Cartilage storage at 4 °C with regular culture medium replacement benefits chondrocyte viability of osteochondral grafts in vitro

Abstract

Maintenance of articular cartilage allografts in culture media is a common method of tissue storage; however, the technical parameters of graft storage remain controversial. In this study, we examined the optimal temperature and culture medium exchange rate for the storage of osteochondral allografts in vitro. Cylindrical osteochondral grafts (n = 120), harvested from the talar joint surface of ten Boer goats, were randomly classified into four groups and stored under the following conditions: Group A1 was maintained at 4°C in culture medium that was refreshed every 2 days; Group A2 was maintained at 4°C in the same culture medium, without refreshing; Group B1, was maintained at 37°C in culture medium that was refreshed every 2 days; Group B2, was maintained at 37°C in the same culture medium, without refreshing. Chondrocyte viability in the grafts was determined by ethidium bromide/fluorescein diacetate staining on days 7, 21, and 35. Proteoglycan content was measured by Safranin-O staining. Group A1 exhibited the highest chondrocyte survival rates of 90.88 %, 88.31 % and 78.69 % on days 7, 21, and 35, respectively. Safranin O staining revealed no significant differences between groups on days 21 and 35. These results suggest that storage of osteochondral grafts at 4°C with regular culture medium replacement should be highly suitable for clinical application.

Assessment of Cell Viability of Fresh Osteochondral Allografts in N-Acetylcysteine-Enriched Medium

“…Moreover, other studies have evaluated the use of conventional culture media such as DMEM as an allograft storage medium, presenting cell viability at 28 days of close to 30% to 60%. 27,28 A relevant factor in the maintenance of cell viability in these studies was the need to replace the culture medium every 2 to 3 days, 15,29 which represents a disadvantage with respect to the medium evaluated in our study, which did not require any replacement, at least for 28 days. This could be explained by NAC's blockade of the cellular signaling pathway of nitric oxide in the regulation of chondrocyte apoptosis, 21 which could be maintained over time with a NAC concentration of 1 mM.…”

Chondrocyte Viability of Particulated Porcine Articular Cartilage Is Maintained in Tissue Storage After Cryoprotectant Exposure, Vitrification, and Tissue Warming

“…30,31 This finding is consistent with previous studies that show a correlation of prolonged storage time at 4 °C with a decrease in chondrocyte viability, proteoglycan loss, cellular apoptosis, and weakened matrix integrity, some of which can occur within 7 to 14 days of storage. 10,[32][33][34][35][36] Transplantation of particulated AC allografts has been developed over the past 2 decades as a viable option for the treatment of full thickness chondral defects. 7,37 Fresh allografts currently used are commercially provided as De Novo by Zimmer.…”

A Comparative Study Using Fluorescent Confocal Microscopy and Flow Cytometry to Evaluate Chondrocyte Viability in Human Osteochondral Allografts

“…Although several scientific studies have explored the best method for preserving fresh osteochondral grafts, no consensus has yet been reached regarding the ideal preservation media [ 4 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. Considering these previous studies as a whole, several preservation media appear to be suitable for maintaining the viability of osteochondral allografts, but it is unclear which medium facilitates better chondrocyte survival, owing to the heterogeneity of results among studies.…”

“…Evaluation of cell viability by confocal fluorescence imaging enables analysis of cartilage samples in their real state of preservation because cartilage does not require significant processing steps prior to analysis. Conversely, the post-analysis methodology used to count living and dead cells is less systematic, producing variations between studies [ 4 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. Mathematical treatment of fluorescence images to obtain a live-dead ratio involves a hand-made design of macro templates that include particle cluster fragmentation, and it establishes a fluorescence signal threshold from which the signal is considered a particle (and counted as a cell) or is discarded.…”