11 kGy gamma irradiated demineralized bone matrix enhances osteoclast activity.

BACKGROUND: Demineralized bone matrix (DBM) allografts are widely used in orthopaedic clinics. However, the biological impact on its osteoinductivity after its sterilization process by gamma irradiation is not well studied. Furthermore, little is known about the relationship between residual calcium levels on osteoinductivity. HYPOTHESIS: We hypothesize that low-dose gamma irradiation retains the osteoinducitivity properties of DBM and causes ectopic bone formation. MATERIALS AND METHODS: A randomised animal trial was performed to compare tissue and molecular responses of low-dose (11 kGy) gamma irradiated and non-irradiated human DBM at 6 weeks post-intramuscular implantation using an athymic rat model. In addition, we correlated residual calcium levels and bone formation in gamma irradiated DBM. RESULTS: A modified haematoxylin and eosin stain identified ectopic bony capsules at all implanted sites with no significant difference on the amount of new bone formed between the groups. Statistically significantly lower ratio of alkaline phosphatase expression over tartrate-resistant acid phosphatase and/or cathepsin K expressions was found between the groups. DISCUSSION: This study found that low-dose gamma irradiated DBM, which provides a sterility assurance level of 10(-6) for bone allografts, retained osteoinductivity but exhibited significantly enhanced osteoclastic activity. Furthermore, this is the first study to find a positive correlation between residual calcium levels and bone formation in gamma irradiated DBM.

Outcome of revision total knee arthroplasty with bone allograft in 30 cases.

Revision Total Knee Arthroplasty is often complicated by large bone defects in the distal femur and proximal tibia. These defects can be managed in a variety of ways including the use of allograft bone. The purpose of this study was to retrospectively evaluate the clinical outcome of revision total knee arthroplasty cases where allograft bone was used. Thirty revision TKA's (27 patients) performed between 1994 and 2009 were followed for a mean of 5 years (1-14 years). Preoperative bone defects were classified using the Anderson Orthopaedic Research Institute classification system. Patient follow-up entailed calculation of the Knee Society Score and radiological assessment of the revision joint replacement in addition to review of complications. Kaplan Meier analysis predicted survivorship at 5 years as 93%, with further revision surgery as end point. The average Knee Society Score was 76.4, with 19 (63%) of knees scoring "excellent" results, 4 (14%) "good", 1 (3%) "fair" and 6 (20%) were "poor". The overall complication rate was 233%. Radiological lucency was demonstrated on recent radiographs for one patient. Three knees were re-revised at 1 year, 6 years and 8 years respectively. Our study demonstrates promising short to medium term results with the use of allograft bone in revision total knee replacement presenting with significant bone loss.

Reducing the radiation sterilization dose improves mechanical and biological quality while retaining sterility assurance levels of bone allografts.

BACKGROUND: Bone allografts carry a risk of infection, so terminal sterilization by gamma irradiation at 25kGy is recommended; but is deleterious to bone quality. Contemporary bone banking significantly reduces initial allograft bioburden, questioning the need to sterilize at 25kGy. METHODS: We inoculated allograft bone with Staphylococcus epidermidis and Bacillus pumilus, then exposed them to gamma irradiation at 0, 5, 10, 15, 20 and 25kGy. Mechanical and biological properties of allografts were also assessed. Our aim was to determine an optimal dose that achieves sterility assurance while minimizing deleterious effects on allograft tissue. RESULTS: 20-25kGy eliminated both organisms at concentrations from 10(1) to 10(3)CFU, while 10-15kGy sterilized bone samples to a bioburden concentration of 10(2)CFU. Irradiation did not generate pro-inflammatory bone surfaces, as evidenced by macrophage activation, nor did it affect attachment or proliferation of osteoblasts. At doses ≥10kGy, the toughness of cortical bone was reduced (P<0.05), and attachment and fusion of osteoclasts onto irradiated bone declined at 20 and 25kGy (P<0.05). There was no change in collagen cross-links, but a significant dose-response increase in denatured collagen (P<0.05). CONCLUSIONS: Our mechanical and cell biological data converge on 15kGy as a threshold for radiation sterilization of bone allografts. Between 5 and 15kGy, bone banks can undertake validation that provides allografts with an acceptable sterility assurance level, improving their strength and biocompatibility significantly. CLINICAL RELEVANCE: The application of radiation sterilization doses between 5 and 15kGy will improve bone allograft mechanical performance and promote integration, while retaining sterility assurance levels. Improved quality of allograft bone will promote superior clinical outcomes.

The use of Gamma-irradiated proximal femoral allografts for bone stock reconstruction in complex revision hip arthroplasty.

We have followed a consecutive series of 49 revision hip arthroplasties, performed for severe femoral bone loss using Gamma-irradiated anatomic-specific proximal femoral allografts longer than five centimetres. The patients were followed for a median 10.2 years, with a five year minimum follow-up. The median preoperative Harris Hip Score (HHS) improved from 42 points to 77 points postoperatively. In four hips the femoral component was further revised for non-union of the allograft and aseptic failure. In one hip the allograft and the femoral component were removed because of infection. In one hip the allograft and the femoral component were re-revised for host step-cut fracture. Junctional-union was observed in 44/49 hips. By defining success as an increase of HHS by 20 points or more, a stable implant and no need for any subsequent re-operations related to the allograft and /or the implant, a success rate of 76% was observed. Kaplan-Meier survivorship analysis predicted 79% rate of survival at 10 years and 75% rate of survival at 17 years, with the need for further revision of the allograft and/or implant as the end point. Three hips underwent re-attachment of the greater trochanter for trochanteric escape. Asymptomatic non-union of the greater trochanter was noticed in another three hips. Moderate allograft resorption was observed in four hips. Two fractures of the host step-cut occurred. There were four dislocations. Good long-term results with the use of large anatomic-specific femoral allografts justify their continued use in cases of revision hip arthroplasty complicated with severe femoral bone loss.

Sponge swabs increase sensitivity of sterility testing of processed bone and tendon allografts.

Sterility testing is the final, and critical, step in quality control of tissue banking. It informs the decision whether to release the tissue allografts for clinical use, or not. The most common method for sterility testing of structural bone and tendon allografts is to swab using cotton tip streaks. This method provides low recovery efficiency; and therefore may pass allografts with low bioburden, providing false negatives. Our pilot data revealed organism recovery efficiencies of 60, 30 and 100% from cotton swab, membrane filtration and sponge swaps, respectively. Our aim was to develop a high sensitivity sterility test for structural bone and tendon allografts using a sponge sampling method. Eighty-one bone and tendon allograft samples were inoculated with organism suspensions (10(2) or less organisms per 0.1 mL) of Clostridium sporogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Bacillus subtilis, Aspergillus niger, Staphylococcus epidermidis and Micrococcus spp. Nasco sponges (4 × 8 cm) were used to aseptically sample the whole surface of allograft samples. The sponges were cut in half and cultured in either tryptone soya or fluid thioglycollate broths for 14 days. Positive culture samples were further examined for microbial morphology. The results showed that the sensitivity of the method, and negative predictive value, is 100% for all inoculated organisms incubated with thioglycollate. We conclude that this sponge sampling method should be applied as the standard for sterility testing of structural bone and tendon allografts.

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.

Computer-assisted vs conventional mechanical jig technique in hip resurfacing arthroplasty.

To compare the effectiveness of computer-assisted surgery (CAS) and mechanical jig technique in hip resurfacing arthroplasty, we reviewed 176 hip resurfacing arthroplasty performed in 158 patients. The initial 131 hips were resurfaced using the conventional mechanical jig technique, and the remaining 45 hips with the CAS technique. The demographic data of the patients were similar for both techniques. Follow-up radiographs taken 2 months after the surgery showed patients in the CAS technique having a significantly better alignment of the femoral component on the frontal and sagittal planes. There was no difference in the risk of implant notching on the femoral neck for both techniques. The implant sizes were similar for both techniques; thus, the amount of bone stock preserved was not technique specific.

Validation of 15 kGy as a radiation sterilisation dose for bone allografts manufactured at the Queensland Bone Bank: application of the VDmax 15 method.

BACKGROUND: ISO 11137-2006 (ISO 11137-2a 2006) provides a VDmax 15 method for substantiation of 15 kGy as radiation sterilisation dose (RSD) for health care products with a relatively low sample requirement. Moreover, the method is also valid for products in which the bioburden level is less than or equal to 1.5. In the literature, the bioburden level of processed bone allografts is extremely low. Similarly, the Queensland Bone Bank (QBB) usually recovers no viable organisms from processed bone allografts. Because bone allografts are treated as a type of health care product, the aim of this research was to substantiate 15 kGy as a RSD for frozen bone allografts at the QBB using method VDmax 15-ISO 11137-2: 2006 (ISO 11137-2e, Procedure for method VDmax 15 for multiple production batches. Sterilisation of health care products - radiation - part 2: establishing the sterilisation dose, 2006; ISO 11137-2f, Procedure for method VDmax 15 for a single production batch. Sterilisation of health care products - radiation - part 2: establishing the sterilisation dose, 2006). MATERIALS: 30 femoral heads, 40 milled bone allografts and 40 structural bone allografts manufactured according to QBB standard operating procedures were used. METHOD: Estimated bioburdens for each bone allograft group were used to calculate the verification doses. Next, 10 samples per group were irradiated at the verification dose, sterility was tested and the number of positive tests of sterility recorded. If the number of positive samples was no more than 1, from the 10 tests carried out in each group, the verification was accepted and 15 kGy was substantiated as RSD for those bone allografts. RESULTS: The bioburdens in all three groups were 0, and therefore the verification doses were 0 kGy. Sterility tests of femoral heads and milled bones were all negative (no contamination), and there was one positive test of sterility in the structural bone allograft. Accordingly, the verification was accepted. CONCLUSION: Using the ISO validated protocol, VDmax 15, 15 kGy was substantiated as RSD for frozen bone allografts manufactured at the QBB.

Sterilization of allograft bone: is 25 kGy the gold standard for gamma irradiation?

For several decades, a dose of 25 kGy of gamma irradiation has been recommended for terminal sterilization of medical products, including bone allografts. Practically, the application of a given gamma dose varies from tissue bank to tissue bank. While many banks use 25 kGy, some have adopted a higher dose, while some choose lower doses, and others do not use irradiation for terminal sterilization. A revolution in quality control in the tissue banking industry has occurred in line with development of quality assurance standards. These have resulted in significant reductions in the risk of contamination by microorganisms of final graft products. In light of these developments, there is sufficient rationale to re-establish a new standard dose, sufficient enough to sterilize allograft bone, while minimizing the adverse effects of gamma radiation on tissue properties. Using valid modifications, several authors have applied ISO standards to establish a radiation dose for bone allografts that is specific to systems employed in bone banking. These standards, and their verification, suggest that the actual dose could be significantly reduced from 25 kGy, while maintaining a valid sterility assurance level (SAL) of 10(-6). The current paper reviews the methods that have been used to develop radiation doses for terminal sterilization of medical products, and the current trend for selection of a specific dose for tissue banks.

Sterilization of allograft bone: effects of gamma irradiation on allograft biology and biomechanics.

Gamma irradiation from Cobalt 60 sources has been used to terminally sterilize bone allografts for many years. Gamma radiation adversely affects the mechanical and biological properties of bone allografts by degrading the collagen in bone matrix. Specifically, gamma rays split polypeptide chains. In wet specimens irradiation causes release of free radicals via radiolysis of water molecules that induces cross-linking reactions in collagen molecules. These effects are dose dependent and give rise to a dose-dependent decrease in mechanical properties of allograft bone when gamma dose is increased above 25 kGy for cortical bone or 60 kGy for cancellous bone. But at doses between 0 and 25 kGy (standard dose), a clear relationship between gamma dose and mechanical properties has yet to be established. In addition, the effects of gamma radiation on graft remodelling have not been intensively investigated. There is evidence that the activity of osteoclasts is reduced when they are cultured onto irradiated bone slices, that peroxidation of marrow fat increases apoptosis of osteoblasts; and that bacterial products remain after irradiation and induce inflammatory bone resorption following macrophage activation. These effects need considerably more investigation to establish their relevance to clinical outcomes. International consensus on an optimum dose of radiation has not been achieved due to a wide range of confounding variables and individual decisions by tissue banks. This has resulted in the application of doses ranging from 15 to 35 kGy. Here, we provide a critical review on the effects of gamma irradiation on the mechanical and biological properties of allograft bone.

Structural allografts for bone stock reconstruction in two-stage revision for infected total hip arthroplasty: good outcome in 16 of 18 patients followed for 5-14 years.

BACKGROUND: The use of massive bone allografts in cases of revision of failed total hip arthroplasties (THAs) due to infection is controversial. PATIENTS AND METHODS: 18 patients presented with infection at the site of a THA and were treated with a two-stage protocol. In the first stage, the prosthesis was removed together with all necrotic tissues and cement material if present. A custom-made mold of Palacos R cement containing 1 g of gentamicin was then inserted in 17 of the 18 patients. Systemic antibiotics were used during the interval period. In the second stage, the patients had either acetabular or femoral reconstruction using bulk allograft bone. RESULTS: Mean follow-up was 9 (5-14) years. 1 patient presented with recurrent infection and underwent a Girdlestone resection arthroplasty as definitive treatment. Another patient had a mechanical failure of the acetabular component, which was revised 10 years after the second stage of the reconstruction. The mean Harris Hip Score improved from 34 points preoperatively to 71 points at the last review. By our definition, 16/18 of the patients had a successful outcome. INTERPRETATION: Our results support the use of massive allografts in staged reconstructions of infected THAs complicated by considerable bone loss.

Mechanical properties of gamma irradiated morselized bone during compaction.

This study examined the effects of gamma irradiation on the compressive properties of morselized cancellous bone from human femoral heads. Twelve bone samples, mean age of 68 years (range 92-39), were divided into 3 groups (N=12) of varying irradiation level (0, 15 and 25 kGy). Each specimen was compacted in a controlled fashion in steps of 0.5 mm at 0.5 mm/min (up 12 mm). The load and stiffness increased with compaction, but this relationship was not linear. There was no statistical significant difference in the compacting load or stiffness between groups (p>0.05) until the last 1 mm of compaction, where the 25 kGy group were significantly stiffer compared to controls but not different to the 15 kGy group. This may be due to decreased interlocking of bone particles caused by higher irradiation levels resulting in a stiffer graft.

Role of core biopsy in diagnosing infection before revision hip arthroplasty.

A prerevision core biopsy from a failed hip joint was performed in 41 hips (38 patients) with a high index of suspicion for sepsis to determine its efficacy in diagnosing sepsis. Seven hips were known septic failures, and core biopsy was undertaken to confirm resolution of sepsis. Forty hips were revised, whereas 1 hip had excision of heterotopic bone. Cultures and permanent histologic sections were obtained during subsequent surgeries, and findings were compared with the results of core biopsy. The commonest organisms isolated were coagulase-negative staphylococci and Propionibacterium acne. In patients without a history of sepsis, core biopsy diagnosed infection with sensitivity of 80%, specificity of 100%, accuracy of 97%, and positive predictive value of 100% using histologic evidence of sepsis at revision as the standard. Core biopsy, however, had low sensitivity and accuracy in establishing resolution of infection in patients with a history of septic failure. Cultures from the aspirate had 44% sensitivity and positive predictive value of 57%. Core biopsy is useful for investigating failed arthroplasties with suspected sepsis. It has advantages of hip aspiration and allows examination of periprosthetic tissue histology. Its role in diagnosing resolution of infection in previously septic failures, however, is limited.