Effects of different methods of demineralized dentin matrix preservation on the proliferation and differentiation of human periodontal ligament stem cells

Demineralized dentin matrix (DDM) is used as a tissue regeneration scaffold. Effective preservation of DDM benefits clinical applications. Cryopreservation and freeze-drying may be effective methods to retain DDM mechanical properties and biological activity.Human periodontal ligament stem cells (hPDLSCs) isolated using enzymatic dissociation were identified by multidirectional differentiation and flow cytometry. DDM was prepared with EDTA and divided into four groups: fresh DDM (fDDM), room temperature-preserved DDM (rtDDM), cryopreserved DDM (cDDM) and freeze-dried DDM (fdDDM). The DDM surface morphology was observed, and microhardness was detected. Transforming growth factor-β1 (TGF-β1), fibroblast growth factor (FGF) and collagen-Ⅰ (COL-Ⅰ) concentrations in DDM liquid extracts were detected by enzyme-linked immunosorbent assay (ELISA). The hPDLSCs were cultured with DDM liquid extracts. The effect of DDM on cells proliferation was examined by CCK-8 assay. The effect of DDM on hPDLSC secreted phosphoprotein-1 (SPP1), periostin (POSTN) and COL-Ⅰ gene expression was examined by real-time qPCR.cDDM dentinal tubules were larger than those of the other groups. The three storage conditions had no significant effect on DDM microhardness and COL-Ⅰ concentration. However, TGF-β1 and FGF concentrations decreased after storage, with the greatest change in rtDDM, followed by fdDDM and cDDM. The liquid extracts of fDDM, cDDM and fdDDM slightly inhibited hPDLSCs proliferation, but those of rtDDM had no significant effect. The hPDLSCs cultured with fDDM, cDDM and fdDDM liquid extracts showed increased SPP1, POSTN and COL-Ⅰ gene expression.Cryopreservation and freeze-drying better maintain the mechanical properties and biological activity of DDM.