Biological Evaluation Of Chondrogenic Differentiation Of MSCs/iPSCs With Modiifed Glucosamine

Glucosamine(GA) and its acidylated derivative, which has many important physiologicalfunctions in vivo, is the basic constitution unit of many important polysaccharides in cells.Researches on the effectiveness of glucosamine and its derivatives, especially the effect on thechondrogenesis ability mediated by cells or tissue engineering scaffolds and the exploration ofcorresponding mechanism, have become more and more attracting. In our study, we firstsynthesized N-acryloyl-glucosamine (AGA), then we compared its chondrocytes inducedcapacity with the raw materials Glucosamine hydrochloride (GAH) and the other GAderivaties N-acetyl-glucosamine (GlcN.Ac) by induced Mesenchymal Stem Cells (MSCs) tochondrocytes, preliminary study results showed that there is a new kind of seed cell incartilage engineering and the effectiveness of GA and its derivatives is worthy to beinvestigated.In this study, GAH was chemically modified with acryloyl chloride. The syntheticproduct AGA of high purity was acquired by regulating the amount of acetone added in thereaction as solvent and the yield was32%. The process avoided the procedure of purificationby column chromatography and optimized the synthetic conditions. In our study, the cellcompatibility and chondrogenic induction capacity is determined by adding GAH, GlcN.Acand AGA of different molar mass into the culture medium. Result of Live/Dead assaysuggested that hMSCs in culture medium of different groups adhereed well and proliferatednormally. However, GAH produced higher toxicity when in higher concentration, resulting incell death during induced culture. Meanwhile, GlcN.Ac group had a greater impact on themodality of MSCs, resulting in atypical cell hypertrophy. Water-soluble tetrazolium (WST)suggested that compounds of different molar mass had a dose-dependent effect on cellproliferation. With increasing the concentration of the three GA compounds, cell densityincreased at corresponding time points. However, toxicity caused by high concentration ofGAH and atypical cell hypertrophy caused by high concentration of GlcN.Ac still remained.miRNA level of cells in different groups was characterized after cultured in chondrogenicinduction medium for7days. The results suggested that the three GA compounds were able topromote the cartilage differentiation and expression of MSCs, to some extent. AGA groupexpressed the best, and its expression levels of three target genes, Sox9, Collagen II andAggrecan, surpassed the other two and the control group. However, induced culture didn’t lastfor long, while the expressions of Collagen II and Aggrecan asked for a long time,. As a result,the two genes didn’t express obviously. Alcian blue and Collagen II staining of different groups presented positive, suggesting that hMSCs was successfully induced into chondrocytes.And different groups held different abilities, for instance,, chondrogenic induction of thesystem became more obviously with the content of AGA in chondrogenic induced mediumincreased. However, glycosaminoglycans (GAG) and the relative content of DNA inexperimental groups showed little help in explaining the differentiation of MSCs intocartilage induced by the three GA compounds. The differentiation might also be influenced bythe hours of chondrogenic induction. Chondrogenic induction experiment in vitro alsosuggested that TGF--β3(Transforming growth factor beta3) might promote the expression ofCollagen II and Aggrecan of host cells and thus the formation of cartilage cells. Moreover, theeffect became more obviously in the presence of AGA, suggesting that the synergies of AGAand TGF-β3to promote the chondrogenic capacity of MSCs was obvious.Induced pluripotent stem cells (iPSCs) are widely used nowadays. They not only, tosome extent, settle the ethical issues in medical caused by embryonic stem cells (ESCs) andmesenchymal stem cells (MSCs), but also overcome the shortcomings caused by differentgenetic background.We hope to induce hiPSCs into hMSCs by adding three growth factor:platelet-derived growth factor AB (PDGF AB), fibroblast growth factor (bFGF), epidermalgrowth factor (EGF). And by directly induced to chondrocytes just as MSCs, we found thatiPSCs harvested higher Collagen II and Aggrecan than MSCs. Preliminary study on the iPSCschondrogenic capacity showed that there is a possibility that we can find an effective way ofusing new cartilage tissue engineering seed cells for cartilage repair.