Effects of FK506 on the healing of diaphyseal, critical size defects in the rat femur.

There is much interest in understanding the influence of the immune system on bone healing, including a number of reports suggesting a beneficial effect of FK506 (tacrolimus) in this regard. The influence of FK506 in a rat, femoral, critical size defect was examined using locally implanted, recombinant, human (rh) BMP-2 and adenovirally-transduced, autologous, adipose-derived mesenchymal stromal cells (AD-MSCs) expressing BMP-2. FK506 was delivered systemically using an implanted osmotic pump. Empty defects and those implanted with unmodified AD-MSCs did not heal in the presence or absence of FK506. Defects treated with rhBMP-2 healed with a large callus containing thin cortices and wispy trabeculae; this, too, was unaffected by FK506. A third of defects implanted with adenovirally-transduced AD-MSCs healed, but this improved to 100 % in the presence of FK506. New bone formed in response to BMP-2 synthesised endogenously by the genetically modified cells had a slimmer callus than those healed by rhBMP-2, with improved cortication and advanced reconstitution of marrow. These results suggest that FK506 may have had little effect on the intrinsic biology of bone healing, but improved healing in response to adenovirally-transduced cells by inhibiting immune responses to the first-generation adenovirus used here. Because the genetically modified cells produced bone of higher quality at far lower doses of BMP-2, this approach should be explored in subsequent research.

Effects of freeze-thaw on the biomechanical and structural properties of the rat Achilles tendon.

Rodent models are commonly used to investigate tendon healing, with the biomechanical and structural properties of the healed tendons being important outcome measures. Tendon storage for later testing becomes necessary when performing large experiments with multiple time-points. However, it is unclear whether freezing rodent tendons affects their material properties. Thus the aim of this study was to determine whether freezing rat Achilles tendons affects their biomechanical or structural properties. Tendons were frozen at either -20 °C or -80 °C directly after harvesting, or tested when freshly harvested. Groups of tendons were subjected to several freeze-thaw cycles (1, 2, and 5) within 3 months, or frozen for 9 months, after which the tendons were subjected to biomechanical testing. Additionally, fresh and thawed tendons were compared morphologically, histologically and by transmission electron microscopy. No major differences in biomechanical properties were found between fresh tendons and those frozen once or twice at -20 °C or -80 °C. However, deterioration of tendon properties was found for 5-cycle groups and both long-term freezing groups; after 9 months of freezing at -80 °C the tear resistance of the tendon was reduced from 125.4 ±â€¯16.4N to 74.3 ±â€¯18.4N (p = 0.0132). Moreover, tendons stored under these conditions showed major disruption of collagen fibrils when examined by transmission electron microscopy. When examined histologically, fresh samples exhibited the best cellularity and proteoglycan content of the enthesis. These properties were preserved better after freezing at -80 °C than after freezing at -20 °C, which resulted in markedly smaller chondrocytes and less proteoglycan content. Overall, the best preservation of histological integrity was seen with tendons frozen once at -80 °C. In conclusion, rat Achilles tendons can be frozen once or twice for short periods of time (up to 3 months) at -20 °C or -80 °C for later testing. However, freezing for 9 months at either -20 °C or -80 °C leads to deterioration of certain parameters.