The role of different macrophages-derived conditioned media in dental pulp tissue regeneration.

Macrophages have been reported to play important roles in tissue repair and regeneration. While it is known that macrophages are present in the dental pulp, their role in dental pulp regeneration is not fully understood. In the present study, we investigated the effects of different phenotype macrophages conditioned medium on the cellular behaviors of hDPSCs and their extracellular matrix (cell sheets) in vitro. Moreover, twenty-four root fragments inserted with cell sheets cultured with different conditioned media were placed into the back subcutaneous space of 6-8-week-old male BALB/c nude mouse. The regenerated tissues in the root fragments were assessed via histologic analysis after 8 weeks of transplantation. M2 macrophages could promote the proliferation, migration, and osteogenic differentiation of hDPSCs. Dental pulp-like tissue with an odontoblast-like layer lining the dentinal surface and well-arranged collagen fibers was harvested in root fragment combined with M2 conditioned medium cultured cell sheet, whereas a large amount of calcium salt deposition and disorganization of collagen fibers were observed in root fragments combined with M1 conditioned medium cultured cell sheet. Therefore, promoting the transformation of M1 into M2 macrophage in dental pulp tissue regeneration may be a potential way for dental pulp regeneration via functional healing.

The Role of Small Extracellular Vesicles Derived from Lipopolysaccharide-preconditioned Human Dental Pulp Stem Cells in Dental Pulp Regeneration.

INTRODUCTION: Regenerative endodontics has created a desirable shift in the treatment paradigm despite current limitations of regenerative outcomes. Mesenchymal stem cells (MSCs) facilitate tissue regeneration and repair in a mild inflammatory environment. Small extracellular vesicles (sEVs) derived from MSCs play an imperative role in the paracrine modulation of regenerative responses modulated by MSCs. However, it remains unknown whether MSCs enhance dental pulp regeneration or whether this enhancement is mediated by sEVs in a mild inflammatory environment. The present study aimed to elucidate the effects of sEVs originated from lipopolysaccharide (LPS)-preconditioned human dental pulp stem cells (hDPSCs) on dental pulp regeneration. METHODS: All sEVs were isolated from hDPSCs cultured with or without LPS (ie, N-sEVs and L-sEVs, respectively). The effect of N-sEVs and L-sEVs on proliferation, migration, angiogenesis, and differentiation of rat bone marrow MSCs was identified in vitro. Moreover, N-sEVs or L-sEVs were implanted into rat pulpless root canal models, and the regenerated tissue in root canals was assessed via hematoxylin-eosin staining, Masson staining, and immunohistochemistry after 30 days of transplantation. RESULTS: Both N-sEVs and L-sEVs could modulate BMSC proliferation, migration, angiogenesis, and differentiation. Both kinds of sEVs enhanced the structure of the regenerated tissue closer to that of a normal dental pulp in vivo. L-sEVs had a more significant effect than N-sEVs. CONCLUSIONS: sEVs released by hDPSCs in a mild inflammatory microenvironment are capable of facilitating the regeneration of dental pulp through functional healing instead of scar healing, which has potential applications in regenerative endodontics.