Xenogenous implanted dental follicle stem cells promote periodontal regeneration throughinduces N2 phenotype of neutrophils

Abstract

Background Periodontal tissue loss is the main reason of tooth mobility and loss caused by periodontal disease. Dental follicle stem cells (DFSCs) have significant therapeutic potential in periodontal regeneration, which maybe mainly depends on its potent immunomodulatory capacity. Consequently, this study aims to elucidate the impact of implanted xenogenous DFSCs on innate immune responses during early and late stage in periodontal defect repair period. Methods To trace and investigate the immunomodulation mechanisms of DFSCs in vivo, DFSCs were engineered (E-DFSCs) using lentiviral vectors expressing CD63-enhanced green fluorescent protein (CD63-EGFP) and β-Actin-mCherry protein (ACTB-mCherry) to exhibit green and red fluorescence. The biological characteristics and functions of E-DFSCs were verified by proliferation, differentiation and co-culture experiments in vitro. In vivo, the periodontal regeneration capacity of E-DFSC was detected by implantation of murine periodontal defect model, and the response of innate immune cells were detected at 1st ,3rd, 5th day (early stage) and 4th week (late stage) after implantation. Results In vitro assessments showed that E-DFSCs retain similar properties to their non-engineered counterparts but exhibit enhanced macrophage immunomodulation capability. In mice models, four-week micro-CT and histological evaluations indicated that E-DFSCs have equivalent efficiency to DFSCs in periodontal defects regeneration. At the early stage of repair in mice periodontal defect, fluorescence tracking showed that implanted E-DFSCs might primarily activate endogenous cells through direct contact and indirect actions, and most of these cells is myeloperoxidase positive neutrophils. Additionally, compared with the control group, the neutrophils infiltration and conversion of N2-type were significantly increased in the E-DFSC group. At the late stage of defect regeneration, more M2-type macrophages, fewer TRAP + osteoclasts and upregulated OPG/RANKL ratio were detected in the E-DFSC group compared to the control group, which indicated that immune balance tilt toward healing and bone-formation. Conclusion The xenogenous implanted DFSCs can induce N2 phenotype of neutrophils in early stage, which can activate the innate immune mechanism of the host to promote periodontal tissue regeneration.