Bmp4-regulated Human Dental Pulp Stem Cells Promote Pulp-like Tissue Regeneration In A Decellularized Dental Pulp Matrix Scaffold

OBJECTIVE: This experiment aims to study the structure and function of decellularized human dental pulp extracellular matrix(dECM),and to investigate whether Bone Morphogenetic Protein 4(BMP4)regulates human dental pulp stem cells(hDPSCs)-mediated pulp regeneration combined with dECM.METHODS:In vitro studies: Dental pulp isolated from healthy third molars was decellularized with 10% sodium dodecyl sulfate(SDS)and Triton X-100,H&E,DAPI staining and electron microscopy were used to observe the structure.Cell Counting Kit-8 analyzes cell proliferation growth curve.Recombinant adenovirus was used to overexpress BMP4 in hDPSCs.Cells were cultured in dECM and dECM+three-dimensional(3D)Vitrogel systems,and the expression of bone/dentin/angiogenesis markers was evaluated by real-time polymerase chain reaction(RT-PCR)and ALP staining.In vivo studies: To evaluate pulp-like tissue formation,h DPSC mixed with dECM and BMP4 was transplanted into nude mice to evaluate the formation of pulp-like tissue.RESULTS: hDPSCs were successfully collected and indentified by flow cytometry,dECM were produced.Cell Counting Kit-8 assays showed that dECM can support h DPSC growth and proliferation without obvious cytotoxicity.The cells were cultivated in dECM and a three-dimensional(3D)Vitrogel system,and the expression of osteo-/odonto-/angiogenic markers was evaluated by real-time polymerase chain reaction.And pulplike tissue was formed in vivo.CONCLUSIONS: Thus,dECM promotes h DPSC proliferation,and BMP4+dECM accelerates pulp-like tissue formation by hDPSCs.