Role of Periodontal Ligament Stem Cells in BLXA4-induced Periodontal Regeneration

Abstract

Periodontal ligament stem cells (PDLSCs) maintain periodontal tissue homeostasis and play a critical role in tissue regeneration and the resolution of inflammation. They release Specialized Pro-resolving Lipid Mediators (SPMs) to regulate inflammation, but less is known about how SPMs stimulate PDLSCs to promote regeneration. This study aims to determine the role of Göttingen Miniature PDLSCs (mpPDLSCs) in Benzo-lipoxin A4 (BLXA4)- mediated periodontal regeneration. The study was conducted on four Göttingen female minipigs (~30 kg; 12-36 months old). This study was designed as a single experimental arm for each interventional group, where each minipig had eight surgically created two-wall bony defects in all four posterior quadrants to establish the periodontal disease model. The interventional groups were allocated as Control, BLXA4, BLXA4+PDLSC, and PDLSCs. After surgical debridement, the groups receiving the BLXA4 treatment were administered 0.05 μg/defect in 18 μl of the BLXA4 lipid nanoparticle, while those receiving PDLSCs had 1x106 cells of the cell suspension following the surgical debridement and the control group received only surgical debridement. Bone regeneration was assessed by microCT. The mpPDLSCs were isolated from freshly extracted lateral incisors from the same host pig. Stem cell markers were characterized by using flow cytometry (CD11b, CD163, CD90, CD105). The multilineage differentiation of mpPDLSCs was assessed by osteogenic, adipogenic, and fibroblastic differentiation. Alizarin Red S staining was used to evaluate osteogenic differentiation; Oil-Red O staining was used for adipogenic differentiation; and Picro Sirius Red staining was used for fibroblastic differentiation after 21 days. A colony-forming unit-fibroblast (CFU-f) assay was utilized to assess cell proliferation using 0.5% crystal violet staining. Furthermore, the in vitro impact of BLXA4 on mpPDLSCs were investigated concerning cell viability along with SPM receptor expression (FPR2 and GPR32) using immunocytochemistry. The isolated mpPDLSCs expressed mesenchymal stem cell markers and lacked hematopoietic stem cell markers. mpPDLSCs were multipotent and differentiated along osteogenic, adipogenic, and fibroblastic lineages with appropriate stimulation. Seeding 100 cells per well in a 6-well plate resulted in colony formation after 10 days. Additionally, mpPDLSCs exhibited enhanced proliferation when stimulated with BLXA4, with or without E. coli LPS stimulation. Moreover, BLXA4-stimulated mpPDLSCs expressed FPR2 and GPR32. The lipidomic results indicated that BLXA4 and PDLSCs exhibited distinct effects on the lipid mediator profiles, leading to differential modulation of pro-inflammatory and pro-resolving pathways. It is noteworthy that their combination did not invariably result in additive effects, suggesting intricate and potentially synergistic interactions between BLXA4 signaling and PDLSC-mediated mechanism. The microCT data, in conjunction with the qualitative histological results, demonstrated that all interventional groups exhibited a significantly improved periodontal regeneration (P.05) in comparison to debridement alone. In summary, this study demonstrates the potential of mpPDLSCs in enhancing periodontal regeneration, alone or when stimulated with BLXA4. The findings indicate that BLXA4 promotes cell proliferation and stimulates the expression of receptors associated with inflammatory resolution (FPR2 and GPR32) in mpPDLSCs. The significant improvements in bone regeneration observed across all interventional groups underscore the therapeutic potential of combining SPMs with stem cell therapy. These results provide a strong foundation for a larger randomized clinical trial designed to further evaluate and translate SPM-enhanced stem cell therapy for periodontal regeneration in humans, potentially offering a novel approach to managing periodontal diseases.