chondroitin-sulfates and tetramethylpyrazine

Cryoprotective agents (CPAs) are used in cryopreservation protocols to achieve vitrification. However, the high CPA concentrations required to vitrify a tissue such as articular cartilage are a major drawback due to their cellular toxicity. Oxidation is one factor related to CPA toxicity to cells and tissues. Addition of antioxidants has proven to be beneficial to cell survival and cellular functions after cryopreservation. Investigation of additives for mitigating cellular CPA toxicity will aid in developing successful cryopreservation protocols. The current work shows that antioxidant additives can reduce the toxic effect of CPAs on porcine chondrocytes. Our findings showed that chondroitin sulphate, glucosamine, 2,3,5,6-tetramethylpyrazine and ascorbic acid improved chondrocyte cell survival after exposure to high concentrations of CPAs according to a live-dead cell viability assay. In addition, similar results were seen when additives were added during CPA removal and articular cartilage sample incubation post CPA exposure. Furthermore, we found that incubation of articular cartilage in the presence of additives for 2 days improved chondrocyte recovery compared with those incubated for 4 days. The current results indicated that the inclusion of antioxidant additives during exposure to high concentrations of CPAs is beneficial to chondrocyte survival and recovery in porcine articular cartilage and provided knowledge to improve vitrification protocols for tissue banking of articular cartilage.

Topics: Animals; Ascorbic Acid; Cartilage, Articular; Cell Physiological Phenomena; Cell Survival; Chondrocytes; Chondroitin Sulfates; Cryopreservation; Cryoprotective Agents; Glucosamine; Pyrazines; Swine; Tissue Banks; Vitrification

High concentrations of cryoprotective agents are required for cryopreservation techniques such as vitrification. Glycerol is a common cryoprotective agent used in cryopreservation protocols but this agent is toxic at high concentrations. This work is an attempt to mitigate the toxic effects of high concentrations of glycerol on intact chondrocytes in human knee articular cartilage from total knee arthroplasty patients by simultaneous exposure to glycerol and a variety of additive compounds. The resulting cell viability in the cartilage samples as measured by membrane integrity staining showed that, in at least one concentration or in combination, all of the tested additive compounds (tetramethylpyrazine, ascorbic acid, chondroitin sulphate, glucosamine sulphate) were able to reduce the deleterious effects of glycerol exposure when examination of membrane integrity took place on a delayed time frame. The use of additive compounds to reduce cryoprotectant toxicity in articular cartilage may help improve cell recovery after cryopreservation.

Topics: Aged; Aged, 80 and over; Ascorbic Acid; Cartilage, Articular; Cell Survival; Chondrocytes; Chondroitin Sulfates; Cryopreservation; Cryoprotective Agents; Glucosamine; Glycerol; Humans; Middle Aged; Pyrazines; Vitrification