Biomechanical evaluation of retrieved massive allografts: preliminary results.

Allograft bone is the primary source of graft material for large skeletal defects. No study has determined the physical characteristics of such grafts after various periods of time in vivo for incorporation and remodeling. The purpose of this pilot study was to obtain allograft tissue and biomechanically evaluate the tissue to assess allograft bone material properties. The mechanical properties of the retrieved allograft tissue were compared to allograft bone prior to transplantation. Histological analysis of the retrieved allograft tissue is currently underway to correlate degree of incorporation, allograft porosity, and microfracture density with allograft material properties. After allograft retrieval, radiographs were used to plan sectioning for histological and biomechanical analyses. Rectangular sections of uniform dimensions (50 x 3 x 3 mm) were mapped and machined from the bulk specimens. The samples were loaded in bending in the medial to lateral direction using a 4-point bending fixture to obtain flexural elastic modulus and breaking strength. Preconditioning was applied to each specimen by cycling through 5 submaximal loading cycles (maximum deflection = 1% specimen length). After preconditioning, the specimens were loaded to failure at a rate of 1 mm/min. Retrieved specimens consisted of 1 tibia, 2 femurs, and 2 humeri ranging from 2 to 13 years in vivo. Two control tibia specimens were also tested. Assuming that material properties of cortical bone are consistent regardless of skeletal site, the preliminary data indicates that allograft modulus and strength decline with time in vivo. Testing and analysis of more specimens continue in order to corroborate these initial results.

Evaluation of fluorescein diacetate for flow cytometric determination of cell viability in orthopaedic research.

Accurate estimation of cellular viability is important both in research and in aspects of orthopaedic clinical practice. We have been interested in the potential for flow cytometric application of fluorescein diacetate (FDA) in evaluating chondrocyte survival following cryopreservation of osteochondral allografts as well as in the assessment of sarcoma necrosis following preoperative chemotherapy. In order to evaluate the suitability of this method for cell viability assays, this study compared FDA with more traditional methodology (trypan blue, clonigenic assay, metabolic activity analysis, measurement of DNA synthesis, and histological assessment of necrosis). Both chondrocytes and sarcoma cells were exposed to various experimental injuries prior to viability analysis. Although it is evident from these experiments that FDA accurately reflects cell survival after physical injury, it underestimates the effect of chemotherapy on cell reproductive potential in vitro. However, FDA is highly correlated with histological assessment of tumor viability after chemotherapy in vivo. It is apparent that the methodology chosen for determination of viability should be appropriate for the type of experimental injury and should analyze the cell function (i.e., metabolic activity or reproductive capacity) that is appropriate for the experimental model.