RESUMO
The aim of this study was to adapt a reliable, reproducible and simple viability assay for cartilage and osteochondral studies. The previous assays (radioisotope uptake, assessment of matrix components, histological methods, oxygen consumption etc.) were complex, laborious, time consuming or suffer from difficulty of interpretation. MTT assay was chosen because it has been widely and successfully used in different cell and tissue studies, but has not been published on human solid articular cartilage. Fresh intact cartilage samples of human tali were tested to investigate the assay. The reliability of the MTT assay was also tested by an fluorescent dye combination. The MTT assay is based on the production of purple formazan pigment from methyltetrazolium salt by the mitochondrial enzymes of viable chondrocytes. The enzyme kinetics of the reaction was also investigated because it was unknown in the case of cartilage. The amount of pigment formed can be measured by spectrophotometry after extraction by methyl cellosolve. The color density is proportional to mitochondrial enzyme activity, reflecting the number of viable chondrocytes. The optimal reagent concentration, biopsy size, and incubation period were established. There is a linear relationship between the cartilage weight and the pigment production activity. A 9.8% nonspecific raction was observed in the negative controls. The enzyme kinetics of the reaction was also investigated. The MTT clevage up to 0.1% (w/v) follows the Michaelis kinetics. We calculated the Michaelis constant (2835 +/- 130 microM), the maximal velocity (36 +/- 3.2 x 10(-5)microMsec(-1)) and the velocity constant (1.27 +/- 0.2 x 10(-7)sec(-1)) of the reaction. The latter is a significant marker for each tissue type. The viability of cartilage was also assessed and calculated by a fluorescent dye combination comprising 1 microg/ml propidium iodide (PI) and 4 microM/ml SYTO-16 stains. The PI stains dead cells (red fluorescence), the SYTO-16 stains live cells (green fluorescence). The staining can be visualised simultaneously, and the live/dead ratio can be calculated by image analysis software from saved image files. The MTT assay is a simple, non-expensive, efficient, reliable, reproducible, sensitive viability test for cartilage studies. The MTT reduction assay and the staining method were corrobative.
RESUMO
One of the most important factors concerning the successful clinical outcome after transplantation of osteochondral allografts is the viability of the cartilage.The viability of cryopreserved cartilage is quite poor, 20-30% cell survival has been published. The purpose of this study was to develop a new storage method which improves the chondrocyte viability. The talus of cadaveric donors was used as a model tissue to compare human osteochondral allograft cartilage viability following cryopreservation with that remaining after prolonged refrigerated storage. Full-thickness cartilage punch biopsies had been cryopreserved, and tali were divided into two matched groups and stored in TCM for 60 days at +4 degrees C, either with or without regular medium replacement. The cartilage of each graft was biopsied and assayed for viability on every third day by the MTT reduction assay. During 4 degrees C storage, a recurring pattern of large fluctuations in apparent cartilage viability was observed in every stored graft, with or without medium replacement. These fluctuations did not appear in control specimens of either fresh or cryopreserved human skin that were assayed in parallel with the cartilage biopsies, so the viability fluctuation seems an intrinsic property of the cartilage in these conditions. Cartilage stored for 60 days at +4 degrees C showed significantly higher viability (35.2 +/- 3.3 %) than fresh cartilage that had been cryopreserved (21.6 +/- 1.8 %). This was true even when cryopreserved and thawed cartilage was subjected to a 3 day post thaw incubation under presumably favorable conditions (17.7 +/- 1.6 %). These viability assay results, (reflective of intracellular metabolic activity), were corroborated by the fluorescent dye mixture SYTO-16 and propidium iodide. The data indicate that long-term stored refrigerated cartilage appears to retain a viability higher than that of cryopreserved cartilage for up to and perhaps beyond 60 days of storage. There was no viability index difference between the medium replaced and non-replaced groups. Although an exceptional result, in one individual case, more than 65% viable cells could be detected in the talar cartilage after 60 days storage at +4 degrees C.
