Effect of radiation sterilization on the osteoinductive properties and the rate of remodeling of bone implants preserved by lyophilization and deep-freezing.

The effect of various doses of ionizing radiation on the osteoinductive properties of decalcified bone matrices implanted heterotopically and on the rate of remodeling of nondecalcified bone grafts implanted orthotopically in allogeneic systems was studied. Decalcified bone matrices and nondecalcified bone grafts were preserved by lyophilization or by deep-freezing and were subsequently irradiated with appropriate doses at room temperature or at -72 degrees. Lyophilized matrices irradiated at room temperature with 35 and 50 kGy, respectively, were completely resorbed five weeks after heterotopic implantation into the muscles and did not induce osteogenesis, whereas the resorption of deep-frozen ones irradiated with the same doses at -72 degrees was slower and new bone formation was induced. The preservation of the osteoinductive capacity of irradiated, deep-frozen matrices may depend on two factors: reduction of radiation damage on the inducing agents and collagen irradiated in the presence of water, which may diminish the rate of matrix resorption. The rate of remodeling of undecalcified deep-frozen bone implants irradiated at -72 degrees and grafted orthotopically was higher than that of lyophilized ones irradiated at room temperature. It is possible that the temperature during irradiation plays a critical role in protection against radiation damage.     

Free radical scavenging alleviates the biomechanical impairment of gamma radiation sterilized bone tissue.

Terminal sterilization of bone allografts by gamma radiation is often essential prior to their clinical use to minimize the risk of infection and disease transmission. While gamma radiation has efficacy superior to other sterilization methods it also impairs the material properties of bone allografts, which may result in premature clinical failure of the allograft. The mechanisms by which gamma radiation sterilization damages bone tissue are not well known although there is evidence that the damage is induced via free radical attack on the collagen. In the light of the existing literature, it was hypothesized that gamma radiation induced biochemical damage to bone's collagen that can be reduced by scavenging for the free radicals generated during the ionizing radiation. It was also hypothesized that this lessening of the extent of biochemical degradation of collagen will be accompanied by alleviation in the extent of biomechanical impairment secondary to gamma radiation sterilization. Standardized tensile test specimens machined from human femoral cortical bone and specimens were assigned to four treatment groups: control, scavenger treated-control, irradiated and scavenger treated-irradiated. Thiourea was selected as the free radical scavenger and it was applied in aqueous form at the concentration of 1.5 M. Monotonic and cyclic mechanical tests were conducted to evaluate the mechanical performance of the treatment groups and the biochemical integrity of collagen molecules were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native mechanical properties of bone tissue did not change by thiourea treatment only. The effect of thiourea treatment on mechanical properties of irradiated specimens were such that the post-yield energy, the fracture energy and the fatigue life of thiourea treated-irradiated treatment group were 1.9-fold, 3.3-fold and 4.7-fold greater than those of the irradiated treatment group, respectively. However, the mechanical function of thiourea treated and irradiated specimens was not to the level of unirradiated controls. The damage occurred through the cleavage of the collagen backbone as revealed by SDS PAGE analysis. Irradiated specimens did not exhibit a noteworthy amount of intact alpha-chains whereas those irradiated in the presence of thiourea demonstrated intact alpha-chains. Results demonstrated that free radical damage is an important pathway of damage, caused by cleaving the collagen backbone. Blocking the activity of free radicals using the scavenger thiourea reduces the extent of damage to collagen, helping to maintain the mechanical strength of sterilized tissue. Therefore, free radical scavenger thiourea has the potential to improve the functional life-time of the allograft component following transplantation.     

Freeze-dried irradiated bone brittleness improves compactness in an impaction bone grafting model

Background?Defatted bone chips with or without freeze-drying and irradiation have mechanical advantages as compared to fresh-frozen controls in in vitro models of impaction. These improved results have been ascribed to replacement of viscous bone marrow by saline and embrittlement of the freeze-dried bone by irradiation.Material and methods?To determine which of these hypotheses is correct, we compared the development of stiffness and compactness of morselized bone graft that had been: 1) fat-reduced with saline, and 2) fresh-frozen, solvent-detergent defatted, 3) freeze-dried irradiated and 4) not irradiated. We used 12 osteo-arthrotic femoral heads to prepare these four batches of morselized bone, and impacted 18 samples from each batch in a cylinder. The frozen bone grafts were tested after thawing at room temperature for 2 hours and the freeze-dried grafts were tested after 30 minutes of rehydration. We monitored the development of compactness and stiffness of the material during impaction.Results?The stiffness of the freeze-dried irradiated bone was greater than that of the other three series after 10, 50 and 150 impactions. The freeze-dried bone chips that were not irradiated and the chips defatted with saline alone were less stiff than the fresh-frozen control after 150 impactions.Interpretation?The brittleness of freeze-dried irradiated bone, caused by loss of the capacity to absorb energy in a plastic way, increases the compactness and stiffness of the morselized grafts. Washing bone with saline alone or treating bone with solvent-detergent but no irradiation had no similar mechanical advantage and the bone did not impact better than fresh-frozen undefatted bone in our model.     

Bone embrittlement and collagen modifications due to high-dose gamma-irradiation sterilization

Bone allografts are often used in orthopedic reconstruction of skeletal defects resulting from trauma, bone cancer or revision of joint arthroplasty. γ-Irradiation sterilization is a widely-used biological safety measure; however it is known to embrittle bone. Irradiation has been shown to affect the post-yield properties, which are attributed to the collagen component of bone. In order to find a solution to the loss of toughness in irradiated bone allografts, it is important to fully understand the effects of irradiation on bone collagen. The objective of this study was to evaluate changes in the structure and integrity of bone collagen as a result of γ-irradiation, with the hypothesis that irradiation fragments collagen molecules leading to a loss of collagen network connectivity and therefore loss of toughness.Using cortical bone from bovine tibiae, sample beams irradiated at 33 kGy on dry ice were compared to native bone beams (paired controls). All beams were subjected to three-point bend testing to failure followed by characterization of the decalcified bone collagen, using differential scanning calorimetry (DSC), hydrothermal isometric tension testing (HIT), high performance liquid chromatography (HPLC) and gel electrophoresis (SDS-PAGE). The carbonyl content of demineralized bone collagen was also measured chemically to assess oxidative damage. Barium sulfate staining after single edge notch bending (SEN(B)) fracture testing was also performed on bovine tibia bone beams with a machined and sharpened notch to evaluate the fracture toughness and ability of irradiated bone to form micro-damage during fracture.Irradiation resulted in a 62% loss of work-to-fracture (p ≤ 0.001). There was significantly less micro-damage formed during fracture propagation in the irradiated bone. HPLC showed no significant effect on pentosidine, pyridinoline, or hydroxypyridinoline levels suggesting that the loss of toughness is not due to changes in these stable crosslinks. For DSC, there was a 20% decrease in thermal stability (p < 0.001) with a 100% increase (p < 0.001) in enthalpy of denaturation (melting). HIT testing also showed a decrease in thermal stability (20% lower denaturation temperature, p < 0.001) and greatly reduced measures of collagen network connectivity (p < 0.001). Interestingly, the increase in enthalpy of denaturation suggests that irradiated collagen requires more energy to denature (melt), perhaps a result of alterations in the hydrogen bonding sites (increased carbonyl content detected in the insoluble collagen) on the irradiated bone collagen.Altogether, this new data strongly indicates that a large loss of overall collagen connectivity due to collagen fragmentation resulting from γ-irradiation sterilization leads to inferior cortical bone toughness. In addition, notable changes in the thermal denaturation of the bone collagen along with chemical indicators of oxidative modification of the bone collagen indicate that the embrittlement may be a function not only of collagen fragmentation but also of changes in bonding.     

Freeze-dried irradiated bone brittleness improves compactness in an impaction bone grafting model

BACKGROUND: Defatted bone chips with or without freeze-drying and irradiation have mechanical advantages as compared to fresh-frozen controls in in vitro models of impaction. These improved results have been ascribed to replacement of viscous bone marrow by saline and embrittlement of the freeze-dried bone by irradiation. MATERIAL AND METHODS: To determine which of these hypotheses is correct, we compared the development of stiffness and compactness of morselized bone graft that had been: 1) fat-reduced with saline, and 2) fresh-frozen, solvent-detergent defatted, 3) freeze-dried irradiated and 4) not irradiated. We used 12 osteoarthrotic femoral heads to prepare these four batches of morselized bone, and impacted 18 samples from each batch in a cylinder. The frozen bone grafts were tested after thawing at room temperature for 2 hours and the freeze-dried grafts were tested after 30 minutes of rehydration. We monitored the development of compactness and stiffness of the material during impaction. RESULTS: The stiffness of the freeze-dried irradiated bone was greater than that of the other three series after 10, 50 and 150 impactions. The freeze-dried bone chips that were not irradiated and the chips defatted with saline alone were less stiff than the fresh-frozen control after 150 impactions. INTERPRETATION: The brittleness of freeze-dried irradiated bone, caused by loss of the capacity to absorb energy in a plastic way, increases the compactness and stiffness of the morselized grafts. Washing bone with saline alone or treating bone with solvent-detergent but no irradiation had no similar mechanical advantage and the bone did not impact better than fresh-frozen undefatted bone in our model.     

Electron spin resonance investigation of laser and heated metal-tip–induced free radical formation in various tissues

The short-lived free radical formation accompanying laser irradiation and laser-heated metal tip contact was examined using electron spin resonance (ESR) spin-trap methodology. Various tissues (canine myocardium, human aorta, liver, and spleen) were irradiated by argon-ion (continuous wave [CW] and pulse) and YAG (CW) lasers employing both naked fiber and a laser-heated metal tip. All showed the formation of primarily carbon-centered, not oxygenated, free radicals, together with some minor unidentified species. This implies that the backbones of amino acids, lipids, or other biological building blocks are cleaved during the irradiation or thermal treatment. Different tissues produce similar radicals but with different amounts when irradiated by argon-ion, YAG, and laser-heated metal tip sources. The amount of the free radicals formed depends on the laser power (within 5–15 W). Compared to the naked fiber, the laser-heated metal tip shows the generation of at least twice the amount of free radicals. The possible relationship of the free radical formation to tissue injury is briefly discussed.     

Effects of Zoledronate on Irradiated Bone In Vivo: Analysis of the Collagen Types I, V and Their Cross-links Lysylpyridinoline, Hydroxylysylpyridinoline and Hydroxyproline

Radiotherapy can lead to a reduction of bone density with an increased risk of pathological fractures. Bisphosphonates may represent a preventive treatment option by increasing the density of anorganic bone mineral. Yet it is unknown how bisphosphonates act on irradiated collagen cross-links, which play an essential role for the mechanical stability of bone. The aim of this study was to evaluate the effects of zoledronate on bone collagens and their cross-links after irradiation. The right femur of 37 rats was irradiated with a single dose of 9.5 Gy at a high dose rate using an afterloading machine. Half of the rats (n = 18) received additionally a single dose zoledronate (0.1 mg/kg body weight). Fourteen and 100 days after irradiation the femora were collected for histologic evaluation and determination of the collagen cross-links lysylpyridinoline, hydroxylysylpyridinoline, and hydroxyproline. The collagen types were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Fourteen days after treatment the lysylpyridinoline levels of all treatment groups were significantly lower compared to the untreated control. After 100 days, in the combined radiotherapy + zoledronate group significantly lower lysylpyridinoline values were determined (p = 0.009). Radiotherapy and/or zoledronate did not change significantly the level of hydroxylysylpyridinoline. The concentration of hydroxyproline was 14 days after irradiation significantly higher in the combined treatment group compared to the control. No significant differences were observed 100 days after treatment. Zoledronate does not have the ability to restore the physiological bone collagen cross-link levels after radiotherapy. However, this would be necessary for regaining the physiological mechanical stability of bone after irradiation and therefore to prevent effectively radiation-induced fractures.     

Bone Material Properties in Osteogenesis Imperfecta

Osteogenesis imperfecta entrains changes at every level in bone tissue, from the disorganization of the collagen molecules and mineral platelets within and between collagen fibrils to the macroarchitecture of the whole skeleton. Investigations using an array of sophisticated instruments at multiple scale levels have now determined many aspects of the effect of the disease on the material properties of bone tissue. The brittle nature of bone in osteogenesis imperfecta reflects both increased bone mineralization density—the quantity of mineral in relation to the quantity of matrix within a specific bone volume—and altered matrix‐matrix and matrix mineral interactions. Contributions to fracture resistance at multiple scale lengths are discussed, comparing normal and brittle bone. Integrating the available information provides both a better understanding of the effect of current approaches to treatment—largely improved architecture and possibly some macroscale toughening—and indicates potential opportunities for alternative strategies that can influence fracture resistance at longer‐length scales. © 2016 American Society for Bone and Mineral Research.     

In vivo oxidation of retrieved cross-linked ultra-high-molecular-weight polyethylene acetabular components with residual free radicals

Wear of ultra-high-molecular-weight polyethylene (UHMWPE) contributes to debris that can lead to periprosthetic osteolysis in total hip arthroplasty. Irradiation not only decreases wear of UHMWPE but also generates residual free radicals that can oxidize the UHMWPE in the long term. Melting or annealing is used to quench the free radicals. Melting is more effective than annealing. We hypothesized that the postirradiation annealed UHMWPE components would oxidize in vivo and that postirradiation melted ones would not. We analyzed surgical explants of UHMWPE acetabular liners. The irradiated and annealed explants showed embrittlement, oxidation, and an increase in crystallinity. The irradiated and melted UHMWPE explants showed no oxidation, no increase in crystallinity, and no embrittlement. To prevent long-term chemical changes in highly cross-linked UHMWPE components, the residual free radicals must be stabilized after irradiation, preferably by melting and not annealing.     

A mixed packing model for bone collagen

Summary A wide variety of physical properties, including sonic velocity, dimensional changes between wet and dried stages, anisotropy of the tissue properties, density, X-ray diffraction, differential microcalorimetry, dielectric constant, and composition (water, mineral, organic content) for the mineralized and demineralized tissue was used to develop a model for the superlattice structure of bone collagen. A mixed model is suggested where the collagen molecules are in register as in SLS type of aggregation within the microfibril, and the microfibrils are staggered in D unit steps according to the Hodge-Petruska scheme. A square packing model with 4 or more molecules per microfibril best fits the HP scheme with the effective molecular diameter of the wet collagen molecule, and allows for the regular array of axial gap filling microcrystallites of 5 nm or larger diameter. It is concluded that: 1. Macroscopic dimensional changes of adult bovine bone matrix closely match molecular dimensional changes of collagen superlattice. 2. Effective molecular diameter of dry collagen is 1.09 nm and that of wet bone collagen is 1.42 – 1.45 nm. 3. Water layer of the wet bone collagen molecule is 0.16 nm thick. 4. Water in the bone collagen molecule is distributed in 5 regimes much like in the tendon collagen molecule. 5. “Hidden” water, 0.10 g water per dry collagen of regimes I and II, is within the triple helix. 6. “External” water incorporated in the collagen molecule provides transition between the highly structured collagen molecule and the intermolecular medium. 7. Water incorporated in the mineralized bone collagen molecule is less than in demineralized bone matrix. 8. For adult bovine cortical bone, 25% by volume is water, 32% dry organic, 43% mineral; 28% by volume of the mineral is axial gap filling, 58% radial intrafibrillar, and 14% radial extrafibrillar.     

A mixed packing model for bone collagen

A wide variety of physical properties, including sonic velocity, dimensional changes between wet and dried stages, anisotropy of the tissue properties, density, X-ray diffraction, differential microcalorimetry, dielectric constant, and composition (water, mineral, organic content) for the mineralized and demineralized tissue was used to develop a model for the superlattice structure of bone collagen. A mixed model is suggested where the collagen molecules are in register as in SLS type of aggregation within the microfibril, and the microfibrils are staggered in D unit steps according to the Hodge-Petruska scheme. A square packing model with 4 or more molecules per microfibril best fits the HP scheme with the effective molecular diameter of the wet collagen molecule, and allows for the regular array of axial gap filling microcrystallites of 5 nm or larger diameter. It is concluded that: 1. Macroscopic dimensional changes of adult bovine bone matrix closely match molecular dimensional changes of collagen superlattice. 2. Effective molecular diameter of dry collagen is 1.09 nm and that of wet bone collagen is 1.42-1.45 nm. 3. Water layer of the wet bone collagen molecule is 0.16 nm thick. 4. Water in the bone collagen molecule is distributed in 5 regimes much like in the tendon collagen molecule. 5. "Hidden" water, 0.10 g water per dry collagen of regimes I and II, is within the triple helix. 6. "External" water incorporated in the collagen molecule provides transition between the highly structured collagen molecule and the intermolecular medium. 7. Water incorporated in the mineralized bone collagen molecule is less than in demineralized bone matrix. 8. For adult bovine cortical bone, 25% by volume is water, 32% dry organic, 43% mineral; 28% by volume of the mineral is axial gap filling, 58% radial intrafibrillar, and 14% radial extrafibrillar.     

Effect of sterilization on bone morphogenetic protein

Demineralized bone matrix and bone morphogenetic protein have been used clinically to accelerate bone regeneration. However, the best method of sterilization has been the subject of controversy. Some investigators have used ethylene oxide, but others have reported that doses adequate for sterilization destroyed the osteoinductivity of demineralized bone matrix and that gamma irradiation was less harmful in this respect. We used partially purified bone morphogenetic protein and type-I collagen to investigate the effects of sterilization by ethylene oxide and gamma irradiation on the activity of bone morphogenetic protein. Osteoinductivity was reduced considerably after sterilization by gamma irradiation at 2.5 Mrad and by ethylene oxide at 37°C for 4 hours and at 55°C for 1 hour; however, the reduction induced by ethylene oxide at 29°C for 5 hours was about half of the control values. This study showed that ethylene oxide at 29°C for 5 hours can be used clinically for sterilization of bone morphogenetic protein. We also investigated the effect of gamma irradiation on bone morphogenetic protein and the collagen carrier separately and found that collagen was far more labile than bone morphogenetic protein.     

In vitro effects of low-level laser irradiation at 660 nm on peripheral blood lymphocytes

BACKGROUND AND OBJECTIVE:The effects of low-level laser light irradiation are still highly contested, and the mechanisms of its action still unclear. This study was conducted to test the effects of low-level laser irradiation at 660 nm on human lymphocytes and to investigate the possible mechanisms by which these effects are produced. STUDY DESIGN/MATERIALS AND METHODS: Whole blood obtained by phlebotomy was irradiated at 660 nm by using energy fluences between 0 and 5.0 J/cm(2). The lymphocytes were isolated after irradiation of the whole blood. For the control experiment, the lymphocytes were first isolated and then irradiated at the same wavelength and energy fluence for comparison. The proliferation of lymphocytes and the formation of free radicals and lipid peroxides were monitored. Hemoglobin was also irradiated in a cell-free environment to test for the production of lipid peroxides. RESULTS: Lymphocyte proliferation was significantly higher (P<0.05) as expressed by a Stimulation Index in samples irradiated in the presence of whole blood compared with lymphocytes irradiated after isolation from whole blood. Free radical and lipid peroxide production also increased significantly when samples were irradiated in the presence of red blood cells. CONCLUSION: The present study supports the hypothesis that one mechanism for the photobiostimulation effect after irradiation at 660 nm is the reaction of light with hemoglobin, resulting in oxygen radical production.     

Selective targeting of protein, water, and mineral in dentin using UV and IR pulse lasers: the effect on the bond strength to composite restorative materials

BACKGROUND AND OBJECTIVES: Previous studies have shown that during the laser irradiation of dentin and bone, thermal damage can be minimized by using a highly absorbed laser wavelength, laser pulses shorter than the thermal relaxation time of the deposited laser energy at that wavelength, and the addition of a layer of water to the tissue surface before ablation. The objective of this study was to investigate the influence of laser pulse duration and wavelength with and without the added water layer on the bond strength of composite to laser prepared dentin surfaces. The specific hypothesis that was tested was that thermal damage to the collagen matrix in dentin compromises the bond strength to composite restorative materials. STUDY DESIGN/MATERIALS AND METHODS: Three laser systems were employed that were tuned to water, collagen, and mineral absorption with pulse durations less than the thermal relaxation time of the deposited energy. The surfaces of human dentin were irradiated by laser irradiation from free-running and Q-switched Er:YSGG lasers, pulsed CO(2) lasers operating at 9.6-microm, and a Q-switched Nd:YAG laser operating at 355-nm. A motion control system and a pressurized spray system incorporating a microprocessor controlled pulsed nozzle for water delivery, were used to ensure uniform treatment of the entire surface. Shear bond testing was used to evaluate the adhesive strength in order to access the suitability of laser treated surfaces for bonding. Bonded interfaces were examined by SEM. RESULTS: All the laser groups had significantly lower bond strengths than the positive acid etch control group. The highest bond strengths were for the short pulse (< 5-microsecond) Er:YSGG and CO(2) laser groups with water. Laser groups without water had significantly reduced bond strengths and thicker layers of thermally damaged dentin. CONCLUSIONS: Thermal damage to the collagen matrix profoundly influences the bond strength to composite restorations.     

Neck fracture femoral heads for impaction bone grafting: evolution of stiffness and compactness during impaction of osteoarthrotic and neck-fracture femoral heads

BACKGROUND: The need for safe bone allografts is increasing and preservation of femoral heads from patients being operated on with hip arthroplasty should be encouraged. However, should we preserve femoral heads from patients operated on for neck fracture as tissue mechanical quality may not be satisfactory? MATERIAL AND METHODS: We compared the evolution of stiffness and compactness of fresh-frozen morselized bone obtained from osteoarthrotic femoral heads and those from neck fractures. Both materials were also compared after freeze-drying and irradiation. We used 6 osteoarthrotic and 6 neck-fracture femoral heads to prepare 4 batches of morselized bone. 18 samples from each batch were impacted in a contained cylinder. Frozen bone grafts were tested after thawing at room temperature for 2 hours and freeze-dried grafts were tested after 30 minutes of rehydration. RESULTS: The stiffness of fresh-frozen neck fracture bone was lower than that of fresh-frozen osteoarthrotic bone at 150 impactions. The stiffness of freeze-dried irradiated bone was higher than that of the fresh-frozen bone and did not differ between osteoarthrotic and neck-fracture bone. INTERPRETATION: Solvent-treated freeze-dried bone from femoral heads procured during arthroplasty for sub-capital hip fractures represents a valuable source of material for allografts, addressing concerns regarding serological testing, medical history and bone quality.     

辐照中脱矿骨基质粒径对胶原结构影响的实验研究

目的探讨不同粒径大小对γ辐照中脱钙骨基质(demineralized bone matrix,DBM)中胶原结构的影响以及辐照保护剂的有效性。方法取同一供体的冻干皮质骨,依据Urist改良法制备不同粒径的(0.5~1.0 mm、1.2~2.8 mm、3.3~4.7 mm及5.7~7.0 mm)DBM样品,按照不同剂量分为:0 kGy、15 kGy、25 kGy及25 kGy(辐照保护剂),真空密封后储存于-80℃冰箱待用。通过扫描电镜观察胶原表面形态,大体观察胶原表面结构损伤的程度;将样品按照0.2 g/ml生理盐水比例在50℃条件下72 h,利用浸提液颜色深度观察胶原被辐照损伤的程度;使用2,4-二硝基苯肼(吸光光度计法)测定样品中羰基含量;十二烷基硫酸钠聚丙烯酰胺钠凝胶电泳法(Sodium dodecyl sulfatepolyacrylamide gel electrophoresis,SDS-PAGE)测定样品中胶原分子量的变化;利用差示热量扫描法(differential scanning calorimetry,DSC)检测样品热变性温度以观察胶原热稳定性。结果样品浸提液颜色与γ辐照剂量相关度较高,未辐照样品浸提液颜色清亮,而在同粒径下随辐照剂量加大浸提液黄色逐渐加深,5.7~7.0 mm粒径组颜色相对较浅;25 kGy组相比于25 kGy+保护剂组浸提液颜色加深。扫描电镜观察到γ辐照导致胶原结构紊乱,纤维断裂,随着辐照剂量增大损伤区域增多,当粒径增大时,损伤区域有减少的趋势;相比于25 kGy组,25 kGy+保护剂组胶原结构性破坏减少。差示热量扫描法得出样品热交换曲线,随着粒径增大,热变性温度有增高的趋势,粒径间对比有统计学差异(F=189.4,P<0.001);同粒径间差异不明显。SDS-PAGE发现同粒径下γ辐照剂量愈大,胶原分子量愈小;同辐照条件下随粒径较小,高分子量胶原含量减少明显;225 kGy+保护剂组相比于25 kGy组,高分子量增多。羰基含量结果显示在同一粒径下,γ辐照使羰基含量增多,0.5~1.0 mm组(F=13.631,P=0.002),1.2~2.8 mm组(F=6.390,P=0.016),3.3~4.7 mm组(F=5.630,P=0.023),5.7~7.0 mm组(F=4.150,P=0.048)的差异均有统计学意义,不同粒径间随着粒径增大羰基含量逐渐减小但差异统计学意义(F=0.560,P=0.650)。结论γ辐照与胶原的氧化损伤具有明显的剂量反应关系,随着γ辐照剂量的增加,胶原损伤程度逐渐增加;DBM的粒径大小影响着胶原对γ辐照的敏感度,随着粒径的减小,DBM颗粒更易被γ辐照损伤;辐照保护剂在辐照过程中对胶原有一定程度的保护作用。     

Resection followed by vascularized bone autograft in patients with possible recurrence of malignant bone tumors after conservative treatment

In conservative treatment of malignant bone tumors, assessment of the local condition is difficult. The radiological changes seen in the irradiated tumor and the frequent occurrence of pathological fractures at this site may give rise to the fear that the tumor has relapsed. Resection of the whole of the involved bone is the best way to assure adequate local control but the extent of the bone defect and the bad local conditions secondary to irradiation make reconstruction hazardous. In two patients (one with Ewing's sarcoma of the femur and one with osteogenic sarcoma of the humerus) the authors used a free, vascularized fibular graft for the reconstruction having obtained consolidation of the limb after resection of the irradiated tumor, with preservation of its function. The encouraging results obtained have suggested a conservative attitude as primary treatment of specific malignant bone tumors.     

Effect of laser soldering irradiation on covalent bonds of pure collagen

Laser tissue welding and soldering is being increasingly used in the clinical setting for defined surgical procedures. The exact induced changes responsible for tensile strength are not yet fully investigated. To further improve the strength of the bonding, a better understanding of the laser impact at the subcellular level is necessary. The goal of this study was to analyze whether the effect of laser irradiation on covalent bonding in pure collagen using irradiances typically applied for tissue soldering. Pure rabbit and equine type I collagen were subjected to laser irradiation. In the first part of the study, rabbit and equine collagen were compared using identical laser and irradiation settings. In the second part of the study, equine collagen was irradiated at increasing laser powers. Changes in covalent bonding were studied indirectly using the sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) technique. Tensile strengths of soldered membranes were measured with a calibrated tensile force gauge. In the first experiment, no differences between the species-specific collagen bands were noted, and no changes in banding were found on SDS-PAGE after laser irradiation. In the second experiment, increasing laser irradiation power showed no effect on collagen banding in SDS-PAGE. Finally, the laser tissue soldering of pure collagen membranes showed virtually no determinable tensile strength. Laser irradiation of pure collagen at typical power settings and exposure times generally used in laser tissue soldering does not induce covalent bonding between collagen molecules. This is true for both rabbit and equine collagen proveniences. Furthermore, soldering of pure collagen membranes without additional cellular components does not achieve the typical tensile strength reported in native, cell-rich tissues. This study is a first step in a better understanding of laser impact at the molecular level and might prove useful in engineering of combined collagen-soldering matrix membranes for special laser soldering applications.     

Sterilization of HIV with irradiation: relevance to infected bone allografts.

BACKGROUND: Bone allograft banks commonly sterilize frozen bone by irradiation. The dose-response relationship for HIV is calculated and the dose required to inactivate the bioburden of virus that may be present in allograft bone is determined. METHODS: A virus titre experiment is performed using irradiated frozen HIV. The virus is maintained on dry ice (approximately -70 degrees C) and is exposed to a cobalt 60 source with 0-40 kGy irradiation at 5 kGy intervals. Lymphocyte cell cultures are exposed to serial dilutions of the irradiated virus. The virus titre is quantified by cytological changes of HIV infection and p24 immunofluorescence. RESULTS: There is a linear relationship between the virus titre and the radiation dose delivered. The inactivation rate of irradiated virus was 0.1134 log10 tissue culture infective doses 50/mL per kGy (95% confidence intervals, 0.1248-0.1020). The irradiation dose required to inactivate the HIV bioburden in allograft bone is 35 kGy. The irradiation dose required to achieve a sterility assurance level of 10(-6) is 89 kGy. This dose exceeds current recommendations for sterilizing medical products and the current practice of many bone banks. CONCLUSIONS: It is concluded that gamma irradiation should be disregarded as a significant virus inactivation method for bone allografts.     

Effects of collagen unwinding and cleavage on the mechanical integrity of the collagen network in bone

The objective of this study was to investigate how molecular level changes in the collagen network affect its mechanical integrity. Our hypothesis is that the cleavage and unwinding of triple helices of collagen molecules significantly reduce the mechanical integrity of the collagen network in bone, whereas collagen crosslinks play a major role in sustaining the structural integrity of the collagen network. To test this hypothesis, the collagen molecular structure was altered in demineralized human cadaveric bone samples in the following two ways: heat induced unwinding and pancreas elastase induced cleavage of collagen molecules. Along with control specimens, the treated specimens were mechanically tested in tension to determine their strength, elastic modulus, toughness, and strain to failure. Also, the percentage of denatured collagen molecules and amounts of two major collagen crosslinks (hydroxylysylpyridinoline and lysylpyridinoline) were determined using high-performance liquid chromatography techniques. It was found that unwinding of collagen molecules may cause more reduction in stiffness (E) but less strain to failure (ef) than cleavage. Both collagen denaturation types cause similar changes in the strength (ss) and work to fracture (Wf) of the collagen network with no significant changes in hydroxylysylpyridinoline and lysylpyridinoline crosslinks. The results of this study indicate that the integrity of collagen molecules significantly affect the mechanical properties of the collagen network in bone, and that collagen crosslinks may play an important role in maintaining the mechanical integrity of the collagen network after collagen denaturation occurs.