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.     

Mechanical strength of cortical allografts with gamma radiation versus ethylene oxide sterilization

We investigated the effects of gamma irradiation versus ethylene oxide (ETO) sterilization on the mechanical strength of cortical bone grafts. Tibias were collected from cadavers of mature goats. Sixty test specimens were randomized into four groups: fresh (no processing), frozen (freezing at -70 degrees C), gamma-irradiated, and ETO-sterilized specimens. Torsion, three-point bending, and compression testing were separately performed with a material testing machine. Parameters studied included maximum stress, strain, deflection, extension, load, shear modulus, and E-modulus. Compared with findings for the fresh specimens, findings were as follows for gamma-irradiated specimens: maximal shear modulus, reduced by 48%; shear stress, by 55%; deflection, by 71%; bending stress, by 51%; bending strain, by 74%; extension, by 60%; and compression strain, by 50%. However, there were no reductions in those parameters for the frozen specimens or the ETO-sterilized specimens. These findings confirm that shear, bending, and compression strength of cortical allografts are weakened by gamma irradiation at room temperature. To maintain optimum mechanical properties, ETO sterilization of allografts is better than gamma sterilization, especially for cortical bone, because it is usually used in load-bearing settings.     

The effect of gamma radiation sterilization on the fatigue crack propagation resistance of human cortical bone

BACKGROUND: Clinical evidence has suggested that the rate of fracture in allografts sterilized with gamma radiation may be higher than that in controls. Gamma radiation sterilization has been shown to affect the post-yield properties of bone but not the elastic modulus. Since most allograft fractures occur with subcritical loads during activities of daily living, it may be that the fatigue properties of irradiated allografts are diminished. In this study, the fatigue crack propagation behavior of cortical bone sterilized with gamma radiation was compared with that of gender and age-matched controls. We hypothesized that gamma radiation significantly reduces the resistance of cortical bone to fatigue crack growth. METHODS: Specimens for fatigue crack propagation testing were machined from four pairs of fresh-frozen human femora obtained from four individuals (a younger male, younger female, older male, and older female donor). Half of the specimens were sterilized with 31.7 kGy of gamma radiation. The specimens were cyclically loaded to failure in a servohydraulic testing system, and crack growth was monitored. The cyclic stress intensity factor and the fatigue crack growth rate were calculated to examine the kinetics of fatigue crack growth. Following testing, the damage zone around the fracture plane was analyzed histologically. RESULTS: The morphology and kinetics of crack growth in irradiated specimens differed from the control data. Overall, the irradiated bone was significantly less resistant to fatigue crack growth than was control tissue (p < 0.05). There was less microdamage associated with fracture in the irradiated specimens than in the control specimens, with the exception of the bone from the older female donor. CONCLUSIONS: Gamma radiation sterilization significantly reduces the fatigue crack propagation resistance of cortical bone. Irradiated specimens also demonstrate a smaller amount of microdamage along the fracture plane. These findings may be due to ultrastructural alterations in the collagen matrix caused by radiation. CLINICAL RELEVANCE: This study suggests that, despite having pre-yield mechanical properties that are similar to those of nonirradiated bone, gamma-radiation-sterilized allograft may be more predisposed to fracture even under the subcritical loads that occur during the activities of daily living.     

同种异体皮质骨板移植免疫学研究

目的: 探讨实验动物和患者接受不同方法处理的同种异体皮质骨板移植后宿主免疫学反应。方法: 48只山羊接受分别经过 -70℃深低温冷冻 4周、 -70℃深低温冷冻 4周 48℃环氧乙烷灭菌、-70℃深低温冷冻 4周 25kGyγ射线辐照处理的同种异体皮质骨板移植,对照组移植异体山羊新鲜皮质骨板。7例髋关节置换术后股骨假体周围骨折患者, 接受深低温冷冻和环氧乙烷处理同种异体皮质骨板移植重建股骨骨折。检测实验动物外周血CD4 、CD8 以及患者外周血T淋巴细胞亚群、抗体、补体和循环免疫复合物。结果: 新鲜骨板移植组CD4 明显升高, 术后 6周达到顶峰。除深低温组术后 6周CD4 升高外, 深低温冷冻、深低温冷冻 环氧乙烷及深低温冷冻 γ射线辐照组T淋巴细胞亚群无明显改变。患者T淋巴细胞亚群、抗体和免疫复合物正常, 切口无感染。结论: 新鲜同种异体皮质骨板具有抗原性, 移植后诱发宿主免疫排斥反应; 深低温冷冻、深冻 环氧乙烷和深冻 γ射线辐照处理同种异体皮质骨板移植受体无明显免疫排斥反应。     

Perforations of cortical bone allografts improve their incorporation

The incorporation of perforated cortical bone allografts was compared with non-perforated allografts. A 5-cm circumferential defect in the middiaphysis at the tibia was created in adult sheep. A frozen tibial allograft was implanted and fixed with a locked nail for 6 months. There was no postoperative immobilization. Group I consisted of eight sheep with non-perforated allografts, whereas Group II was comprised of 10 sheep with perforated allografts. Union was evaluated radiographically, whereas the central part of the allograft had a densitometric evaluation. Creeping substitution was assessed on microradiographs from cross-sections of the central 3 cm of graft by measurement of porosity and percentage of new and old bone area within the confines of the graft. The width of periosteal and endosteal callus also was determined. There was no statistical difference between both groups for the union score and bone density. However, the cortical bone graft porosity and the amount of new bone within the cortical bone differed significantly between the perforated allografts and the non-perforated ones. Periosteal callus was similar in both groups, whereas endosteal callus was significantly more wide and extended in the perforated allografts. Perforation of a cortical bone substantially improved the amount of newly formed bone by the host when compared with a non-perforated bone. The creation of channels seemed to increase the interface between living soft tissues of the host and the allografted bone with a resulting enhanced incorporation.     

Sterilization by gamma radiation impairs the tensile fatigue life of cortical bone by two orders of magnitude.

Cortical bone grafts are utilized frequently for skeletal reconstruction, spinal fusion and tumor surgery. Due to its efficacy and convenience terminal sterilization by gamma radiation is often essential to minimize disease transmission and infection. However, the impairment in the material properties of bone tissue secondary to gamma radiation sterilization is a concern since the mechanical functionality of a bone graft is of primary importance. While the extent of this impairment is well investigated for monotonic loading conditions, there does not seem to exist any information on the effects of gamma radiation sterilization on cortical bone's fatigue properties, the physiologically relevant mode of loading. In this study we investigated the degradation in the high-cycle and low-cycle tensile fatigue lives of cortical bone tissue secondary to gamma radiation sterilization at a dose of 36.4 kGy which approximately falls in the higher end of the standard dose range used in tissue banking. The high-cycle and the low-cycle fatigue tests were conducted under load control at initial strain levels of 0.2% and 0.4%, respectively. Monotonic tensile tests were also conducted to compare the impairment of fatigue properties with the impairment of monotonic properties. Results demonstrated that the impairment in both the high-cycle and the low-cycle fatigue lives were two orders of magnitude following sterilization, a change much more pronounced than that observed for monotonic loading. In conclusion, the results suggest that the impairment of the mechanical function of gamma radiation sterilized allografts is even worse in fatigue than monotonically. Therefore, grafts should be designed to minimize functional strains and avoid stress raisers to prevent premature fatigue failures.     

Infection in massive bone allografts sterilised by radiation

Summary Massive bone allografts sterilised by radiation have been used in our hospital since 1985. The 164 consecutive reconstructions which we carried out before December 1990 were in equal part for tumour resections and revision arthroplasties associated with major bone defects. The allografts were used as intercalary or composite graft-prosthesis reconstructions. Fourteen (8.5%) skin sloughs or infections occurred, all but one after operations for malignant tumours. The grafts never appeared to be responsible. Infection after malignant tumour resection most commonly occurred at the knee when preoperative radiation had been used. Twenty-five reconstructions were performed in the presence of infection, which only recurred in one case. Twelve infections were treated, but 2 other patients died for other reasons within a year. One patient had to have an amputation and one had persistent infection. In the other cases, the infection healed. Only two debridements alone were successful. Removal of the graft and replacement with antibiotic-loaded cement was the most effective treatment.

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Résumé Les auteurs ont utilisé depuis 1985 des allogreffes massives radio-stérilisées. Des 164 reconstructions successives faites jusqu'à Décembre 1990, la moitié concernait des tumeurs et l'autre des reprises de prothèses avec dégâts osseux majeurs (excluant les petites reconstructions faites avec des têtes fémorales de banque). Il s'agissait de reconstructions intercalaires ou composites (allogreffe-prothèse). Quatorze nécroses cutanées ou infections ont été observées (8,5%). Toutes concernaient des reconstructions pour tumeurs malignes, sauf une, survenue après révision d'une prothèse initialement septique. Les greffes n'ont jamais paru être la cause de l'infection. Le risque septique s'est avéré plus important après résection tumorale et pourtant le rôle de la chimiothérapie n'a pu être prouvé. Des lambeaux de couverture auraient dû éviter les nécroses cutanées qui sont toutes survenues au genou. Vingt-cinq reconstructions ont été faites alors qu'il existait des antécédents d'infection; une seule a récidivé. Douze infections ont été traitées (deux malades étant décédés au cours de la première année d'une cause non infectieuse). Un patient ayant reçu une radiothérapie pré-opératoire a dû être amputé et une infection persiste après un nettoyage simple. Dans les autres cas, l'infection est apparemment guérie, le plus souvent après ablation de la greffe, remplacée temporairement par du ciment imprégné d'antibiotiques, avant reconstruction osseuse secondaire.

     

同种异体皮质骨板移植治疗股骨假体周围骨折

目的 评价同种异体皮质骨板移植治疗髋关节股骨假体周围骨折的临床治疗效果。方法 对7例髋关节置换术后股骨假体周围骨折患者应用深低温冷冻和环氧乙烷处理同种异体皮质骨板移植治疗股骨骨折。根据Harris评分,外周血T淋巴细胞亚群、抗体和免疫复合物检测、X线摄片以及核素骨显像评价其治疗效果。结果 患者未发生免疫排斥反应和感染,术后3、6、12、24个月Harris评分分别比术前平均增加21、32、40、40分。术后3个月骨折愈合,一部分移植骨板与宿主骨骨性连接,同位素骨显像骨板移植区放射性分布比对侧浓集。术后12个月,85％移植骨板与宿主骨融为一体,宿主股骨皮质厚度增加3～5mm,平均4．3mm,骨板放射性浓集进一步加深,15％移植骨板出现吸收现象。术后24个月,80％移植骨板重新塑形后恢复宿主股骨皮质骨密度,10％骨板表面吸收停止,股骨假体无松动。结论 深低温冷冻和环氧乙烷处理同种异体皮质骨板移植无菌、抗原性弱、生物相容性好,移植骨板能够维持骨折和假体稳定性,增加股骨局部骨量储备,避免再次手术取出金属内置物,是用来治疗股骨假体周围骨折较理想的方法之一。     

The mechanical and biological properties of demineralised cortical bone allografts in animals

We studied the calcium content and mechanical strength of cortical bone from rats and dogs after different periods of demineralisation, showing that the rate of demineralisation differed considerably between the species. Specimens from the rat were further treated by chemical extraction and autolysis and tested for osteoinductive properties. We showed that partially demineralised cortical bone retained adequate mechanical strength, while retaining the biological effects of completely demineralised bone. This shows that it is possible to prepare allografts which have adequate mechanical strength and still retain osteo-inductive properties.     

The bacteriology of bone allografts.

We report the contamination rate in the Cambridge bone bank of 35 consecutive allograft specimens, all harvested in a clean-air environment, using a strict aseptic technique and antibiotic cover. Five of 27 femoral heads taken from living donors and three of eight massive allografts taken from cadavers were found to be contaminated. The contaminated femoral heads were discarded. All massive allografts were rendered sterile by gamma-irradiation. It is important to exclude bacteriological contamination of harvested and banked bone.     

Morphometric and physical investigations of segmental cortical bone autografts and allografts in canine ulnar defects.

Cortical bone grafts were implanted for six months in mature dogs using an osteoperiosteal 3-cm defect in the ulna to evaluate their respective morphometric and physical values compared with autografts. The bone-grafting material included fresh auto- and allografts, frozen and thimerosal preserved allografts, and partially demineralized bone allografts. The grafts were evaluated by roentgenograms, microradiograms, photon absorptiometry, porosity, fluorescence labeling measurements, and torsional loading at failure. Autografts achieved a better union score than the allografts, but intracortical bone porosity, percentage of cumulative new bone, and mineral apposition rate were not variables with statistical significance. Lamellar bone was found earlier and in greater quantity in autografts. Within the graft, new bone was deposited at a slower rate than in the recipient bone. Autografts showed less peripheral resorption and a greater torsional resistance than allografts. Photon absorptiometry demonstrated that nondemineralized allografts underwent a substantial loss of peripheral bone. This marked reduction in the outer diameter of the graft had more influence on torsional resistance than did the intracortical porosity of the graft. Demineralized allografts were osteoinductive in only 28% of the cases and appeared to respond in an all-or-nothing pattern. Frozen and thimerosal preserved allografts were the most acceptable substitutes to autografts.     

异体皮质骨板在股骨髁部粉碎性骨折内固定术中的应用

目的探讨异体皮质骨板股骨内侧辅助固定结合外侧解剖型钢板内固定治疗股骨髁部粉碎性骨折的临床疗效。方法自2006-03—2013-06采用异体皮质骨板股骨内侧辅助固定结合外侧解剖型钢板内固定治疗股骨髁部粉碎性骨折及该部位术后骨不连、钢板断裂及感染病灶清除术后骨缺损33例。结果本组33例均获平均32(12~69)个月随访。骨折均愈合,平均愈合时间7.6(3~18)个月。无感染、内固定物松动、再骨折、免疫排斥反应等并发症发生。影像学资料示术后6个月异体骨板边缘变圆钝,异体骨板与宿主骨之间存在部分间隙,术后12个月异体骨板边缘进一步变薄、变圆,异体骨板两端与股骨之间间隙消失,可见边缘与宿主骨愈合征象。按Merchan等疗效评定标准进行膝关节功能评定:优16例,良12例,可4例,差1例,优良率84.8%。结论应用异体皮质骨板股骨内侧辅助固定结合外侧解剖型钢板内固定治疗股骨髁部粉碎性骨折,增加了股骨内侧的支撑力度,提高了固定的稳定性,术后可以早期膝关节功能锻炼,减少钢板的疲劳断裂。     

Value of multiple cortical perforations for the rehabilitation of massive deep-frozen bone allografts. Experimental study in sheep]

Incorporation of massive cortical bone allografts in human is slow and remains incomplete. Late biopsies of implanted allografts or histological studies of explanted allografts always show the partial substitution of necrotic bone by new bone from the host. The aim of the present study was to evaluate the effect of drilling the massive deep-frozen cortical allografts in order to induce osteogenesis. Thirteen sheep were operated on and a standard segment of the proximal ulna was removed and the gap filled either by an unperforated allograft or by a perforated one. Based on histological and microradiographic examination, a quite complete substitution of the perforated allografts was observed but in this model no statistically significant difference was observed between perforated and unperforated allografts. Further study is needed to assess the effect of the perforations.     

Graft perforations favor osteoinduction. Studies of rabbit cortical grafts sterilized with ethylene oxide.

The healing of freeze-dried, ethylene oxide sterilized, segmental, allogenic cortical bone grafts was investigated in 15 rabbits using a 2-cm ulnar diaphyseal defect. Five different groups of bone grafts were evaluated: 1) unperforated undemineralized, 2) perforated undemineralized, 3) unperforated demineralized, 4) perforated demineralized, and 5) perforated demineralized grafts enclosed by silicone rubber (Silastic) sheets. There were 3 animals in each group. At 18 days, the study was terminated, and the implants were examined using radiographs and qualitative histologic preparations. We observed that healing of perforated demineralized bone was superior to unperforated demineralized bone, that undemineralized bone was partially sequestered in reactive lacunae, and that perforations in demineralized bone became centers of osteoinduction. Demineralized bone sterilized with ethylene oxide by this method vigorously formed new bone.     

Immunological responses to bone soluble proteins in recipients of bone allografts.

The significance of an immune response in complications of bone allograft procedures is not well understood. This study evaluates the immunological reaction to bone allografts in either cortical or cancellous form. Serological responses from osteoarthritis (OA) patients and normal individuals to soluble proteins extracted from allografts were assessed using Western blotting and ELISA techniques. A large number of patients expressed antibodies to bone proteins extracted from the washed bone as compared with normal controls. Antibodies were present in patients without the use of bone allografts, indicating that OA patients develop antibodies to bone soluble proteins. However, patients receiving allografts exhibited an increased immune reactivity against multiple bone proteins when compared with non-grafted patients. Protein characterization of the immunoreactive proteins revealed that the majority of antigenic targets were fragments of various collagen molecules. The data suggests that OA patients develop antibodies to bone soluble proteins prior to surgery, and these antibodies increase after revision surgery utilizing bone allografts. These findings support the implications that various collagen molecules as well as their degraded fragments represent potential immunogenic proteins within bone allografts. The removal of these antigenic proteins from the allografts prior to surgery may alleviate this immunological reactivity and improve graft outcome.