Key Laboratory of Shannxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi'an Jiaotong University, Xi'an, Shannxi, 710004, China; Clinical Research Center of Shannxi Province for Dental and Maxillofacial Diseases, College of Stomatology, Xi'an Jiaotong University, Xi'an, Shannxi, 710004, China; Department of Advanced Oral Sciences and Therapeutics, University of Maryland Dental School, Baltimore, MD, 21201, USA.
Department of Advanced Oral Sciences and Therapeutics, University of Maryland Dental School, Baltimore, MD, 21201, USA; Department of Orthodontics, School of Stomatology, Capital Medical University, Beijing, China.
J Dent. 2020 Jan;92:103259. doi: 10.1016/j.jdent.2019.103259. Epub 2019 Dec 3.
Human periodontal ligament stem cells (hPDLSCs) are promising for periodontal regeneration. However, to date, there has been no report of hPDLSC differentiation into the fibrogenic lineage. There has been no report demonstrating hPDLSC differentiation into all three (osteogenic, fibrogenic and cementogenic fibrogenic) lineages in the same report. The objectives of this study were to harvest hPDLSCs from the periodontal ligaments (PDL) of the extracted human teeth, and use the same vial of hPDLSCs to differentiate into all three (osteogenic, fibrogenic and cementogenic) lineages for the first time.
hPDLSCs were harvested from PDL tissues of the extracted premolars. The ability of hPDLSCs to form bone, cementum and collagen fibers was tested in culture mediums. Gene expressions were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). Immunofluorescence, alizarin red (ARS), Xylenol orange, picro sirius red staining (PSRS), alcian blue staining (ABS) and alkaline phosphatase (ALP) staining were evaluated.
In osteogenic medium, hPDLSCs had high expressions of osteogenic genes (RUNX2, ALP, OPN and COL1) at 14 and 21 days (15-20 folds of that of control), and produced mineral nodules and ALP activity (5 and 10 folds those of the control). hPDLSCs in fibrogenic medium expressed high levels of PDL fibrogenic genes (COL1, COL3, FSP-1, PLAP-1 and Elastin) at 28 days (20-70 folds of control). They were stained strongly with F-actin and fibronection, and secreted PDL collagen fibers (5 folds of control). hPDLSCs in cementogenic medium showed high expressions of cementum genes (CAP, CEMP1 and BSP) at 21 days (10-15 folds of control) and synthesized mineralized cementum (50 folds via ABS, and 40 folds via ALP staining, compared to those of control).
hPDLSCs differentiated into bone-, fiber- and cementum-forming cells, with potential for regeneration of periodontium to form the bone-PDL-cementum complex.
人牙周韧带干细胞（hPDLSCs）在牙周再生方面具有广阔的应用前景。然而，迄今为止，尚无 hPDLSC 分化为纤维生成谱系的报道。也没有报道表明在同一份报告中 hPDLSC 能同时分化为成骨、纤维生成和矿化纤维生成三个谱系。本研究的目的是从人牙牙周韧带（PDL）中分离 hPDLSCs，首次使用同一瓶 hPDLSCs 将其分化为成骨、纤维生成和矿化纤维生成三个谱系。
从提取的前磨牙 PDL 组织中分离 hPDLSCs。在培养基中检测 hPDLSCs 形成骨、牙骨质和胶原纤维的能力。采用实时定量聚合酶链反应（qRT-PCR）分析基因表达。免疫荧光、茜素红（ARS）、二甲氧唑橙、苦味酸天狼猩红染色（PSRS）、阿利新蓝染色（ABS）和碱性磷酸酶（ALP）染色进行评估。
在成骨培养基中，hPDLSCs 在第 14 天和第 21 天高表达成骨基因（RUNX2、ALP、OPN 和 COL1）（对照的 15-20 倍），并产生矿化结节和 ALP 活性（对照的 5-10 倍）。在纤维生成培养基中，hPDLSCs 在第 28 天高表达牙周纤维生成基因（COL1、COL3、FSP-1、PLAP-1 和弹性蛋白）（对照的 20-70 倍）。它们的 F-肌动蛋白和纤维连接蛋白染色强烈，分泌牙周胶原纤维（对照的 5 倍）。在矿化纤维生成培养基中，hPDLSCs 在第 21 天高表达牙骨质基因（CAP、CEMP1 和 BSP）（对照的 10-15 倍），并合成矿化牙骨质（ABS 染色 50 倍，ALP 染色 40 倍，与对照相比）。
hPDLSCs 分化为骨形成细胞、纤维形成细胞和成牙骨质细胞，具有形成牙周复合体的骨-PDL-牙骨质的再生潜力。
