Irradiation has no effect on the incorporation of impacted morselized bone: a bone chamber study in goats

Background Gamma irradiation has been widely used for sterilization of bone allografts. However, gamma irradiation alters proteins. This is favorable when it reduces immunogenicity, but is undesirable when osteoinductive proteins are damaged. Although the effect of gamma irradiation on BMPs has been studied, the effect of irradiation on the process of incorporation of morselized bone chips remains unclear. We studied the effects of sterilization by gamma irradiation on the incorporation of impacted morselized allografts.

Methods Bone chambers with impacted allografts, rinsed impacted allografts, allografts that were rinsed and subsequently irradiated, and an empty control were implanted in proximal medial tibiae of goats. Incorporation was evaluated using histology and histomorphometry.

Results Histology revealed evidence of bone graft incorporation, which proceeded in a similar way in unprocessed, rinsed, and both rinsed and irradiated bone grafts. After 12 weeks, no difference in bone and tissue ingrowth was found between the unprocessed, the rinsed, and the rinsed and subsequently irradiated allografts. The amount of unresorbed graft remnant was highest in the unprocessed bone grafts.

Interpretation We conclude that sterilization with gamma irradiation does not influence the incorporation of impacted rinsed bone allografts.     

Experimental study of the thermic effect on bone at 60 degrees C, as applied to bone allograft]

Bone reconstruction methods increasingly often require using bank bone. These massive bone fragments can be taken only from dead subjects or those in irreversible coma. Surgical sterility of the samples does not always guarantee the absence of an HIV infection, for which seroconversion often occurs very late. B. Spire's work has shown the effectiveness of a 30-minute heat treatment at 56 degrees C to inactivate HIV in blood products. Our study has therefore evaluated the effectiveness of a heat treatment method for bone to inactivate HIV on one hand, and the mechanical and histologic consequences of this treatment on rabbit bone. All bone fragments in this study were frozen at -80 degrees C to reproduce the same conditions of use as in current bone banks. Heating deep-frozen fragments of spongious tissue and of bone marrow from seropositive subjects in a 60 degrees C humid heat allowed confirming thermal sterilization of HIV, but a greater number of case is required to support this technique, as well as a verification with cortical bone. Thermal sterilization of bone allografts does not alter the mechanical properties nor the possibilities of bone regrowth in allografts. Application to human bone allografts should be confirmed by a greater number of cases, but it appears as a simple means to suppress the current disadvantage of late seroconversion control.     

几种制备因素对同种骨移植物的生物力学特性的影响

本文研究了超声清洗,冷冻干燥和Co~(60)r射线辐照灭菌几项制备因素对同种骨移植物的生物力学特性的影响。结果表明,实验组和对照组之间无显著性差异,即这些制备因素对同种骨移植物的生物力学特性无影响。     

深度冷冻大段同种骨的制备及应用

目的：建立现代骨组织库，观察深度冷冻大段同种骨移植的临床效果。方法：采用深度冷冻、物理脱脂、真空包装和辐照灭菌等现代技术处理人类大段骨组织，临床应用24例，包括创伤性肢体骨重建、半侧骨盆重建、半侧骶骨移植、四肢大段骨或骨关节移植等，观察近期临床结果。结果：骨移植材料的无菌检测合格，生物力学强度无明显下降，可远程运输和短期保存．全部病例无明显的免疫排斥反应，成功率为100％.结论：深度冷冻大段同种骨临床使用安全可靠，保存和运输方便.     

冷冻干燥同种骨临床应用的初步报告

目的：建立现代骨组织库，观察系列冷冻干燥同种骨移植的临床效果。方法：采用深度冷冻、真空冷冻干燥和辐照灭菌等现代技术处理人类骨组织，临床应用系列冻干骨94例，包括骨缺损填充、骨不连植骨、脊柱和关节融合、人工关节翻修、髋臼造盖等，观察近期临床结果。结果：冻干同种骨的理化性能、无菌检测和生物性能完全符合体内植入物的相关国家标准，常温下可保存2年。94例中的90例无明显的免疫排斥反应，优良率为95．76％，成功率为97．87％。结论：冷冻干燥同种骨使用安全，保存和运输方便，可替代临床自体骨移植。     

Radiation-sterilized insoluble collagenous bone matrix is a functional carrier of osteogenin for bone induction

Summary The influence of gamma radiation on the role of the collagenous substratum as a carrier for proteins which cause bone induction was examined. Osteoinductive demineralized bone matrix was extracted by 4 M guanidinium hydrochloride. The insoluble collagenous bone matrix (ICBM) obtained was not osteoinductive; however, when reconstituted with partially purified osteogenin, bone induction was restored. In order to apply the principle of bone induction to clinical use, methods of sterilization must be optimized to maintain the osteoinductive activity of bone allografts. The inactive substratum was irradiated and reconstituted with an active, partially purified bone extract and bioassayed. Irradiation of the ICBM by a Cobalt 60 source at a dose of 1 and 3 Mrads had no deleterious effect on the functional role of the substratum.     

Validation of 11 kGy as a Radiation Sterilization Dose for Frozen Bone Allografts

A radiation sterilization dose (RSD) of 25 kGy is deleterious to bone allografts. This study aimed to establish a lower RSD for bone allografts using method 1 of International Standard Organisation 11137.2:2006. This provides a database to select an RSD corresponding to an allograft's bioburden, given that the bioburden's gamma resistance is equal to or less than the standard. This can be verified by irradiating 100 allografts at a dose selected to provide a sterility assurance level of 10⁻². The bioburden of our allografts was 0, which prescribed a verification dose of 1.3 kGy. After irradiating 100 allografts, sterility tests returned no positive cultures. We therefore validated an RSD of 11 kGy for allografts with that bioburden. According to the standard, this RSD provides a sterility assurance level of 10⁻⁶ for bone allografts.     

Fracture resistance of gamma radiation sterilized cortical bone allografts.

Gamma radiation is widely used for sterilization of human cortical bone allografts. Previous studies have reported that cortical bone becomes brittle due to gamma radiation sterilization. This embrittlement raises concern about the performance of a radiation sterilized allograft in the presence of a stress concentration that might be surgically introduced or biologically induced. The purpose of this study was to investigate the effect of gamma radiation sterilization on the fracture resistance of human femoral cortical bone in the presence of a stress concentration. Fracture toughness tests of specimens sterilized at a dose of 27.5 kGy and control specimens were conducted transverse and longitudinal to the osteonal orientation of the bone tissue. The formation of damage was monitored with acoustic emission (AE) during testing and was histologically observed following testing. There was a significant decrease in fracture toughness due to irradiation in both crack growth directions. The work-to-fracture was also significantly reduced. It was observed that the ability of bone tissue to undergo damage in the form of microcracks and diffuse damage was significantly impaired due to radiation sterilization as evidenced by decreased AE activity and histological observations. The results of this study suggest that, for cortical bone irradiated at 27.5 kGy, it is easier to initiate and propagate a macrocrack from a stress concentration due to the inhibition of damage formation at and near the crack tip.     

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.     

Nonallograft osteoconductive bone graft substitutes

An estimated 500,000 to 600,000 bone grafting procedures are done annually in the United States. Approximately (1/2) of these surgeries involve spinal arthrodesis whereas 35% to 40% are used for general orthopaedic applications. Synthetic bone graft substitutes currently represent only 10% of the bone graft market, but their share is increasing as experience and confidence in their use are accrued. Despite 15 to 20 years of clinical experience with various synthetic substitutes, there have been few welldesigned, controlled clinical trials of these implants. Synthetic bone graft substitutes consist of hydroxyapatite, tricalcium phosphate, calcium sulfate, or a combination of these minerals. Their fabrication technique, crystallinity, pore dimensions, mechanical properties, and resorption rate vary. All synthetic porous substitutes share numerous advantages over autografts and allografts including their unlimited supply, easy sterilization, and storage. However, the degree to which the substitute provides an osteoconductive structural framework or matrix for new bone ingrowth differs among implants. Disadvantages of ceramic implants include brittle handling properties, variable rates of resorption, poor performance in diaphyseal defects, and potentially adverse effects on normal bone remodeling. These inherent weaknesses have refocused their primary use to bone graft extenders and carriers for pharmaceuticals. The composition, histologic features, indications, and clinical experience of several of the synthetic bone graft substitutes approved for orthopaedic use in the United States are reviewed.     

Immune response to perforated and partially demineralized bone allografts

Immune responses have been shown to be involved in the pathogenesis of clinical complications of cortical bone allografts. In an attempt to reduce the immunogenicity of these allografts, we evaluated cortical bone allografts modified by laser perforation and partial demineralization transplanted orthotopically into sheep tibiae. The recipient animals were divided into three groups, of eight animals each, according to the type of cortical allograft that was transplanted: group 1, no treatment (control); group 2, demineralization only; and group 3, laser perforation and partial demineralization. All animals were tissue-typed by biochemical definition of MHC class I molecules, using unidimensional isoelectric focusing and Western blotting. Mismatches of donors and recipients were assessed by testing samples of each donor and recipient pair in parallel and by comparing their individual bands. Donor-specific alloantibodies were detected by a similar technique, using an enzyme-linked immunosorbent assay (ELISA) format. Negative controls were included in all tests. All grafts were poorly immunogenic, whether they were untreated, processed by partial demineralization, or processed by both laser perforation and partial demineralization. Only two recipient animals showed a transient, antibody-mediated donor-specific immune response. One of these animals had received a control allograft, whereas the other animal had received a laser-perforated and partially demineralized bone allograft. All of the grafts in this study, including control grafts, were stripped of soft tissues and their bone marrow was removed; cellular sources of alloantibody stimulation may have been eliminated by these processes. The results of this study suggest that immune responses to bone allografts may be reduced by removing the bone marrow and adjacent soft tissues. The processing of cortical bone allografts by laser perforation and partial demineralization appeared to have little effect on immune responses.     

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.     

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.     

Ethylene oxide sterilization of bone grafts: Residual gas concentration and fibroblast toxicity

We examined the concentration of ethylene oxide in bone allografts after gas sterilization. Chips of the human femoral head were investigated. Residual gas concentration was determined by gas chromatography after the bone chips had been subjected to defatting and freeze-drying, followed by ethylene oxide gas sterilization. Bones were prepared in various ways in an attempt to reduce the concentration of residual ethylene oxide. The concentration was higher when gas sterilization was performed before freeze-drying than when it was done afterwards. An experiment performed with fibroblasts showed the high toxicity of residual ethylene oxide in bone chips, even when the concentration was very low. The growth of fibroblast was reduced more in medium which had been shaken with bones sterilized with ethylene oxide before freeze-drying than in medium which had been shaken with bones sterilized after freeze-drying. The higher residual ethylene oxide concentrations resulted in a decrease in fibroblastic culture activity. Our experiment showed the importance of reducing the residual ethylene oxide gas concentration. Defatting and freeze-drying result in lower residual ethylene oxide concentrations.     

Frozen musculoskeletal allografts. A study of the clinical incidence and causes of infection associated with their use

A retrospective study was performed to determine the clinical incidence and causes of infection related to the use of frozen musculoskeletal allografts. The results of this study of 324 grafts, prepared and supplied by our hospital bone bank, showed that the patients in whom femoral head grafts and other small bone and soft-tissue allografts were used had a negligible clinical incidence of infection. The incidence of infection related to the use of large allografts, such as osteoarticular or diaphyseal grafts, was approximately 5 per cent in patients who had treatment for a bone tumor and 4 per cent in those who had revision of a hip arthroplasty. These rates of infection were not substantially different from those that have been reported in similar series in which large allografts or sterilized prosthetic devices were used. The causes of the infections were difficult to determine, but contamination of the allograft was probably not a factor in most patients.     

Natural history of autografts and allografts

The clinical outcome of bone grafting procedures depends on many factors, including type and fixation of the bone graft as well as the site and status of the host bed. Bone grafts serve one or both of two main functions, as a source of osteogenetic cells and as a mechanical support. Autografts, both cancellous and cortical, are usually implanted fresh and are often osteogenetic, whether by providing a source of osteoprogenitor cells or by being osteoinductive. The latter is a process whereby the transplanted tissue induces mesenchymal cells of the recipient to differentiate into osteoblastic cells. Cortical grafts, whether autogeneic or allogeneic, at least initially act as weight-bearing space fillers or struts. All bone grafts are initially resorbed, but cancellous grafts are completely replaced in time by creeping substitution, while cortical grafts remain an admixture of necrotic and viable bone for a prolonged period of time. The three-dimensional framework, which supports invasion of the bone grafts by capillaries and osteoprogenitor cells, termed "osteoconduction", is another important function of both autografts and allografts. Fresh allografts are more slowly and less completely replaced by host bones because they invoke both local and systemic immune responses that diminish or destroy the osteoinductive and conductive processes. Although freezing or freeze-drying of allografts improves acceptance, their failure rate is still too high. These processes are also influenced by the vascularity and composition of the host bed. Thus, the interaction of the host and the bone graft determines the success of these procedures, which ultimately is to provide a mechanically efficient support structure.     

Immune responses to osteochondral allografts: nature and significance.

Osteochondral allografts evoke immune responses. The nature of these immune responses and their biologic significance are still only partially understood. It is clear, however, that cell surface antigens of the major histocompatibility complex represented on the cellular elements of bone grafts cause T-cell activation, specifically those of the suppressor/cytotoxic phenotype. In numerous animal models, the most immunogenic bone allografts (mismatched, fresh) have demonstrated the poorest clinical and biologic outcomes, while more closely matched and/or grafts treated to reduce immunogenicity (frozen, freeze-dried) have more successfully incorporated. These observations support the hypothesis that immune responses against bone-graft related antigens have biologic significance and that reducing these responses may improve clinical results.     

Influence of aeration, storage, and rinsing conditions on residual ethylene oxide in freeze-dried bone allograft

Bone allografts sterilized with ethylene oxide gas (EO) are used in the field of orthopedic surgery, and the reduction of the EO residual concentration is an urgent clinical matter. We therefore investigated the efficacy of aeration and the effects of varied preservation periods and rinsing conditions on the reduction of EO residuals in freeze-dried bone allografts in the present study. Before aeration, the EO residual level was 12.6 ppm, and, after the repeating of aeration at 60°C once, two times, and three times, the level decreased to 10.9 ppm, 3.1 ppm, and 0.47 ppm, respectively. Regarding the duration of preservation at room temperature, the mean EO residual level was 10.5 ppm, 4.9 ppm, and 4.6 ppm, 1, 2, and 3 weeks after EO sterilization, respectively. By rinsing with physiological salt solution, the level was decreased to 6.9 ppm by 5-min rinsing with 100 ml. Rinsing with 500 ml of this solution decreased the levels to 3.9 ppm, 2.8 ppm, and 2.0 ppm when done for 1, 5, and 10 min, respectively. Rinsing with 2000 ml of this solution decreased the levels to 3.6 ppm, 2.6 ppm, and 1.7 ppm when done for 1, 5, and 10 min, respectively. These experimental results with chip bone allografts lead us to recommend repeated preoperative aeration and more than 2 weeks' preservation before use for reducing the residual EO concentration. It was also evident that intraoperative rinsing with 500 ml of physiological saline for 10 min reduced the EO residual level. Received: June 13, 2001 / Accepted: November 5, 2001     

大段同种异体骨移植愈合的实验研究

目的　研究大段同种异体骨移植愈合过程和特点。方法　采用新西兰大白兔股骨中段切除 1 5cm骨干和骨膜实验动物模型。将 36只兔随机分成实验组和对照组。实验组植入深低温保存的同种异体骨 ,对照组植入自体新鲜骨 ,均用直径 3mm的髓内针固定。于术后第 1、 2、 3个月分别行X线摄片、ECT、大体标本、组织学观察 (四环素荧光标记、HE染色 )以及BMP免疫组织化学染色。结果　深低温保存的同种异体骨移植愈合与自体骨相似 ,ECT显示术后第 1个月骨代谢活跃 ,2、 3个月趋于稳定。移植骨BMP表达阴性 ,新生骨及其周围类基质表达阳性。其愈合是从宿主骨向移植骨 ,从周围向中央 ,从哈佛氏管向其四周逐渐进行爬行替代的过程。结论　同种异体骨移植愈合过程是移植骨全方位活化的过程 ,即全方位再血管化、新骨形成和宿主与移植骨接连的过程。其主要通过骨传导实现成骨 ,骨诱导亦发挥积极作用