenger treated-irradiated specimens exhibited lamellar extrusions, indicating microstructural level resistance to fracture, as shown in Fig. 12. In contrast, fracture surfaces of irradiated specimens were flat, indicating that failure occurred at the ultrastructural scale without microstructrual involvement. SEM fractography of fracture surfaces from monotonic and fatigue specimens revealed different fracture mechanisms between the treatment groups. Failure surfaces of unirradiated control specimens (C and 1.5C) were tortuous at the microscale such that there were lamellar extrusions, indicating the involvement of the microstructure in the fracture process (Fig. 12). In contrast, fracture surfaces of irradiated specimens were flat without any meandering of the surface features at the microstructural level, indicating that the failure occurred at the ultrastructural scale and the final failure propagated without any regard to the microscopic architecture. Fractographic analyses of thiourea treated and irradiated specimens revealed that the failure pattern was qualitatively similar to tortuous fracture surface of the control specimens as opposed to the featureless fracture surface of irradiated specimens, suggesting that thiourea treatment shifts the failure process from the ultrastructural level back to the microstructural level. SDS-PAGE gel electrophoresis of specimens from C and 1.5C treatment groups demonstrated the α_n, α_J2 and β bands of the collagen molecules as revealed from gel profile analyses (Fig. 13). These three bands were almost imperceptible in the lanes of irradiated specimens despite similar amounts of solubilized collagen were loaded in all lanes, indicating that collagen molecules were cleaved along their backbones and, resultingly, the number of intact collagen molecules was greatly diminished. Furthermore, the lanes of irradiated specimens exhibited a smear of stain across its length, indicating that gamma radiation cleaves collagen molecules randomly along its backbone. Thiourea treatment helps to alleviate this cleavage, as evidenced from the reappearance of intact ctπ, α₎₂ and β bands in the lanes of irradiated specimens treated with thiourea. The described pattern of behavior was consistent for all of the four aliquots which were run on SDS-PAGE.

Example III - Biomechanical and Biochemical Analysis of Biological Materials Preparation of test specimens Four fresh frozen bovine ulnae (3-4 years old) were machined into tensile test specimens. All specimens were obtained from the anterior mid-diaphyseal region. A low-speed metallurgical saw (South Bay Tech, San Clemente, CA USA) and a table- top milling machine (Sherline, CA USA) were used to machine specimens. The gage region measured 16 mm in length, 2 mm in width, and 1mm in thickness. Specimens were kept in calcium supplemented saline solution and stored at -40°C. Treatment of Specimens Thirty specimens were randomly placed into six treatment groups (n=5, each): control (C), irradiated (I), 0.5 [M] thiourea treatment (0.5 [M]), 1.5 [M] thiourea treatment (1.5 [M]), 0.5 [M] thiourea treatment and irradiation (0.5 [M] -I) and 1.5 [M] thiourea treatment and irradiation (1.5 [M] - 1). Specimens were kept in aqueous thiourea solution supplemented with calcium at 4°C for two-weeks. The solution was supplemented with protease inhibitors to prevent microbial degradation and was changed every three days. Gamma radiation sterilization was performed using Co source at an average dose of 30 kGy (Steris Corporation, Mentor, OH USA). Biomechanical Tests Specimens were monotonically loaded to failure under tension using an electromagnetic testing machine (ELF 3200, Enduratec, Minnetonka, MN USA). The loading was displacement controlled at a rate of 1%/s. Energy to fracture parameter was obtained from stress-strain curves. Biochemical Analysis The integrity of collagen molecules were assessed by SDSPAGE analysis

(n=2). The purification involved demineralization by formic acid, salt precipitation, dialysis to remove demineralization products, and solubilization of collagen molecules through pepsin digestion. In between purification steps of specimens, samples were lyophilized. Solubilized collagen molecules were loaded on 5% acrylimide-bis SDS- PAGE gels to determine the amount of intact alpha and beta chains. Results There were no statistically significant differences between the mechanical properties of any treatment groups for the bovine cortical bone. However, the trend in the data was as expected for most mechanical properties observed such that the irradiated group demonstrated smaller energy to fracture than controls. Thiourea treated control groups were similar to controls showing that thiourea alone does not alter mechanical properties of bone. There was improvement in the mechanical properties of irradiated specimens treated with thiourea such that mechanical properties of irradiated specimens treated with 1.5 [M] thiourea was greater than irradiated specimens treated with 0.5 [Ml thiourea and irradiated alone. Also, the mechanical properties of irradiated specimens treated with 0.5 [M] thiourea were greater than those specimens irradiated alone. The mechanical properties of irradiated specimens treated with 7.5 [M] thiourea were similar to the mechanical properties of unirradiated controls, showing that thiourea has a radioprotective effect on the mechanical properties of bone. Biochemical results showed that there was a decrease in the intensity of Q- chains following sterilization. It is shown in lane profiling that the irradiated samples treated with thiourea demonstrated intact o-chains. It is to be understood, however, that since it is known that gamma radiation embrittles bone tissue by impairing the post-yield properties rather than the pre-yield properties, and since bovine bone already suffers in terms of post-yield deformability in its natural state, further embrittlement due to gamma radiation sterilization is highly limited. Therefore, it is not suggested that bovine ulna be use as a model for investigation of gamma radiation induced embrittlement.

Example IV - Nucleic Acid Targeting Radiosensitizer (NTR) Compositions Referring now to Figs. 16a-16d, various steps are schematically shown. The free radical scavenger (FRS) protects the integrity of the collagen phase in the organic tissue that is sterilized, and thus its mechanical integrity. In order to prevent the FRS from also protecting any undesirable contaminants the following method is also within the contemplated scope of the present invention. To increase the likelihood that any contaminants will be damaged, at least one "nucleic acid targeting radiosensitizer (NTR)" composition is used. The NTR complex comprises two parts: one part selectively binds to the DNA and/or the RNA of the contaminant (i.e., for example, virus, bacteria, fungi, and the like) but NOT to the collagen; and the other part generates damaging free radical species upon radiation. In certain embodiments, the NTR comprises a phenantridine-nitroimidazole complex. In another aspect, the present invention provides a method for improving the biomechanical performance of at least one sterilized biological material. The method also provides for minimizing, and in certain embodiments, reversing the extent of damage to the biological materials such that the sterilized biological material have with an associated recovery of their mechanical strength. The graft is rinsed to remove excess unbound NTR from the graft leaving only those NTR bound to the contaminants. Then, the free radical scavenger (FRS) is applied as in the above examples. The FRS is present in both the contaminants and the collagen; however, since the contaminants have the NTR complex in addition to the FRS they will be more likely to be killed. Prior to irradiation the oxygen level of the product is reduced by packing in an airtight package under vacuum. When irradiated the NTR complex generates oxygen free radicals in the vicinity of the DNA/RNA of contaminants and renders the contaminants selectively vulnerable to irradiation. In certain embodiments, the NTR comprises a composition that has a desired level of biocompatiblity. The DNA binding part of the composition can comprise any of such difference chemicals as platinum, acridine based nitro compounds, nitracrine, quinoline analogs, phenanthridines, and the like. The free radical generating part of the NTR can comprise any of such many different chemicals as 2-nitroimidazoles, mitomycin C, tirapazamine, and the like. Furthermore, there are clinically available cancer drugs which are useful as NTRs as well. The present invention does show that the suppression of free radical generation has a radioprotective effect on the mechanical and biochemical properties of gamma radiation sterilized cortical bone tissue. The suppression of free radical damage via thiourea treatment yield stronger and more durable grafts. While in the preferred embodiment of the invention the damage to the collagen phase is blocked by the introduction of free radical scavengers into the sterilization process, it must be understood that the present invention contemplates the use of substances that inhibits free radical damage to the collagen molecules, including the use of direct chemical reactions with the free radicals, or by minimization the formation of the free radicals. For example, an antioxidant, such as vitamin E, vitamin C, or beta-carotene, can be used to react with the free radicals before the free radicals react with the collagen molecules. Additionally, while the preferred embodiment has been described with the sterilization of a bone tissue fragment, it must be understood that the present invention contemplates the sterilization of any organic tissue fragment that includes collagen, such as demineralized bone tissue, cartilage tissue and tendon tissue, or any connective tissue that includes collagen and any other organic extracellular matrix materials such as proteoglycans, glycosaminoglycans and elastin. Further, while the preferred embodiment has been described with the use of an aqueous solution for the delivery of free radical scavengers, it must be understood that the present invention contemplates the use of any medium, such as an oil based solution, that is suitable to deliver to the bone tissue a substance for inhibiting free radical damage. The present invention improves the biomechanical performance of bone transplants and thus, it increases the functional lifetime of the bone transplants following surgery, through improved ductility, as compared to conventional bone transplants. Increased long-term survival of the bone transplant, i.e. the ability of the bone transplant to withstand longer duration before failure than conventional bone transplants, provides additional valuable time for the host bone structure to recover its strength. Consequently, clinical complications related to premature failure of bone transplants are alleviated and/or prevented. Overall, the invention improves the current practice of gamma radiation sterilization of bone grafts, including improving the mechanical strength of the grafts. All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of the foregoing illustrative embodiments, it will be apparent to those skilled in the art that variations, changes, modifications, and alterations may be applied to the compositions and/or methods described herein, without departing from the true concept, spirit, and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

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Claims

What is claimed is: 1. A composition comprising at least one irradiated, sterilized and biomechanically strengthened biological material, wherein the biological material has been treated with at least one scavenging substance that has a high affinity for free radicals, the biological material having a molecular or supramolecular level of porosity which allows for the penetration of molecules of the high affinity free radical scavenging substance, the biological material having retained biomechanical values that are improved when compared with a biological material exposed to the radiation in the absence of the high affinity free radical scavenging substance.

2. The composition of Claim 1 wherein the biological material comprises at least one of bone tissue, demineralized bone tissue, cartilage tissue, collagen tissue, ligament, tendon tissue or combinations thereof.

3. The composition of Claim 2 wherein the high affinity free radical scavenging substance comprises at least one antioxidant.

4. The composition of Claim 2 wherein the high affinity free radical scavenging substance comprises at least one free radical scavenger having a high solubility and high diffusibility into the biological material.

5. The method of Claim 1 wherein the free radical scavenger comprises thiourea.

6. The composition of Claim 2 wherein the biological material comprises bone tissue that has a ductility value within the range of from about 0.8 Joules to about 1.6 Joules.

7. The composition of Claim 4 wherein the high affinity free radical scavenging substance has a molecular weight less than about 300 Da.

8. The composition of Claim 1 wherein the biological material irradiated in the presence of the free radical affinity scavenging substance demonstrates intact α- chains.

9. The composition of Claim 1 comprising at least one gamma-irradiated sterilized and biomechanically strengthened biological material.

10. A composition comprising: i) at least one irradiated, sterilized and biomechanically strengthened biological material, wherein the biological material has been treated with at least one scavenging substance that has a high affinity for free radicals, the biological material having a molecular or supramolecular level of porosity which allows for the penetration of molecules of the high affinity free radical scavenging substance, the biological material having retained biomechanical values that are improved when compared with a biological material exposed to radiation in the absence of the substance having the high affinity for free radicals; and, ii) at least one aqueous solvent, wherein the aqueous solvent and the free radical affinity scavenging substance are present in a combined amount effective to inhibit biomechanical degradation of the biological material.

11. The composition of Claim 10 wherein the biological material comprises at least one of bone tissue, demineralized bone tissue, cartilage tissue, ligament, tendon tissue or combinations thereof.

12. The composition of Claim 10 wherein the high affinity free radical scavenging substance comprises at least one antioxidant.

13. The composition of Claim 12 wherein the high affinity free radical scavenging substance comprises at least one free radical scavenger having a high solubility and high diffusibility into the biological material

14. The composition of Claim 10 wherein the biological material comprises bone tissue that has a ductility value within the range of from about 0.8 Joules to about 1.6 Joules.

15. The composition of Claim 10 wherein the high affinity free radical scavenging substance has a molecular weight less than about 300 Da.

16. The composition of Claim 10 wherein the biological material irradiated in the presence of the free radical affinity scavenging substance demonstrates intact α- chains.

17. A method for improving the biomechanical performance of at least one irradiated biological material and for minimizing damage to the biological material with an associated recovery in the mechanical strength of such irradiated biological material, comprising: (a) providing at least one biological material having a molecular or supramolecular level of porosity which allows for the penetration of into the biological material; optionally, treating at least one biological material with at least one nucleic acid targeted radiosensitizer; (b) treating the at least one biological material with at least one scavenging substance that has a high affinity for free radicals; and (c) exposing the at least one biological material to radiation.

18. The method of Claim 17 wherein the biological material comprises at least one of bone tissue, demineralized bone tissue, cartilage tissue, collagen tissue, ligament, tendon tissue and combinations thereof.

19. The method of Claim 17 wherein the high affinity free radical scavenging substance comprises at least one antioxidant.

20. The method of Claim 17 wherein the high affinity free radical scavenging substance comprises at least one free radical scavenger having a high solubility and high diffusibility into the biological material.

21. The method of Claim 20 wherein the free radical scavenger substance comprises thiourea.

22. The method of Claim 17 wherein the treating step comprises immersing the at least one biological material in a solution including the free radical scavenger substance.

23. The method of Claim 22 wherein the immersing step is for a period of at least 4 to about 8 hours.

24. The method of Claim 22 wherein the immersing step is for a period of at least 14 days.

25. The method of Claim 17 wherein the free radical scavenger substance comprises thiourea.

26. The method of Claim 25 wherein a concentration of thiourea in the solution is at least about 0.5 M.

27. The method of Claim 25 wherein a concentration of thiourea in the solution is at least about 1.5 M.

28. The method of Claim 17 wherein the biological material is exposed to gamma radiation.

29. A method for improving the biomechanical performance of at least one irradiated biological material and for minimizing damage to the at least one irradiated biological material with an associated recovery in the mechanical strength of such at least one irradiated biological material, comprising optionally, treating at least one biological material with at least one nucleic acid targeted radiosensitizer; administering at least one free radical scavenger substance, via an aqueous solution, to the biological material; optionally, adjusting the aqueous free radical scavenger solution to a pH of about 7.0; optionally, adding at least one proteolytic inhibitor to the solution; immersing the biological material in the aqueous free radical scavenger solution under vacuum; optionally, storing the immersed at least one biological material under cold conditions for a suitable length of time; optionally, preserving the biological material by cryopreservation; and, exposing the at least one biological material to an effective dosage of radiation.

30. The method of Claim 29 wherein the biological material comprises at least one of bone tissue, demineralized bone tissue, cartilage tissue, collagen tissue, ligament, tendon tissue and combinations thereof.

31. The method of Claim 29 wherein the high affinity free radical scavenging substance comprises at least one antioxidant.

32. The method of Claim 29 wherein the high affinity free radical scavenging substance comprises at least one free radical scavenger having a high solubility and high diffusibility into the biological material.

33. The method of Claim 32 wherein the free radical scavenger substance comprises thiourea.

34. The method of Claim 29 wherein the treating step comprises immersing the at least one biological material in a solution including the free radical scavenger substance.

35. The method of Claim 34 wherein the immersing step is for a period of at least 4 to about 8 hours.

36. The method of Claim 34 wherein the immersing step is for a period of at least 14 days.

37. The method of Claim 29 wherein the free radical scavenger substance comprises thiourea.

38. The method of Claim 37 wherein a concentration of thiourea in the solution is at least about 0.5 M.

39. The method of Claim 37 wherein a concentration of thiourea in the solution is at least about 1.5 M.

40. The method of Claim 29, wherein the radiation comprises gamma radiation.

41. The method of Claim 29, wherein the at least one proteolytic inhibitor comprises at least one of a disinfecting and bioburden-reducing amount of at least one antibiotic agent, antiviral agent and/or antimycotic agent.

42. The method of Claim 29, wherein the molarity of the solution is returned to its original molarity by adding further free radical scavenger substance.

43. A method for prophylaxis or treatment of a condition or disease or malfunction of at least one tissue in a mammal comprising introducing into a mammal in need thereof one or more biological materials sterilized according to the method of Claim 17.

44. A method for prophylaxis or treatment of a condition or disease or malfunction of at least one tissue in a mammal comprising introducing into a mammal in need thereof one or more biological materials sterilized according to the method of Claim 29.

45. A method for prophylaxis or treatment of a condition or disease or malfunction of at least one tissue in a mammal comprising introducing into a mammal in need thereof one or more biological materials of claim 1.