17. Vitrification of intact human articular cartilage

Abstract

Large articular cartilage joint defects result in joint degeneration and osteoarthritis. The only method currently able to biologically deal with this problem is osteochondral allografting but existing storage techniques are unable to maintain cell viability for long periods of time. Successful vitrification of intact articular cartilage would allow storage of intact articular cartilage with live cells indefinitely enabling the establishment of a tissue bank that can support a joint transplantation program. We hypothesized that sequentially exposing intact human articular cartilage to a number of cryoprotective agents (CPAs) at lowering temperatures would enable successful vitrification with high cell viability. Intact human articular cartilage on its bone base was harvested from total knee arthroplasty donors and deceased donor after ethical approval from our local institution. The cartilage was exposed to four CPAs (Me 2 SO, glycerol, propylene glycol, and ethylene glycol) at lowering temperatures (0 °C, −10 °C, −15 °C) for mathematically determined times to achieve a desired concentration of each CPA at the cartilage-bone interface. After full equilibration in the final vitrification solution the samples were stored in liquid nitrogen for a minimum of 30 min and up to 3 months. The samples were warmed in a 37 °C water bath and cell viability was determined using membrane integrity stains while cell functionality was documented using a mitochondrial assay as well as pellet cultures to demonstrate collagen II and sulphated proteoglycan production. Eighteen 10 mm diameter dowels from 10 knee arthroplasty patients demonstrated 75.4 + 12.1% cell recovery as determined by membrane integrity stains. Two further samples demonstrated maximal mitochondrial function that was 95% of fresh control sample maximal function although there was a five day incubation period required prior to achieving these results. Pellet cultures demonstrated production of sulphated proteoglycans and collagen II shown by safranin O histology staining and immunohistochemistry, respectively, which was similar to fresh controls. Different sample types such as larger tissue sections, tissue from deceased human donors (both small and large sections) and one dowel stored for 3 months all demonstrated similar cell recovery as determined by membrane integrity stains. This is the first report of successful vitrification of intact human articular cartilage on its bone base – a requirement for transplantation. We have demonstrated good to excellent cell recovery as determined by membrane integrity stains. The surviving cells are functional and can produce cartilage specific macromolecules such as sulphated proteoglycans and collagen II indicating that these cryopreserved cells should be able to maintain the cartilage matrix over the long term after transplantation. The work in this abstract has been submitted for journal publication.