Xenogenous implanted dental follicle stem cells promote periodontal regeneration through inducing the N2 phenotype of neutrophils

Abstract

Background Periodontal tissue loss is the main reason for 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 their potent immunomodulatory capacity. Consequently, this study aims to elucidate the impact of implanted xenogenous DFSCs on innate immune responses during early and late stages in the 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-DFSCs was detected by implantation of murine periodontal defect model, and the response of innate immune cells was detected at the 1st, 3rd, and 5th days (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 defect 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 are myeloperoxidase-positive neutrophils. Additionally, compared with the control group, the neutrophilic infiltration and conversion of N2-type were significantly increased in the E-DFSC group. At the late stage of defect regeneration, more M2-type

1. Introduction

Periodontitis, a highly prevalent oral infectious disease, is characterized by the formation of periodontal pockets, attachment loss, and alveolar bone resorption, ultimately leading to tooth loss [1]. Additionally, periodontal infection is linked to systemic diseases such as cardiovascular diseases, diabetes [2], and Alzheimer’s disease [3], posing significant risks to overall health. Although current clinical treatments effectively manage pathogenic bacteria and inflammation, they are insufficient for the repair of defects in soft and hard periodontal tissues caused by periodontitis [4]. The presence of periodontal defects will repeatedly induce periodontitis activity and promote disease progression. Traditional surgical interventions employ primitive stem cells to create new periodontal attachments; however, the local inflammatory microenvironment often impairs the functionality of these cells, hindering periodontal regeneration [5, 6]. Recently, cell therapy has emerged as a prominent direction in periodontal tissue engineering research, with the local transplantation of exogenous stem cells showing efficacy in enhancing periodontal regeneration [6].

Dental follicle stem cells (DFSCs), recognized as ideal seed cells, are harvested from tissues in the tooth germ developmental stage and exhibit capabilities such as self-renewal, multi-differentiation, migration, colonization, and immunoregulation [7]. They also offer advantages for periodontal regeneration due to their abundant sources, ease of expansion, and low immunogenicity [8]. The efficacy of DFSCs in promoting periodontal regeneration in animal models of periodontitis or periodontal defects has been confirmed, although the specific mechanisms remain to be elucidated [7, 9–11]. Numerous studies have explored the functional mechanisms of mesenchymal stem cells (MSCs) post-implantation in vivo, proposing various theories [12]. As research advances, the initial trans-differentiation mechanism struggles to account for the brief survival of implanted xenogeneic and allogeneic MSCs, as well as the disparity between the sites of periodontal regeneration and cell implantation [13]. Recent investigations have shifted focus toward the paracrine and immunomodulatory roles of MSCs, which are now considered critical for tissue regeneration. This study aims to elucidate the impact of implanted xenogenous DFSCs on innate immune responses during early and late stages in the periodontal defect repair period, providing insights into the mechanisms underlying DFSC-mediated periodontal regeneration.