Metformin-Enhanced Secretome from Periodontal Ligament Stem Cells Promotes Functional Recovery in an Inflamed Periodontal Model: In Vitro Study.

OBJECTIVE: Secretory factors, termed the secretome, in the conditioned medium (CM) from dental mesenchymal stem cells (MSCs) have shown anti-inflammatory, anti-apoptotic, and tissue regenerative potential. This cell-free product could be further developed by preconditioning cells with various biochemical agents, which lead to a change in secretome and CM profiles. Among the favorable candidates for CM production, metformin as an anti-diabetic medication is currently considered a potential agent for dental hard tissue and periodontal regeneration. Here, we aimed to assess the composition of CM from periodontal ligament stem cells (PDLSCs) grown in metformin-preconditioned media (Met-CM) compared to normal PDLSC-CM and assess the ability of Met-CM to recover the function of inflamed PDLSCs. METHODS: Met-CM and normal CM were collected from PDLSCs grown with or without 50 µM metformin, respectively, under healthy culture conditions. Mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were performed to comparatively evaluate the proteomic profiles in PDLSC-CM versus Met-CM. We then treated the PDLSC cultures with lipopolysaccharide (LPS) from Porphyromonas gingivalis to induce inflammation and evaluated the osteogenic/cementogenic differentiation in the presence of Met-CM or normal PDLSC-CM by assessing alkaline phosphatase activity, intracellular calcium levels, and mRNA expression of osteogenic and cementogenic factors, including RUNX2, OCN, OSX, and CEMP-1. Subsequently, we performed RNA sequencing to identify transcriptomic changes in the treated cells. RESULTS: We identified 202 differentially expressed proteins, 175 of which were significant, in Met-CM versus normal PDLSC-CM. Among the analyzed groups, the top three protein classes were protein-binding activity modulator, cytoskeletal protein, and extracellular matrix (ECM) protein. Treatment of PDLSCs with LPS significantly attenuated ALP activity, [Ca(2+)](i), and the mRNA expression levels of RUNX2, OCN, OSX, and CEMP-1, whereas treatment with Met-CM alone markedly enhanced PDLSC differentiation activity compared with the control. Moreover, osteogenic/cementogenic differentiation of the LPS-treated PDLSCs was recovered through incubation in Met-CM. Transcriptomic analysis identified 511 and 3591 differentially expressed genes in the control versus Met-CM and LPS versus LPS + Met-CM groups, respectively. The enrichment of biological processes includes positive regulation of DNA-templated transcription and skeletal system morphogenesis in the control versus Met-CM comparison, as well as positive regulation of transcription from the RNA polymerase II promoter and negative regulation of the apoptotic process in the LPS versus LPS + Met-CM comparison. Molecular function analysis demonstrated the enrichment of protein-binding terms among the DEGs from each comparison. CONCLUSIONS: Metformin preconditioning enhanced the recovery effect of PDLSC-CM on LPS-induced inflamed PDLSCs. These findings suggest that metformin preconditioning could represent a practical formula for PDLSC-secretome, which may contribute to the development of future cell-free periodontal regenerative strategies.