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.

Abstract:To clarify the distribution of bone-marrow-derived cells in fractures treated by plate fixation, fracture models were created using the green fluorescent protein (GFP) chimeric mouse. We observed 2 types of fracture healing processes with different types of callus formation and cellular events by using Mouse Fix™, a device allowing plate fixation on the mouse femur, and differences in the distribution of bone-marrow-derived cells between the 2 types. The GFP chimeric mice were created by bone marrow transplantation. Fractures were created on the left femurs of mice and stabilized with either rigid (Group R) or flexible (Group F) plates to prepare undecalcified fresh-frozen sections. In Group F, a large external callus and a large intramedullary callus were formed mostly by endochondral ossification. The cells that made up the intramedullary callus and callus in the fracture gap were GFP positive, but most cells of the external callus were not. In Group R, bone union was achieved mostly without external callus formation, bone apposition occurred directly in the gap, and a small intramedullary callus was formed. As observed in Group F, this group had GFP-positive cells in the callus within the fracture gap and in the intramedullary calluses. The results of this study provided direct evidence of the distribution of bone-marrow-derived cells in the callus of fractures treated by plate fixation under different stability conditions.

To eliminate the potential for infection, many tissue banks routinely process and terminally sterilize allografts prior to transplantation. A number of techniques, including the use of scanning electron microscopy, bone graft models, and mechanical property tests, are used to evaluate the properties of allograft bone. However, as these methods are time consuming and often destroy the bone sample, the quality assessment of allograft bones are not routinely performed after processing and sterilization procedures. Raman spectroscopy is a non-destructive, rapid analysis technique that requires only small sample volumes and has recently been used to evaluate the mineral content, mineral crystallinity, acid phosphate and carbonate contents, and collagen maturity in human and animal bones. Here, to establish a quality assessment method of allograft bones using Raman spectroscopy, the effect of several common sterilization and preservation procedures on rat femoral bones were investigated. We found that freeze–thawing had no detectable effects on the composition of bone minerals or matrix, although heat treatment and gamma irradiation resulted in altered Raman spectra. Our findings suggest Raman spectroscopy may facilitate the quality control of allograft bone after processing and sterilization procedures.

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To gain a better understanding of the causative factors of hyperinsulinaemia, pancreatic oxidative stress in STR/ Ort mice and a type 2 diabetic mouse model (db/db) was evaluated. Expression of oxidative stress marker HSP32 in the pancreas of STR/Ort mice was significantly higher than that in the liver, kidney and spleen. The expression level of HSP32 in STR/Ort was significantly higher than in db/m at 5, 15 and 20 weeks of age (P B 0.05) and tended to be higher than that in db/m (P 0 0.069). The expression level of HSP47 in STR/Ort was also significantly higher than in db/m at 5 and 10 weeks (P B 0.05) and tended to be higher than that of db/m at 15 (P 0 0.086) and 20 weeks (P 0 0.052). Taken together, these results suggest that elevation of oxidative stress in the pancreas during early developmental periods may induce hyperinsulinaemia in STR/Ort mice through impaired insulin metabolism and contribute to the initiation and progression of osteoarthritis.