Radioprotection provides functional mechanics but delays healing of irradiated tendon allografts after ACL reconstruction in sheep

Successful protection of tissue properties against ionizing radiation effects could allow its use for terminal sterilization of musculoskeletal allografts. In this study we functionally evaluate Achilles tendon allografts processed with a previously developed radioprotective treatment based on (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) crosslinking and free radical scavenging using ascorbate and riboflavin, for ovine anterior cruciate ligament reconstruction. Arthroscopic anterior cruciate ligament (ACL) reconstruction was performed using double looped allografts, while comparing radioprotected irradiated and fresh frozen allografts after 12 and 24 weeks post-implantation, and to control irradiated grafts after 12 weeks. Radioprotection was successful at preserving early subfailure mechanical properties comparable to fresh frozen allografts. Twelve week graft stiffness and anterior-tibial (A-T) translation for radioprotected and fresh frozen allografts were comparable at 30 % of native stiffness, and 4.6 and 5 times native A-T translation, respectively. Fresh frozen allograft possessed the greatest 24 week peak load at 840 N and stiffness at 177 N/mm. Histological evidence suggested a delay in tendon to bone healing for radioprotected allografts, which was reflected in mechanical properties. There was no evidence that radioprotective treatment inhibited intra-articular graft healing. This specific radioprotective method cannot be recommended for ACL reconstruction allografts, and data suggest that future efforts to improve allograft sterilization procedures should focus on modifying or eliminating the pre-crosslinking procedure.     

Indonesia: Statistical Sampling Technique in Validation of Radiation Sterilisation Dose of Biological Tissue

The aim of the work is to find the best solution for statistical sampling technique in validation of radiation sterilization dose (RSD) for biological tissues, according to ISO standard. As a model for sampling are biological tissues retrieved from one cadaver donor which consist of frozen bone grafts (18 packets), lyophilized allografts (68 packets) and demineralized bone powder grafts (40 packets). The size and type of products vary from long bones, cancellous chips to bone powders, tendons and facia lata, that make the number of bioburden per product could not be treated equally. Frozen samples could not be considered as the same production batch with lyophilized samples, because of different processing and irradiation temperature. The minimum number of uniformed samples needed for validation per production batch size, according to ISO 13409, is from 20 to 79 and 20 of them will be used for the test sample size, i.e. 10 for bio-burden determination and the remaining 10 for verification dose. Based on the number of uniformed grafts, statistical sampling can be carried out on lyophilized and demineralized bone grafts, but not on frozen bone grafts. Bioburden determinations were carried out and validated according to ISO 11737-1. Results of average bioburden determination (cfu/per packet), using sample item portion (SIP) = 1, are 5 cfu/packet for lyophilized bone grafts and 4 cfu/packet for demineralized bone powder grafts. Verification doses obtained were 2.40 kGy for lyophilized grafts and 2.24 kGy for demineralized bone powder grafts. The results of verification dose were accepted and the RSD of 25 kGy is substantiated It can be concluded that a statistical sampling technique can be applied if all the grafts produced in the same process such as lyophilized, demineralized as well as frozen are assumed to be in one production batch regardless of sample uniformity such as size, type and weight; for this ISO 13409 can be applied for the validation of RSD.     

The use of deep frozen and irradiated bone allografts in the reconstruction of tibial plateau fractures

To investigate the clinical behavior of deep frozen and irradiated bone allografts in the treatment of depressed tibial plateau fractures. Twenty-two patients with a tibial plateau fracture were treated with cancellous bone allografts. The bone allograft preparation process included fresh-freezing at ?70 °C for 4 weeks and gamma-irradiation at 25 kGy. All of the patients were followed for 1–2 years. The clinical effects were assessed using the Rasmussen score for tibial head fractures and X-rays. Postoperatively, the average excellent and fair Rasmussen scores were 88.9 %. Only one patient developed an infection, with no integration between allograft and recipient bone observed. All of the other bone allografts were incorporated successfully, and no osteoporosis or sclerosis was observed. The frozen and gamma-irradiated bone allograft is a good alternative in the treatment of tibial plateau fractures, which we have shown can integrate with the surrounding host bone.     

Development of a bacteriophage model system to investigate virus inactivation methods used in the treatment of bone allografts

Bone allografts are commonly used in a variety of surgical procedures, to reconstruct lost bone stock and to provide mechanical support during the healing process. Due to concerns regarding the possibility of disease transmission from donor to recipient, and of contamination of grafts during retrieval and processing procedures, it is common practice to sterilise bone allografts prior to issue for clinical use. It is vital that the sterilisation processes applied to allografts are validated to demonstrate that they achieve the required level of bioburden reduction, and by extension that validated models are used for these studies. Two common sterilisation protocols applied to bone allografts are gamma irradiation and ethylene oxide gas sterilisation, and there are currently no validated models available for measuring the anti-viral efficacy of ethylene oxide treatment with regard to bone allografts or readily useable models for assessing the anti-viral efficiency of gamma irradiation treatment. We have developed and validated models for both these sterilisation processes, using the bacteriophage ϕ×174, and utilised the models to measure the antiviral activity of the standard ethylene oxide and gamma irradiation sterilisation processes applied to bone allografts by the National Blood Service. For the irradiation model, we also utilised bacterial spores (Bacillus pumilus). Our results show that ethylene oxide sterilisation (which can only be applied to lyophilised grafts) inactivated >6.1log₁₀ of the model virus, and gamma irradiation (at 25–40 kGy and applied to frozen allografts) inactivated 3.6–4.0log₁₀ of the model virus and >4log₁₀ of the bacterial spores. Gamma irradiation at this dosage is therefore not in itself a sterilisation process with respect to viruses.     

Very long-term radiographic and bone scan results of frozen autograft and allograft bone grafting in 17 patients (20 grafts) a 30- to 35-year follow-up

In the early 1950s, 48 patients received bone implants from a bone bank in Tel-Hashomer Hospital that stored frozen autograft and allograft bones at temperatures less than -17 degrees C. Seventeen (35%) of these patients (20 implants), 10 men and 7 women, with a mean age of 52.4 (34-69) years were available for follow-up after a mean period of 32.5 (30-35) years. They underwent clinical examination, radiographs and bone scans to evaluate their surgical results. Fracture healing, non-union, graft resorption, osteoporosis and bone sclerosis were used as radiographic criteria for bone incorporation, and normal, increased and decreased uptake served to assess the bone scan. Based on the above criteria, the results were satisfactory in 17 (85%) and poor in 3 (15%). The three failures were after shelf operation for hip dysplasia that used two allografts and one autograft. Allogenous or a combination of allogenous with autogenous frozen bone grafts proved to be a satisfactory and durable method for filling bone defects.     

Freeze-dried microarterial allografts

Rehydrated freeze-dried microarterial allografts were implanted to bridge arterial defects using New Zealand White rabbits as the experimental model. Segments of artery from the rabbit ear and thigh were harvested and preserved for a minimum of 2 weeks after freeze-drying. These allografts, approximately 1 mm in diameter and ranging from 1.5 to 2.5 cm in length, were rehydrated and then implanted in low-pressure and high-pressure arterial systems. Poor patency was noted in low-pressure systems in both allografts and autografts, tested in 12 rabbits. In the high-pressure arterial systems, allografts that were freeze-dried and reconstituted failed in a group of 10 rabbits with an 8-week patency rate of 30 percent. Gamma irradiation in an effort to reduce infection and antigenicity of grafts after freeze-drying was associated with a patency rate of 10 percent at 8 weeks in this system in another group of 10 rabbits. Postoperative cyclosporin A therapy was associated with a patency rate of 22.2 percent in the high-pressure arterial system in a 9-rabbit group. Control autografts in this system in a group of 10 rabbits showed a 100 percent patency at 8 weeks. Microarterial grafts depend on perfusion pressure of the vascular bed for long-term patency. Rehydrated freeze-dried microarterial allografts do not seem to function well in lengths of 1 to 2.5 cm when implanted in a high-pressure arterial system. Freeze-dried arterial allografts are probably not antigenic.     

Lyophilized allogeneic bone tissue as an antibiotic carrier

The rising number of primary joint replacements worldwide causes an increase of revision surgery of endoprostheses due bacterial infection. Revision surgery using non-cemented implants seems beneficial for the long-term outcome and the use of antibiotic-impregnated bone grafts might control the infection and give a good support for the implant. In this study we evaluated the release of antibiotics from fresh-frozen and lyophilized allogeneic bone grafts. Lyophilized bone chips and fresh frozen bone chips were mixed with gentamicin sulphate, gentamicin palmitate, vancomycin, calcium carbonate/calcium sulphate impregnated with gentamicin sulphate, and calcium carbonate/calcium sulphate bone substitute material impregnated with vancomycin. The efficacy of each preparation was measured by drug release tests and bacterial susceptibility using B. subtilis, S. aureus and methicillin-resistant Staphylococcus aureus. The release of gentamicin from lyophilized bone was similar to the release rate from fresh frozen bone during all the experimental time. That fact might be related to the similar porosity and microstructure of the bone chips. The release of gentamicin from lyophilized and fresh frozen bone was high in the first and second day, decreasing and keeping a low rate until the end of the second week. Depending on the surgical strategy either polymethylmethacrylate or allogeneic bone are able to deliver sufficient concentrations of gentamicin to achieve bacterial inhibition within two weeks after surgery. In case of uncemented revision of joint replacements allogeneic bone is able to deliver therapeutic doses of gentamicin and peak levels immediately after implantation during a fortnight. The use of lyophilized and fresh frozen bone allografts as antibiotic carriers is recommended for prophylaxis of bone infection.     

The differentiation of bone marrow cells to functional T lymphocytes following implantation of thymus grafts and thymic stroma in nude and AT×BM mice

Cardiac allografts were used to compare the immunologic capacity of nude mice and adult, thymectomized, lethally irradiated, bone marrow-reconstituted (AT × BM) mice. Neither nude nor AT × BM mice were able to reject cardiac allografts of any party. However, both rejected grafts of any party following implantation of neonatal thymus or thymus from 3-week-old syngeneic mice. Irradiated syngeneic thymus grafts (800 R) were equally effective in restoring host responsiveness against allografts. In contrast, allogeneic thymus grafts restored the capacity to reject second-party heart grafts only in AT × BM mice. Second-party grafts persisted indefinitely when placed on nude mice implanted with an allogeneic, unirradiated thymus graft. Third-party grafts transplanted 17 weeks after reconstitution, however, were rejected. Irradiated nude mice given normal littermate bone marrow and simultaneously grafted with second-party thymus and heart allografts also failed to reject their second-party heart grafts. The difference in ultimate capacity to respond between AT × BM and nude mice suggests that a maturational defect exists in the nude mouse enviroment which impedes development of precursor T lymphocytes.     

Preserved Tissue Allografts in Reconstructive Surgery

Results of treatment with three various kinds of allografts: lyophilized bone, deep frozen bone and cartilage preserved in physiological solution, all of them radiation-sterilized are presented. We believe that this presentation may be helpful in estimating the tissue bank's allografts and in establishing indications and contraindications in the application of allografts in surgery. The ‘indices of coincidence’ were compared in a group of 1014 patients after bone (lyophilized and radiation-sterilized) transplantation. It seems that such a variable as ‘rebuilding of graft’ may be of prognostic value in analysing the results of treatment in this group. The application of frozen and radiation-sterilized allogenic bone grafts for reconstructions is also described. An analysis of the results of treatment in 1125 patients reveals that the use of preserved bone reduces the extent and duration of surgery. Almost total substitution of grafts may be seen in 3–8 months after surgery. Allogenic, preserved cartilage is often used in facial reconstructions of face. Human costal cartilage, preserved in 0.9% NaCl and radiation-sterilized, was used for reconstruction. The patients were examined 24–190 months after surgery (in several clinical units) and results were collected in a special questionnaire by the team that performed surgery. In an analysed group of 437 patients after cartilage transplantation, 42.2% were operated because of posttraumatical changes, 29.0% because of congenital malformations and in 16.7% non-specific inflammations were the cause of reconstructive operations. Malformations were located mainly in the nose (59%), the ear concha (16.5%) and 10.9% were mandible. The results of treatment were compared with ages of patients, diagnosis and the locations of the changes. Very good results were achieved in 33.5% of the patients, and satisfactory in 41.8% of the patients. However, in 19.9% of the patients the result of treatment was unsatisfactory. Correlation between some clinical and biological characteristics and the result of treatment is under discussion. This revised version was published online in July 2006 with corrections to the Cover Date.     

Long term follow-up of reconstruction with allogeneic mandibular bone crib packed with autogenous particulate cancellous bone marrow

Allogeneic mandibular bone processed by the deep frozen method was used as a biologic crib for mandibular reconstruction. The allogeneic mandible is biocompatible, bioresorbable, of low antigenicity and provides the morphology for symphysis contour, angle of mandible and dental arch form. The particulate cancellous bone marrow (PCBM) contains marked osteogenic potential of hematopoietic marrow, which promotes osteogenesis. The cancellous marrow graft lacks structure and requires a crib to house it during the bone regeneration to the consolidation phase. Fresh frozen mandible was hollowed out for packing with PCBM prior to securing it to the defect by a rigid fixation method. Four cases of large mandibular defects resulting from treatment of benign odontogenic tumors were reconstructed utilizing this technique. All cases showed excellent facial contour and function satisfactorily in mastication and pronunciation. Complete graft incorporation and restoration of the osseous continuities were observed for four to 12 years after the operation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Vascularized bone allografts: in vitro assessment of cell-mediated and humoral responses

The immunologic consequences of transplantation of vascularized bone allografts have not been previously characterized. In this study, knee allografts, both vascularized and nonvascularized, were transplanted from Lewis rats to Brown Norway rats across a strong histocompatibility barrier. A total of 66 transplants and 8 control animals were evaluated. The vascularized knee grafts consisted of 1 cm of proximal tibia and distal femur with a minimal muscular cuff isolated on the femoral vessels, and these were transplanted to a heterotopic, subcutaneous position on the abdominal wall of the recipient rat. Nonvascularized allografts (identical but without anastomoses) were transplanted for comparison. The cell-mediated response was measured by lymphocytotoxicity assay, and the humoral response was measured by cytotoxic antibody assay, both employing 51Cr-labeled target cells. The timing and intensity of the immune response differed according to the type of graft. The vascularized bone allografts generated significant cell-mediated and humoral responses as early as 5 days posttransplant. A significant humoral response in nonvascularized bone allografts was not apparent until day 14, while cell-mediated response in these grafts was variable. These findings were correlated with the histologic appearance of the grafted tissue. Cyclosporine, which was administered to one group of vascularized bone allografts, resulted in the suppression of both types of immune responses. The histologic appearance of this group resembled that of isografts transplanted as controls. The clinical application of vascularized bone allografts may offer significant advantages over nonvascularized allografts in the reconstruction of massive bone defects. Complications such as nonunion, fracture, and collapse of articular segments seen in nonvascularized allograft transplantation may be avoided by preservation of the blood supply to the graft. Characterization of the immune response to vascularized bone allografts may subsequently allow the manipulation of the host and/or graft tissue and promote graft incorporation.     

Particle size influence in an impaction bone grafting model. Comparison of fresh-frozen and freeze-dried allografts

BackgroundImpaction bone grafting with large particles is considered as mechanically superior to smaller morsels. Interest of freeze-dried irradiated bone for impaction bone grafting has been observed with small particles. Influence of bone process on other particle sizes still needed to be assessed.Material and methodsTwenty-four osteoarthrotic femoral heads were used to prepare fresh-frozen and freeze-dried irradiated cancellous bone. Each group was divided into four batches of different particle sizes, each batch containing 18 samples. The different particle sizes were obtained with a Retsch Cross Beater Mill SK 100, Noviomagus rotating bone mills with two sizes of rasps and a Luer bone rongeur. Bone grafts were impacted in a contained cylinder. Stiffness was monitored during impaction.ResultsFreeze-dried irradiated grafts showed higher stiffness than fresh-frozen bone whatever the size of the particles. Large particles obtained with the rongeur and the large rasp from the Noviomagus bone mill were mechanically superior than small particles up to 30 impactions.InterpretationLarge particles offer better mechanical performance as a greater magnitude of force would be required to deform and break the particles. Freeze-dried irradiated bone brittleness reduces this advantage after 30 impactions. Large particles embrittlement leads to similar mechanical results as small particles at higher impaction rate. This may account for partial collapse of the graft layer in clinical situation when impaction rate is lower. This model supports the use of small particles obtained with thin rasp bone mill when freeze-dried irradiated bone for impaction bone grafting and large particles obtained with the Rongeur when fresh-frozen bone is available.     

Remodeling of heat-treated cortical bone allografts for posterior lumbar interbody fusion: serial 10-year follow-up

We have selected heat-treated bone allografts as the graft material since the Tokai Bone Bank, the first regional bone bank in Japan, was established in 1992. In this study, we examined changes in bone mineral density (BMD), and morphology observed by magnetic resonance imaging (MRI), and histological findings of bone grafts in cases followed up for 7-10?years after bone grafting to grasp the remodeling of heat-treated cortical bone allografts for posterior lumber interbody fusion (PLIF). BMD of bone grafts was reduced by half at 10?years after grafting. MRI revealed that bone grafts were indistinguishable initially in only 22.2% of cases, whereas after a lengthy period of 10?years distinguishable in many cases. Histologically, new bone formation at the graft-host interface was observed earlier, at 1?year after grafting, than that at the periphery of canals in the specimens. The laminated structure of the cortical bone eroded over time, and fragmented bone trabeculae were observed in the specimens at 8?years or longer after grafting, though necrotic bone still remained in some sites.     

Lyophilized veins as arterial interposition allografts

Venous allografts were evaluated in two models. Lyophilized allograft veins used as interposition grafts in the infrarenal aorta of the canine were studied and found to be patent at 1 year. Pathologic examination of the grafts revealed mild intimal hyperplasia and persistence of the basic structure of the lyophilized vessel. The ability of venous tissue to elicit an antibody response when transplanted into an allogeneic recipient was studied in the rat using the lymphocyte cytotoxicity assay. Fresh and Me₂SO-cryoprotected frozen veins produced circulating antibody when used as interposition grafts in the infrarenal aorta of the rat. Lyophilized and noncryoprotected frozen veins did not induce measurable antibody. Lyophilized allograft veins are a nonimmunogenic vascular graft material with acceptable long-term patency.     

Effect of two cleaning processes for bone allografts on gentamicin impregnation and in vitro antibiotic release

Bone allografts are a useful and sometimes indispensable tool for the surgeon to repair bone defects. Microbial contamination is a major reason for discarding allografts from bone banks. To improve the number of safe allografts, we suggest chemical cleaning of the grafts followed by antibiotic impregnation. Comparison of two chemical cleaning processes for bone allografts aiming for antibiotic impregnation and consequently delivery rates in vitro. Bone chips of 5–10 mm were prepared from human femoral heads. Two cleaning methods (cleaning A and cleaning B) based on solutions containing hydrogen peroxide, paracetic acid, ethanol and biological detergent were carried out and compared. After the cleaning processes, the bone chips were impregnated with gentamicin. Bacillus subtilis bioassay was used to determine the gentamicin release after intervals of 1–7 days. Differences were compared with non-parametric Mann–Whitney U tests. The zones of inhibition obtained from the bone grafts cleaned with both cleaning processes were similar between the groups. The concentration of the released antibiotic was decreasing gradually over time, following a similar pattern for both groups. The cleaning procedure A as well as the cleaning procedure B for bone allografts allowed the impregnation with gentamicin powder in the same concentrations in both groups. The delivery of gentamicin was similar for both groups. Both cleaning procedures were easy to be carried out, making them suitable for routine use at the bone banks.     

Prolongation of skin allograft survival after neonatal injection of donor bone marrow and epidermal cells

Composite-tissue (e.g., hand allograft) allotransplantation is currently limited by the need for immunosuppression to prevent graft rejection. Inducing a state of tolerance in the recipient could potentially eliminate the need for immunosuppression but requires reprogramming of the immunological repertoire of the recipient. Skin is the most antigenic tissue in the body and is consistently refractory to tolerance induction regimens using bone marrow transplantation alone. It was hypothesized that tolerance to skin allografts could be induced in rats by injecting epidermal cells with bone marrow cells during the first 24 hours of life of the recipients. Brown Norway rats (RT1n) served as donors for the epidermal cells, bone marrow cells, and skin grafts. Epidermal cells were injected intraperitoneally and bone marrow cells were injected intravenously into Lewis (RT1l) newborn recipient rats. In control groups, recipients received saline solution with no cells (group I, n = 12), bone marrow cells only (group II, n = 15), or epidermal cells only (group III, n = 15). In the experimental group (group IV, n = 18), recipients received epidermal and bone marrow cells simultaneously. Skin grafts were transplanted from Brown Norway (RT1n) rats to the Lewis (RT1l) rats 8 weeks after cell injections. Skin grafts survived an average of 8.5 days in group I (10 grafts), 9.2 days in group II (12 grafts), and 12 days in group III (14 grafts). Grafts survived 15.5 days (8 to 26 days) in group IV (15 grafts). The difference was statistically significant (p < 0.05). Hair growth was observed in some accepted grafts in group IV but never in the control groups. This is the first report of prolonged survival of skin allografts in a rat model after epidermal and bone marrow cell injections. Survival prolongation was achieved across a major immunological barrier, without irradiation, myeloablation, or immunosuppression. It is concluded that the presentation of skin-specific antigens generated a temporary state of tolerance to the skin in the recipients that could have delayed the rejection of skin allografts.     

The impact of the International Atomic Energy Agency (IAEA) program on radiation and tissue banking in Indonesia

In 1986, the National Nuclear Energy Agency (Batan) in Jakarta started the research and development for the setting up of a tissue bank (Batan Research Tissue Bank/BRTB) by preserving fresh amnion or fetal membranes by lyophilisation and then sterilising by gamma irradiation. During the period of 1990 and 2000, three more tissue banks were set up, i.e., Biomaterial Centre in Surabaya, Jamil Tissue Bank in Padang, and Sitanala Tissue Bank in Tangerang. In 1994, BRTB produced bone allografts. The banks established under the IAEA program concentrated its work on the production of amnion, bone and soft tissues allografts, as well as bone xenografts. These tissues (allografts and xenografts) were sterilised using gamma irradiation (about 90%) and the rest were sterilized by ETO and those products have been used in the treatment of patients at more than 50 hospitals in Indonesia. In 2004, those tissue banks produced 8,500 grafts and 5,000 of them were amnion grafts for eye treatment and wound dressing. All of those grafts were used for patients as well as for research. In 2006, the production increased to 9,000 grafts. Although the capacity of those banks can produce more grafts, we are facing problems on getting raw materials from suitable donors. To fulfill the demand of bone grafts we also produced bone xenografts. The impact of the IAEA program in tissue banking activities in Indonesia can be summarised as follows: to support the national program on importing substitutes for medical devices. The price of imported tissues are between US$ 50 and US$ 6,000 per graft. Local tissue bank can produce tissues with the same quality with the price for about 10–30% of the imported tissues.     

The effects of prolonged deep freezing on the biomechanical properties of osteochondral allografts

Musculo-skeletal allografts sterilized and deep frozen are among the most common human tissue to be preserved and utilized in modern medicine. The effects of a long deep freezing period on cortical bone has already been evaluated and found to be insignificant. However, there are no reports about the influences of a protracted deep freezing period on osteochondral allografts. One hundred osteochondral cylinders were taken from a fresh specimen and humeral heads of 1 year, 2 years, 3 years and 4 year old bones. Twenty chips from each period, with a minimum of 3 chips per humeral head. Each was mechanically tested by 3 point compression. The fresh osteochondral allografts were significantly mechanically better than the deep frozen osteochondral allografts. There was no statistical significant time dependent difference between the deep frozen groups in relation to the freezing period. Therefore, we conclude that, from the mechanical point of view deep freezing of osteochondral allografts over a period of 4 years, is safe without further deterioration of the biomechanical properties of the osteochondral allografts.     

Vascularized bone allograft transplantation in a genetically defined rat model

A heterotopic subcutaneous model for experimental vascularized bone allograft transplantation has been presented. This model uses genetically defined rats and allows serial assessment of graft viability. The reliability of this model has been proven by successful isograft transplantation. This model was used to study the effect of matching at the major histocompatibility complex on vascularized bone allograft survival. Whereas grafts transplanted across a minor histocompatibility barrier survived until sacrifice, grafts transplanted across a major histocompatibility barrier were victims of an acute rejection process. This study, therefore, showed genetic disparity to be a critical determinant of vascularized bone allograft survival. It indicates that primary vascularized bone allografts are as susceptible to rejection as heart and kidney allografts. For these reasons, it can be anticipated that genetic matching will be important in clinical vascularized bone allograft transplantation. The model used in this study should be useful for obtaining further fundamental immunologic information concerning vascularized bone allograft transplantation.     

Repeated freeze–thaw cycles reduce the survival rate of osteocytes in bone-tendon constructs without affecting the mechanical properties of tendons

Frozen bone-patellar tendon bone allografts are useful in anterior cruciate ligament reconstruction as the freezing procedure kills tissue cells, thereby reducing immunogenicity of the grafts. However, a small portion of cells in human femoral heads treated by standard bone-bank freezing procedures survive, thus limiting the effectiveness of allografts. Here, we characterized the survival rates and mechanisms of cells isolated from rat bones and tendons that were subjected to freeze–thaw treatments, and evaluated the influence of these treatments on the mechanical properties of tendons. After a single freeze–thaw cycle, most cells isolated from frozen bone appeared morphologically as osteocytes and expressed both osteoblast- and osteocyte-related genes. Transmission electron microscopic observation of frozen cells using freeze-substitution revealed that a small number of osteocytes maintained large nuclei with intact double membranes, indicating that these osteocytes in bone matrix were resistant to ice crystal formation. We found that tendon cells were completely killed by a single freeze–thaw cycle, whereas bone cells exhibited a relatively high survival rate, although survival was significantly reduced after three freeze–thaw cycles. In patella tendons, the ultimate stress, Young’s modulus, and strain at failure showed no significant differences between untreated tendons and those subjected to five freeze–thaw cycles. In conclusion, we identified that cells surviving after freeze–thaw treatment of rat bones were predominantly osteocytes. We propose that repeated freeze–thaw cycles could be applied for processing bone-tendon constructs prior to grafting as the treatment did not affect the mechanical property of tendons and drastically reduced surviving osteocytes, thereby potentially decreasing allograft immunogenecity.