Development of a KLD-12 polypeptide/TGF-β1-tissue scaffold promoting the differentiation of mesenchymal stem cell into nucleus pulposus-like cells for treatment of intervertebral disc degeneration

摘要

Objective: To develop tissue engineering scaffolds consisting of self-assembling KLD-12 polypeptide/TGF-β1 nanofiber gel, for the induction of mesenchymal stem cell (MSCs) differentiation into nucleus pulposus (NP)-like cells. Methods: The release of TGF-β1 from KLD-12 polypeptide gels containing varying TGF-β1 concentrations was detected by ELISA. MSCs were isolated with a density gradient method and their differentiation into NP-like cells was analyzed in KLD-12 polypeptide/TGF-β1- or KLD-12 polypeptide control nanofiber-gel 3D-cultures. The Alcianblue method, Real-time quantitative PCR (RT-qPCR), and immunocytochemistry were used to measure the expression of extracellular matrix (ECM) molecules, such as aggrecan, glycosaminoglycans (GAGs), and type II collagen. Results: ELISA results documented favorable time-dependent release characteristics of TGF-β1 in the KLD-12 polypeptide/TGF-β1 gel scaffolds. The results of CCK-8 cell proliferation assay showed the TGF-β1 containing scaffolds induced higher growth rate in MSCs compared to the control group. Calcein-AM/PI fluorescent staining showed: the cells in the gel grew well, maintaining the circular shape of cells, and the spindle and fusiform shape of cells on the gel edges. The cell viability displayed a survival rate of 89.14% ± 2.468 for the TGF-β1 group with no significant difference between the two groups at 14 d of culture. The production of ECM was monitored showing higher expression of GAGs in the TGF-β1 group (P < 0.01) with highest amounts at 10 d and 14 d compared to 4 d and 7 d (P < 0.05). Real-time PCR results revealed that the expression levels of collagen II and aggrecan mRNA were higher in the TGF-β1 group (P < 0.05). Finally, immunocytochemical staining of collagen II confirmed the higher expression levels. Conclusion: A scaffold containing a KLD-12 polypeptide/TGF-β1-nanofiber gel and MSCs differentiated into NP-like cells is able to produce ECM and has the potential to serve as a three-dimensional (3-D) support scaffold for the filling of early postoperative residual cavities and the treatment of intervertebral disc degeneration.