Transforming growth factor β1-enriched secretome up-regulate osteogenic differentiation of dental pulp stem cells, and a potential therapeutic for gingival wound healing: A comparative proteomics study.

Current study aimed at comparing the human dental pulp-derived stem cell (hDPSC) secretome (Control secretome) and transforming growth factor beta1 (TGF-β1)-transfected hDPSC secretome (TGF-β1 Secretome), which have the potential to be therapeutic in terms of regenerative dentistry, in terms of osteogenesis, adipogenesis and gingival wound healing with proteomic analyses. pCMV-TGF-β1 plasmid was transfected into hDPSCs by electroporation. hDPSC and TGF-β1 transfected hDPSC secretomes were collected for LC-MS/MS. Protein contents in control secretome and TGF-β1 secretome were analyzed by tandem mass spectrometry-based shotgun proteomic method. Bioinformatic evaluations for canonical pathways, upstream regulators and networks were completed via Ingenuity Pathway Analysis (IPA, QIAGEN) software. Surface marker expressions between groups, treated secretome were measured by flow cytometry. To support the proteomic data morphologically, we performed osteogenic-adipogenic differentiation in hDPSCs treated with control secretome and TGF-β1 secretome, and scratch wound healing assay in gingival fibroblasts. Statistical analyses were performed by GraphPad Prism 8.02. Venn diagram classification showed us 174 common proteins were identified from each group. In the control secretome 140 unique proteins were identified and 66 entries were exclusive for TGF-β1 secretome. TGF-β1 secretome was found to have therapeutic effect on MSC-specific immunophenotypes. TGF-β1 secretome was determined to up-regulate osteogenesis-related molecules and pathways while down-regulating adipogenesis-related pathways. Analysis of canonical pathways showed that TGF-β1 secretome is associated with the wound healing pathway. Our study provided the first evidence that proteins identified in TGF-β1-transfected hDPSC secretomes are potential regulators of osteogenic/adipogenic differentiation and fibroblast wound healing. Based on these results, TGF-β1 secretome may have a therapeutic effect in repairing osteoporosis-related bone injuries, wound healing of oral mucosa and gingival tissue. TGF-β1 secretome may be a potential cell-free therapeutic in orthopedics and regenerative dentistry.

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