Coordinated regulation of mirnas to induce chondrogenesis and inhibit angiogenesis in stem cells and ECM materials under hypoxic environment

摘要

Background: Currently, cartilage repair biomaterials loaded with or without cytokines have been widely studied. But few have achieved complete and stable cartilage regeneration. One of the crucial reasons is that the mesenchymal stem cells (MSCs) seeded in cartilage repair biomaterials would be affected in the microenvironment under hypoxia. Hypoxia would potentially induce angiogenesis, resulting cartilage secondary degeneration. To explore the antagonism of chondrogenesis and angiogenesis, we investigated the coordinated regulation of epigenetic mechanisms in MSCs and ECM materials under hypoxic environment. The present study is aimed to guide the design and synthesis of biological adaptive cartilage repair biomaterials. Materials and Methods: C3H10T1/2 cell line was used in this study. C3H10T1/2 cell line seeded as pellet or in ECM materials was added chondrogenic medium (with 10 ng/mL TGFP3) or DMEM medium for 21 days under hypoxia (2% 02) or normoxia (21% 02). Genes and miRNAs related with chondrogenesis and angiogenesis were detected by RT-qPCR technique on Day 7,14,21. Dual-luciferase report system was used to explore the regulating mechanisms of miRNAs on angiogenesis. Result: Pellet culture showed higher expression of Sox9, Col2a1, Acan, and lower expression of Mmp13 and Adamts5 with chondrogenic medium than with none chondrogenic medium (P＜0.05), which indicated chondrogenesis of C3H10T1/2 cell line. As previously reported, HIF1α was up-regulated under hypoxia environment compared with normoxia environment (P＜0.05). Meanwhile, Sox9, Col2a1 and Acan were higher expressed and Adamts5 and Mmp13 were lower expressed (P＜0.05). As a key regulator, miR-140 exhibited higher expression and miR-146 lower expression. ECM materials were mainly composed of hyaluronic acid and chondroitin sulfate crosslinking the form of hydrogel. Hoechst 33258 fluorescent staining validated its cytocompatibility with few dead cells and immunohistochemical staining showed abundant Collagen type Ⅱ on Day 21 in the chondrogenic induction of C3H10T1/2 cells. The chondrogenic phenotype was more stabilized in the hydrogel of ECM materials with chondrogenic medium than DMEM medium, showing higher Sox9, Col2a1, Acan while lower Adamts5, Mmp13 expressed (P＜0.05). At the same time, miR-140 was up-expressed and miR-146 down-expressed. DuaMuciferase report assays demonstrated that miR-140 directly targets VEGFα by binding its 3-UTR. Conclusions: Bio-adaptation of chondrogenesis and anti-angiogenesis would be coordinated by means of regulation of MSCs differentiation with biological active factors, ECM materials and hypoxia. Up-regulation of miR-140 and down-regulation of miR-146 probably played important regulating effects on promoting chondrogenesis as well as inhibiting angiogenesis. Bioactive modification with these regulatory molecules would enhance the bio-adaptation of cartilage repair materials.