Substance P-induced RAD16-I scaffold mediated hUCMSCs stereo-culture triggers production of mineralized nodules and collagen-like fibers by promoting osteogenic genes expression.

Tissue engineering has become an important therapeutic method for injuries. This study aimed to generate collagen-like matrix constructed by hUCMSCs combining self-assembled polypeptide and evaluate differentiated capacity, safety and biocompatibility. Human umbilical cord tissues were isolated and used to primarily culture hUCMSCs. hUCMSCs were identified using immunofluorescence and flow cytometry. Adipogenic- and osteogenic-differentiation of hUCMSCs were evaluated using Oil-red O and Alizarin-Red staining. Self-assembling collagen peptide RAD16-I hydrogel and substance P (SP) were prepared and combined together to form RAD16-I/SP complex. Surface morphology and ultrastructures were observed with scanning electron microscopic (SEM). hUCMSCs in simulated collagen-like matrix environment were plane-cultured and stereo-cultured. Cell viability was examined using CCK-8 and fluorescent staining assay. Osteogenic genes were detected with qRT-PCR and western blot assay. HE staining and Masson staining were used to assess production of mineralized nodules and collagen-like fibers, respectively. Collagen-like matrix complex by combining RAD16-I/SP complex with stereo-cultured hUCMSCs was successfully generated. hUCMSCs in collagen-like matrix complex demonstrated adipogenic-differentiation and osteogenic-differentiation potential. SP-induced RAD16-I mediated stereo-culture of hUCMSCs demonstrated higher cell activity and proliferation potential. SP-induced RAD16-I mediated stereo-culture of hUCMSCs promoted osteogenesis-related molecules expression. SP-induced RAD16-I mediated stereo-culture of hUCMSCs promoted production of mineralized nodules and triggered formation of collagen-like fibers. Cell-collagen-like matrix complex injection (RAD16-I/SP/hUCMSCs complex) exhibited better biocompatibility and no cytotoxicity. In conclusion, SP-induced RAD16-I mediated stereo-culture of hUCMSCs remarkably promoted osteogenesis-related gene expression, triggered production of mineralized nodules and formation of collagen-like fibers. This established cell-collagen-like matrix complex (RAD16-I/SP/hUCMSCs) injection exhibited better biocompatibility, without cytotoxicity.