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

Yuan Li; Shuyin Zhang; Wantao Li; Xueni Zheng; Yang Xue; Kaijin Hu; Hongzhi Zhou

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

Am J Transl Res. 2021 Apr 15;13(4):1990-2005. eCollection 2021.

Authors

Yuan Li¹, Shuyin Zhang¹, Wantao Li¹, Xueni Zheng¹, Yang Xue¹, Kaijin Hu¹, Hongzhi Zhou¹

Affiliation

¹ State Key Laboratory of Military Stomatology, National Clinical Research Center for Oral Diseases, Shaanxi Clinical Research Center for Oral Diseases, Department of Oral and Maxillofacial Surgery, School of Stomatology, The Fourth Military Medical University Xi'an 710032, PR China.

PMID: 34017371
PMCID: PMC8129421

Abstract

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.

Keywords: Tissue engineering; biocompatibility; hUCMSCs; multidirectional differentiation; self-assembled polypeptide.