The Influence Of Hypoxia On Tenocyte Proliferation And The Preliminary Study On The Mechanism Of Cell Expansion

Objectives:Tenocyte is a kind of seed cells for constructing tissue engineered tendon. The constructed tendon is more similar to the native tendon in morphology, structure and function, etc. However, isolated and cultured tenocytes are not adequate to meet the constructing need because of small size biopsy, poor proliferative capacity, easy drift of their phenotype and loss of function during in vitro expansion. It has been studied that hypoxia can promote tenocyte proliferation. Therefore, we used different oxygen concentrations (2%O2,5%O2,10%O2,21%O2) in this study expanding tenocytes to identify the more suitable oxygen concentration for culture. And the apoptosis and cell cycle of tenocytes cultured in these oxygen concentrations were detected to explore whether the mechanism of tenocyte proliferation in hypoxia was related to the regulation of apoptosis or cell cycle. Considering lactic acid is the product of anaerobic metabolism, the concentration of lactic acid in cultured tenocytes was also examined. Moreover, tenocytes were cultured in the medium containing lactic acid under normoxia in order to observe the effect of lactic acid on tenocyte proliferation and identify whether hypoxia promoted cell proliferation by mediating lactic acid production. Our work will lay a foundation for studying molecular mechanism of hypoxia promoting cell proliferation in the future.Methods:1. Tenocytes were seeded onto 96-wcll plate under oxygen concentration of 2%, 5%,10%(experimental groups) or 21%(control group) for 8 days. Their optical density(OD) of each group was detected by CCK8 assay every day to analyze the effect of different oxygen concentration on passage 2 (P2) tenocyte proliferation.2. Tenocytes of P2 cultured under different O2 conditions were stained by Annexin V-FITC/PI to detect their cell apoptosis ratio.3. Tenocytes of P2 cultured under different O2 conditions were stained by DAPI to detect their cell cycle.4. Tenocytes of P2 cultured under 21% O2 were seeded onto 96-well plate, exposed to different lactate concentrations (5mmol/L, lOmmol/L,15mmol/L, 20mmol/L,25mmol/L) for 7 days. Their optical density(OD) of each group was detected by CCK8 assay every day to analyze the effect of different lactate concentration on tenocyte proliferation.Results:1.2%O2 and 5%O2 could promote P2 tenocyte proliferation compared with 21%O2, but 10%O2 could not promote it.2. The influence of 2%O2,5%O2,10%O2 and 21%O2 in tendon cell apoptosis had no significant differences.3. The influence of 2%C>2,5%O2,10%O2and 21%O2 in tendon cell cycle had no significant differences.4. Compared with no lactic acid in culture, the addition of 5-25mmol/L L-lactate to medium had a slight inhibitory effect on P3 tenocyte proliferation, but the effect was not statistically significant.Conclusion:In the range of 2%O2-10%O2, the expansion ability of P2 tenocytes was enhanced with the decrease of oxygen concentration, that is,2%O2 was the most favorable oxygen concentration in tendon cell proliferation. But tenocyte proliferation promoted by hypoxia was not regulated through cell apoptosis and cell cycle.L-lactic acid as main product in anaerobic metabolism had no effect on the proliferation of tendon cells.