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Abstract

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.

Novel ex-vivo mechanobiological intervertebral disc culture system

“…Cell viability in annulus fibrosus (AF) and NP was assessed using a colorimetric method based on reduction of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at active mitochondria (Csonge et al, 2002). After 2 hour immersion in MTT (1 mg/ml), AF and NP separation, and 24 hour organic extraction using 2-Methoxyethanol (CHROMASOLV® for HPLC, Sigma-Aldrich, Inc., St. Louis, MO), absorbance values (λ = 570 nm) of 200 µl of extract were normalized per tissue sample by wet weight and compared to fresh (t=0) tissue samples.…”

A novel, cryopreserved, viable osteochondral allograft designed to augment marrow stimulation for articular cartilage repair

“…These results indicate that viable chondrocytes continue to secrete growth factors, contributing to the persistence of growth factor secretion over time from the CVOCA and the resulting increase in MSC migration and chondrogenesis. The high percentage of viable cells preserved within the CVOCA (70.5 %) is a significant improvement compared to previous attempts at cartilage cryopreservation that resulted in only 20–50 % viable cells and, due to the known importance of viable cells for graft performance, prevented cryopreserved osteochondral allografts from gaining widespread use in the past [ 35 – 37 ].…”

Ultra friss osteochondralis allograft átültetéssel szerzett 10 éves magyarországi tapasztalataink

“…Ezen felül, a klinikai tapasztalat azt mutatta, hogy a hyalinporcban lévő sejtmentes mátrix a fagyasztott graftokban az idő előrehaladtával degenerálódik, valószínűleg azért, mert a minimális számú túlélő sejt nem képes fenntartani a szöveti homeosztázist (7). Az úgynevezett "cryopreserved" graftok esetében a fagyasztás valamilyen nutritív médiumban történik, ez valamelyest javítja a chondrocyták túlélését, azonban a sejtelhalás ilyen előkezelés mellett is drasztikus mértékű marad (5,12,17). Ezért az ily módon tartósított és előkezelt graftok nem hozták meg a várt eredményt.…”