High-throughput Screening Stepwise Tenogenic Culture System And Tissue Engineering Strategy For Tendon Tissue Repair

Tendon stem/progenitor cells(TSPCs)are potential seed cells in tendon tissue engineering and regeneration,but in vitro expanded TSPCs are prone to lose phenotype.Small molecules are easy to synthesize,save and control quality,we intend to explore the use of high-throughput screening to obtain small molecule compounds for in vitro proliferation and phenotype maintenance of TSPCs,and establish a stable culture system.We used Scx GFP fluorescence visualization report system to high-throughput screen small molecules from FDA approved drug library and bioactive compound library,and obtained candidate molecular groups.Positive small molecules were further verified by secondary screening.The concentration of each molecule was optimized to obtain the best small molecule cocktails which maintain the phenotype and initiate tenogenesis.We found that the small molecule cocktails could not promote cell proliferation while maintaining phenotype.Therefore,we also screened small molecules which promote cell proliferation through high-content cell analysis.Future,we used single cell analysis to confirm the differentiation,and obtained the best combination of small molecules through comparative experiments.Based on the above findings,we have established an effective stepwise strategy for tenogenic induction.The first step is to promote cell proliferation and the second step is to induce tendon differentiation by small molecules.Finally,combined with biocompatible hydrogel,we can achieve the application in vivo.Through 3D printing technology,we can combine the stepwise induced living cells with hydrogel,and promote tendon regeneration and repair in vivo.In summary,the study established a small molecule strategy to induce TSPCs stepwise differentiation,combined the strategy with 3D printing hydrogel in vivo and proved the feasibility and efficacy of small molecule cocktails in the application of tendon repair.