Investigation On The Effect And Regulation Mechanism Of Lactate On Osteochondral Tissue Cells

Tissue engineering and regenerative medicine stratigies have been developed for bone and cartilage injury therapies, poly(lactic-co-glycolic acid)(PLGA)and poly-1-lactate acid (PLLA) are polymers that have been widely used as scaffolds for bone and cartilage injury therapies. Studies have shown that the degradation products of PLGA and PLLA may affect bone and cartialge tissues, however the effect of lactate, which is the main degradation product of PLGA and PLLA, on bone and cartialge tissues is largely unknown. This would warrant further investigation on the biological effects of lactate on bone and cartilage tissue cells. Our study can be divided into two parts:Part Ⅰ Effect of lactate on chondrocytesThe effects of lactate on Osteoarthritic (OA) and normal chondrocytes have been studied in this part, the main findings are as followed:1.1 Lactate regulates osteoarthritis development in OA chondrocytes in vitroThe amounts of lactate and the pH value (acid) of the PLGA and PLLA degradation medium were measured. The effects of PLGA and PLLA degradation medium, as well as different lactate concentrations and timing of exposure on chondrocytes proliferation and cartilage-specific matrix synthesis were investigated by various techniques including global gene expression profiling and gene knockdown experiments. It was shown that PLGA and PLLA degradation medium differentially regulated chondrocyte proliferation and matrix synthesis. Acidic pH caused by lactate inhibited chondrocyte proliferation and matrix synthesis. The effect of lactate on chondrocyte matrix synthesis was both time and dose dependent. A lactate concentration of 100mM and exposure duration of 8h significantly enhanced matrix synthesis. Lactate could also inhibit expression of cartilage matrix degradation genes in osteoarthritic chondrocytes, such as the major aggrecanase ADAMTS5, whilst promoting matrix synthesis simultaneously. Pulsed addition of lactate was shown to be more efficient in promoting COL2A1 expression. Global gene expression data and gene knock down experiments demonstrated that lactate promote matrix synthesis through up-regulation of HIF1A. These observed differential biological effects of lactate on chondrocytes would have implications for the future design of polymeric cartilage scaffolds.1.2 Lactate affects the phenotype maintenance of normal chondrocytes in cultureNormal chondrocytes were passaged from P1 to P5, and the gene expression levels of matrix proteins such as COL2A1, AC AN and SOX9 were detected in control and lactate treated groups. Our resutls showed a significant higher level of COL2A1, AC AN and SOX9 expression in lactate treated group. Further we found that lactate could up-regulate glycolysis related genes expression in chondrocytes, and studies in hypoxia and TGF-β3 treated as well as passaged chondrocytes showed that glycolysis was positively correlated to the phenotype of chondrocytes, which implied that the mechanism that lactate promotes chondrocyte phenotype maintenance may through glycolysis stimulation. Finally, we showed that blockage of glycolysis in chondrocytes by 2-Deoxy-D-glucose markedly reduced lactate-induced gene expression of matrix proteins. Together, these results indicate that lactate promotes phenotype maintenance in normal chondrocytes through glycolysis stimulation.Part Ⅱ Effect of lactate on mesenchymal stem cellsMesenchymal stem cells are main progenitor cells for bone tissue, a remarkable feature of stem cells is the potential of their self-renewal capacity, the role of lactate on the regulation of stem cells self-renewal is unclear. We here studied the effect of lactate on self-renewal capacity of stem cells, and the underlying mechanism involved KDM6B regulation of glycolysis. We found that although lactate inhibits the proliferation of hMSC, low concentration of lactate promotes hMSC self-renewal capacity as well as its stemness gene expression. The mechanism study showed that lactate may up-regulate hMSC glycolysis, and glycolysis is positively correlated to self-renewal capacity of hMSC, inhibition of glycolysis down-regulates lactate induced self-renewal capacity in hMSC. Further we found that glycolysis in hMSC is regulated by KDM6B, and lactate promotes hMSC self-renewal capacity through KDM6B mediated glycolysis up-regulation. These results revealed a new physiological regulatory function of lactate in hMSC.