- The Effects of Multiple Freeze–Thaw Cycles on the Biomechanical Properties of the Human Bone-Patellar Tendon-Bone Allograft
Abstract
Soft tissue allografts, such as the bone-patellar tendon-bone (BPTB) graft, have been frequently used for anterior cruciate ligament (ACL) reconstruction. As allografts are subjected to freezing and thawing for multiple cycles, the objective of this study was to measure the changes of the biomechanical properties of the human BPTB allograft after 4 and 8 freeze–thaw cycles in comparison to a single freeze–thaw cycle. Three BPTB specimens were procured from 21 human donors and divided into three groups: 1, 4, or 8 freeze–thaw cycles. Each freeze–thaw cycle consisted of freezing at −20 ± 10°C for more than 6 h and thawing at 22 ± 3°C for at least 6 h. Tensile testing of the BPTB specimens consisted of loading between 50 N and 250 N for 100 cycles and then loading to failure. Cyclic loading revealed a similar amount of creep (∼0.5 mm) among the three freeze–thaw cycles groups (p = 0.38). The stiffness of the BPTB graft for the 1, 4, and 8 freeze–thaw cycle groups were 244 ± 42 N/mm, 235 ± 39 N/mm, and 231 ± 40 N/mm, respectively (p = 0.43). Similar findings were obtained for the ultimate load of the BPTB graft (p = 0.14) and the tangent modulus of the PT substance (p = 0.41). The results of this study suggest that there would be little measurable effect on the structural properties of the BPTB graft or mechanical properties of the PT tissue substance following 8 freeze–thaw cycles. These BPTB allografts could potentially be re-frozen without a loss in their biomechanical properties, given appropriate storage and care. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1193–1198, 2011
- Demineralization Removes Residual Alendronate in Allograft Bone Procured From Donors With a History of Bisphosphonate Use
Abstract
BACKGROUND: Bisphosphonate-associated osteonecrosis (BON) of the jaw is a growing concern in the dental community, but the possible presence of residual bisphosphonates in demineralized allograft bone from bisphosphonate-using tissue donors and the clinical implications of using such bone are unclear. The objectives of this study are to determine whether alendronate remained in demineralized bone matrix (DBM) procured from donors with a documented history of oral bisphosphonate use and to examine whether the demineralization process removes alendronate from allograft bone.
METHODS: A gas chromatography?mass spectrometry method was developed and validated to quantify residual alendronate in allograft bone. Alendronate levels in DBM procured from tissue donors with a history of oral bisphosphonate use were compared to alendronate levels in DBM procured from donors without a history of bisphosphonateuse. In addition, mineralized and demineralized bone was soaked in alendronate at concentrations of 0.002, 2.0, and 2,000 ng/mg bone and analyzed to examine the effect of the demineralization process.
RESULTS: Residual alendronate was not detected in the DBM from either group, nor was it detected in any of the DBM samples soaked in alendronate solutions. Soaked mineralized bone contained measureable alendronate, but the substance was removed by demineralization.
CONCLUSIONS: The demineralization process effectively removed residual alendronate from allograft bone. These results may relieve anxieties regarding the use of DBM in dental patients because it is unlikely to trigger BON of the jaw.
- Osteoinductivity of Demineralized Bone Matrix (DBM) is Independent of Donor Bisphosphonate Use
Abstract
BACKGROUND: Demineralized bone matrix (DBM) is commonly used as a bone graft substitute because of its osteoinductive properties, either alone or to supplement an osteoconductive material. The aging population has led to an increase in the number of prospective DBM donors who have taken bisphosphonates to prevent osteoclast-mediated bone resorption. The aim of this study was to determine whether oral bisphosphonate usage affects the osteoinductivity of donor DBM.
METHODS: Sex- and age-matched pairs of samples were provided by four tissue banks (3-4 pairs/bank). DBM-donors without bisphosphonates had a mean age of 68.1±2.0 years and the mean age of DBM-donors with bisphosphonates was 69.1±2.5 years. Each pair included one donor known to take bisphosphonates and one who had not taken bisphosphonates. DBM previously confirmed as osteoinductive was the positive control and heat-inactivated DBM was the negative control. DBM incubated with 1ml PBS containing 0, 0.002, 2.0 or 2,000ng/ml alendronate was also tested. Gelatin capsules containing 15mg DBM were implanted bilaterally in the gastrocnemius muscle of male nude mice (n=8 implants/group). Mice were euthanized 35 days post-implantation, and hind limbs were recovered and processed for histology. Osteoinductivity was measured using a qualitative score and by histomorphometry.
RESULTS: 10/15 samples from bisphosphonate donors and 9/15 samples from non-bisphosphonate patients were osteoinductive. Qualitative scores were comparable (1.7±0.4 without; 1.9±0.7with bisphosphonates). Osteoinductive DBMs produced ossicles of comparable size, regardless of bisphosphonate usage. Histomorphometric measurements of the area of new bone formation and residual DBM were also comparable. Addition of alendronate to control DBM did not affect its osteoinductivity.
CONCLUSIONS: DBM from donors with and without bisphosphonates have the same ability to induce bone formation. However, it is not known if the quality of the new bone is affected, with downstream consequences to remodeling.
CLINICAL RELEVANCE: These results show that DBM can be safe and effective as an osteoinductive material, even when the original bone graft is obtained from donors that have used bisphosphonates.
- Guidance for Removal of Fetal Bovine Serum From Cryopreserved Heart Valve Processing
Abstract
Bovine serum is commonly used in cryopreservation of allogeneic heart valves; however, bovine serum carries a risk of product adulteration by contamination with bovine-derived infectious agents. In this study, we compared fresh and cryopreserved porcine valves that were processed by 1 of 4 cryopreservation formulations, 3 of which were serum-free and 1 that utilized bovine serum with 1.4 M dimethylsulfoxide. In the first serum-free group, bovine serum was simply removed from the cryopreservation formulation. The second serum-free formulation had a higher cryoprotectant concentration, i.e. 2 M dimethylsulfoxide, in combination with a serum-free solution. A colloid, dextran 40, was added to the third serum-free group with 2 M dimethylsulfoxide due to theoretical concerns that removal of serum might increase the incidence of tissue cracking. Upon rewarming, the valves were inspected and subjected to a battery of tests. Gross pathology revealed conduit cracking in 1 of 98 frozen heart valves. Viability data for the cryopreserved groups versus the fresh group demonstrated a loss of viability in half of the comparisons (p < 0.05). No significant differences were observed between any of the cryopreserved groups, with or without bovine serum. Neither routine histology, autofluorescence-based multiphoton imaging nor semiquantitative second-harmonic generation microscopy of extracellular matrix components revealed any statistically significant differences. Biomechanics analyses also revealed no significant differences. Our results demonstrate that bovine serum can be safely removed from heart valve processing and that a colloid to prevent cracking was not required. This study provides guidance for the assessment of changes in cryopreservation procedures for tissues.
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