Xiong Yanshan, Shen Ting, Xie Xiaoli
Xiangya Stomatological Hospital, Xiangya School of Stomatology, Central South University, Key Laboratory of Oral Health Research, Hunan 3D Printing Engineering Research Center of Oral Care, Hunan Clinical Research Center of Oral Major Diseases and Oral Health, Changsha, 410008, Hunan, China.
J Dent Sci. 2022 Jul;17(3):1135-1143. doi: 10.1016/j.jds.2022.01.007. Epub 2022 Feb 7.
BACKGROUND/PURPOSE: 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.
背景/目的：脱矿牙本质基质（DDM）用作组织再生支架。有效保存DDM有利于临床应用。冷冻保存和冻干可能是保留DDM力学性能和生物活性的有效方法。
采用酶解法分离人牙周膜干细胞（hPDLSCs），通过多向分化和流式细胞术进行鉴定。用乙二胺四乙酸（EDTA）制备DDM，并分为四组：新鲜DDM（fDDM）、室温保存的DDM（rtDDM）、冷冻保存的DDM（cDDM）和冻干DDM（fdDDM）。观察DDM表面形态，检测显微硬度。采用酶联免疫吸附测定（ELISA）法检测DDM液体提取物中转化生长因子-β1（TGF-β1）、成纤维细胞生长因子（FGF）和Ⅰ型胶原（COL-Ⅰ）的浓度。将hPDLSCs与DDM液体提取物共培养。采用CCK-8法检测DDM对细胞增殖的影响。采用实时定量聚合酶链反应（qPCR）检测DDM对hPDLSCs分泌的磷蛋白-1（SPP1）、骨膜蛋白（POSTN）和COL-Ⅰ基因表达的影响。
cDDM的牙本质小管比其他组的大。三种保存条件对DDM显微硬度和COL-Ⅰ浓度无显著影响。然而，保存后TGF-β1和FGF浓度降低，rtDDM变化最大，其次是fdDDM和cDDM。fDDM、cDDM和fdDDM的液体提取物对hPDLSCs增殖有轻微抑制作用，但rtDDM的液体提取物无显著影响。用fDDM、cDDM和fdDDM液体提取物培养的hPDLSCs显示SPP1、POSTN和COL-Ⅰ基因表达增加。
冷冻保存和冻干能更好地维持DDM的力学性能和生物活性。
