BACKGROUND: Human periodontal ligament stem cells (hPDLSCs) are important candidate seed cells for periodontal tissue engineering, but the presence of lipopolysaccharide(LPS) in periodontal tissues inhibits the self-renewal and osteogenic differentiation of hPDLSCs. Our previous studies demonstrated that TAZ is a positive regulator of osteogenic differentiation of hPDLSCs, but whether TAZ can protect hPDLSCs from LPS is still unknown. The present study aimed to explore the regulatory effect of TAZ on the osteogenic differentiation of hPDLSCs in an LPS-induced inflammatory model, and to preliminarily reveal the molecular mechanisms related to the NF-κB signaling pathway.
METHODS: LPS was added to the culture medium of hPDLSCs. The influence of LPS on hPDLSC proliferation was analyzed by CCK-8 assays. The effects of LPS on hPDLSC osteogenic differentiation were detected by Alizarin Red staining, ALP staining, Western Blot and qRT-PCR analysis of osteogenesis-related genes. The effects of LPS on the osteogenic differentiation of hPDLSCs with TAZ overexpressed or knocked down via lentivirus were analyzed. NF-κB signaling in hPDLSCs was analyzed by Western Blot and immunofluorescence.
RESULTS: LPS inhibited the osteogenic differentiation of hPDLSCs, inhibited TAZ expression, and activated the NF-κB signaling pathway. Overexpressing TAZ in hPDLSCs partly reversed the negative effects of LPS on osteogenic differentiation and inhibited the activation of the NF-κB pathway by LPS. TAZ knockdown enhanced the inhibitory effects of LPS on osteogenesis.
CONCLUSIONS: Overexpressing TAZ could partly reverse the inhibitory effects of LPS on the osteogenic differentiation of hPDLSCs, possibly through inhibiting the NF-κB signaling pathway. TAZ is a potential target for improving hPDLSC-based periodontal tissue regeneration in inflammatory environments.

BACKGROUND: MicroRNAs are differentially expressed in periodontitis tissues. They are involved in cellular responses to inflammation and can be used as markers for diagnosing periodontitis. Microarray analysis showed that the expression level of microRNA-671-5p in periodontal tissues of patients with periodontitis was increased. In this study, we investigated the mechanism of action of microRNA-671-5p in human periodontal ligament stem cells (hPDLSCs) under inflammatory conditions.
RESULTS: HPDLSCs were treated with lipopolysaccharide (LPS) to establish an inflammation model. The cell survival rate was determined using the cell counting kit-8 (CCK8). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses were used to detect the expression of microRNA-671-5p and dual-specificity phosphatase (DUSP) 8 proteins, respectively, Interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α were detected using qRT-PCR and Enzyme-linked immunosorbent assay (ELISA). A dual-luciferase reporter system was employed to determine the relationship between micoRNA-671-5p and DUSP8 expression. Activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway was confirmed using western blot analysis. Following the treatment of hPDLSCs with LPS, the expression levels of microRNA-671-5p in hPDLSCs were increased, cell viability decreased, and the expression of inflammatory factors displayed an increasing trend. MicroRNA-671-5p targets and binds to DUSP8. Silencing microRNA-671-5p or overexpressing DUSP8 can improve cell survival rate and reduce inflammatory responses. When DUSP8 was overexpressed, the expression of p-p38 was reduced.
CONCLUSIONS: microRNA-671-5p targets DUSP8/p38 MAPK pathway to regulate LPS-induced proliferation and inflammation in hPDLSCs.

OBJECTIVE: To evaluate the efficacy of a modified sericin hydrogel scaffold loaded with dexamethasone (SMH-CD/DEX) scaffold for promoting bone defect healing by stimulating anti-inflammatory macrophage polarization.
METHODS: The light-curable SMH-CD/DEX scaffold was prepared using dexamethasone-loaded NH2-β-cyclodextrin (NH2-β-CD) and sericin hydrogel and characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), biocompatibility assessment and drug release test. THP-1 macrophages incubated with the scaffold were examined for protein expressions of iNOS and Arg-1, mRNA expressions of IL-6, Il-10, Arg-1 and iNOS, and surface markers CD86 and CD206 using Western blotting, RT-qPCR, and flow cytometry. In a co-culture system of human periodontal ligament stem cells (HPDLSCs) and THP-1 macrophages, the osteogenic ability of the stem cells incubated with the scaffold was evaluated by detecting protein expressions of COL1A1 and Runx2 and expressions of ALP, Runx2, OCN and BMP2 mRNA, ALP staining, and alizarin red staining. In a rat model of mandibular bone defect, the osteogenic effect of the scaffold was assessed by observing bone regeneration using micro-CT and histopathological staining.
RESULTS: In THP-1 macrophages, incubation with SMH-CD/DEX scaffold significantly enhanced protein expressions of Arg-1 and mRNA expressions of IL-10 and Arg-1 and lowered iNOS protein expression and IL-6 and iNOS mRNA expressions. In the co-culture system, SMH-CD/DEX effectively increased the protein expressions of COL1A1 and Runx2 and mRNA expressions of ALP and BMP2 in HPDLSCs and promoted their osteogenic differentiation. In the rat models, implantation of SMH-CD/DEX scaffold significantly promoted bone repair and bone regeneration in the bone defect.
CONCLUSIONS: The SMH-CD/DEX scaffold capable of sustained dexamethasone release promotes osteogenic differentiation of stem cells and bone defect repair in rats by regulating M2 polarization.

OBJECTIVE: This study aimed to investigate the effects of sitagliptin on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in lipopolysaccharide (LPS)-induced inflammatory microenvironment and its molecular mechanism.
METHODS: hPDLSCs were cultured in vitro and treated with different concentrations of sitagliptin to detect cell viability and subsequently determine the experimental concentration of sitagliptin. An hPDLSCs inflammation model was established after 24 h of stimulation with 1 µg/mL LPS and divided into blank, control, low-concentration sitagliptin (0.5 µmol/L), medium-concentration sitagliptin (1 µmol/L), and high-concentration sitagliptin (2 µmol/L), high-concentrationsitagliptin+stromal cell derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) pathway inhibitor (AMD3100) (2 µmol/L+10 µg/mL) groups. A cell-counting kit-8 was used to detect the proliferation activity of hPDLSCs after 24, 48, and 72 h culture. The apoptosis of hPDLSCs cultured for 72 h was detected by flow cytometry. After inducing osteogenic differentiation for 21 days, alizarin red staining was used to detect the osteogenic differentiation ability of hPDLSCs. The alkaline phosphatase (ALP) activity in hPDLSCs was determined using a kit. The levels of inflammatory factors [tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6] in the supernatant of hPDLSCs culture were detected by enzyme-linked immunosorbent assay. The mRNA expressions of osteogenic differentiation genes [Runt-associated transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN)], SDF-1 and CXCR4 in hPDLSCs were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Western blot analysis was used to determine SDF-1 and CXCR4 protein expression in hPDLSCs.
RESULTS: Compared with the blank group, the proliferative activity, number of mineralized nodules, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in the control group significantly decreased. The apoptosis rate and levels of TNF-α, IL-1β, and IL-6 significantly increased (P<0.05). Compared with the control group, the proliferative activity, number of mineralized nodule, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in low-, medium-, and high-concentration sitagliptin groups increased. The apoptosis rate and levels of TNF-α, IL-1β, and IL-6 decreased (P<0.05). AMD3100 partially reversed the effect of high-concentration sitagliptin on LPS-induced hPDLSCs (P<0.05).
CONCLUSIONS: Sitagliptin may promote the proliferation and osteogenic differentiation of hPDLSCs in LPS-induced inflammatory microenvironment by activating the SDF-1/CXCR4 signaling pathway. Furthermore, it inhibited the apoptosis and inflammatory response of hPDLSCs.
目的: 探讨西格列汀对脂多糖（LPS）诱导的炎症微环境下人牙周膜干细胞（hPDLSCs）增殖、凋亡、炎症和成骨分化的影响及分子机制。方法: 体外培养hPDLSCs，用不同浓度的西格列汀处理后检测细胞活力，以确定后续西格列汀实验浓度。采用1 µg/mL LPS刺激诱导24 h建立hPDLSCs炎症模型并分为空白组、对照组、西格列汀低浓度组（0.5 µmol/L）、西格列汀中浓度组（1 µmol/L）、西格列汀高浓度组（2 µmol/L）、西格列汀高浓度+基质细胞衍生因子-1（SDF-1）/CXC趋化因子受体4（CXCR4）通路抑制剂（AMD3100）组（2 µmol/L+10 µg/mL）。细胞计数试剂盒-8检测培养24、48、72 h后的hPDLSCs增殖活性；流式细胞术检测培养72 h后hPDLSCs凋亡情况；诱导成骨分化21 d后茜素红染色检测hPDLSCs成骨分化能力，试剂盒测定hPDLSCs中碱性磷酸酶（ALP）活性；酶联免疫吸附检测hPDLSCs培养上清液中炎症因子肿瘤坏死因子（TNF）-α、白细胞介素（IL）-1β、IL-6水平；实时荧光定量聚合酶链反应（RT-qPCR）检测hPDLSCs中成骨分化相关基因Runt相关转录因子2（RUNX2）、骨钙素（OCN）、骨桥蛋白（OPN）及SDF-1和CXCR4 mRNA表达；Western blot检测hPDLSCs中SDF-1、CXCR4蛋白表达。结果: 与空白组比较，对照组hPDLSCs增殖活性、矿化结节数量、染色强度、ALP活性和RUNX2、OCN、OPN mRNA及SDF-1、CXCR4 mRNA和蛋白表达水平显著降低，凋亡率、TNF-α、IL-1β、IL-6水平显著升高（P<0.05）；与对照组比较，西格列汀低、中、高浓度组hPDLSCs增殖活性、矿化结节数量、染色强度、ALP活性和RUNX2、OCN、OPN mRNA及SDF-1、CXCR4 mRNA和蛋白表达水平依次升高，凋亡率、TNF-α、IL-1β、IL-6水平依次降低（P<0.05）；AMD3100可部分逆转高浓度西格列汀对LPS诱导的hPDLSCs的作用效果（P<0.05）。结论: 西格列汀可能通过激活SDF-1/CXCR4信号通路促进LPS诱导的炎症微环境下hPDLSCs的增殖和成骨分化，抑制hPDLSCs凋亡和炎症反应。.

OBJECTIVE: This study sought to explore the role of developmental endothelial locus-1 (DEL-1) in osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and investigate the therapeutic effect of DEL-1 in ligature-induced experimental periodontitis with type 2 diabetes mellitus (T2DM).
BACKGROUND: T2DM is a significant risk factor for periodontitis. Treatment modalities for periodontitis with T2DM are being explored. DEL-1 is a versatile protein that can modulate the different stages of inflammatory diseases including periodontitis. The direct effect of DEL-1 on osteogenic differentiation of PDLSCs in periodontitis with T2DM is poorly understood.
METHODS: Primary hPDLSCs were isolated from periodontal ligament tissue and identified by flow cytometry. In osteogenesis experiments, alkaline phosphatase (ALP), Alizarin Red staining and western blot were used to assess the osteogenic effect of DEL-1 on hPDLSCs in high glucose and inflammation environments. The mouse model of ligature-induced experimental periodontitis was established. H&E and Masson\'s trichrome staining were used to assess the change of periodontal tissue after local periodontal injection of DEL-1. Immunohistochemical staining was used to evaluate osteogenic-related protein expression.
RESULTS: hPDLSCs expressed mesenchymal stem cell (MSC)-specific surface markers and were negative for hematopoietic cell surface markers. hPDLSCs had the potential for multidirectional differentiation. DEL-1 could enhance the osteogenic differentiation of hPDLSCs in high glucose and inflammation environments, although it did not return to the control level. Histological staining showed that DEL-1 contributed to alveolar bone regeneration and osteogenic-related protein expression, but the degree of improvement in T2DM mice was lower than in non-T2DM mice.
CONCLUSIONS: In summary, we demonstrated that DEL-1 could promote osteogenic differentiation of hPDLSCs in high glucose and inflammation environment and rescue alveolar bone loss in experimental periodontitis with T2DM, which could provide a novel therapeutic target for periodontitis with T2DM.

This study aims to investigate the effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2VitD3) on osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and the activity of hPDLSC sheets and the differences in the tissue regeneration activity of hPDLSC sheets on tooth root fragment treated by different methods. Healthy caries-free premolars were collected. The hPDLSCs were obtained by enzymatic digestion. Surface markers of stem cells were analyzed by flow cytometry and the multidirectional differentiation ability of hPDLSCs was detected. During the osteogenic differentiation of hPDLSCs, 1,25(OH)2VitD3 was added and the effect of 1,25(OH)2VitD3 on osteogenic differentiation of hPDLSCs was assessed using Western blotting, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay, cell staining, and immunofluorescence. After hPDLSC sheets were prepared, histology and immunofluorescence analysis of the effect of 1,25(OH)2VitD3 on sheet activity were performed. In addition, root fragments were prepared and treated with scaling, 24% EDTA (ethylenediamide tetraacetic acid), and Er,Cr:YSGG lasers, respectively, and the tissue regeneration activity of hPDLSC sheets on different root fragments were observed. 1,25(OH)2VitD3 promoted the high gene and protein expressions of osteogenic markers ALP (alkaline phosphatase), Runx2, and OPN (osteopontin antibody) in hPDLSCs, along with enhanced ALP activity and staining, alizarin red staining, and immunofluorescence staining, indicating that the osteogenic differentiation ability of hPDLSCs was improved. Extracellular matrix secretion was increased in hPDLSC sheets, along with the positive expressions of the protein markers fibronectin and collagen I, suggesting that 1,25(OH)2VitD3 could enhance these effects. In addition, the root fragments treated by Er,Cr:YSGG laser were more suitable for the attachment and regeneration of hPDLSC sheets, demonstrating that 1,25(OH)2VitD3 could improve the tissue regeneration performance of these sheets. 1,25(OH)2VitD3 can promote osteogenic differentiation of hPDLSCs and thus plays an active role in hPDLSC sheet formation and tissue regeneration. In addition, the Er,Cr:YSGG laser can be used as the recommended treatment method for the root surface regenerated by hPDLSCs.

Vascularization plays an important role in dental and craniofacial regenerations. Human periodontal ligament stem cells (hPDLSCs) are a promising cell source and, when co-cultured with human umbilical vein endothelial cells (hUVECs), could promote vascularization. The objectives of this study were to develop a novel prevascularized hPDLSC-hUVEC-calcium phosphate construct, and investigate the osteogenic and angiogenic efficacy of this construct with human platelet lysate (hPL) in cranial defects in rats for the first time.
hPDLSCs and hUVECs were co-cultured on calcium phosphate cement (CPC) scaffolds with hPL. Cell proliferation, angiogenic gene expression, angiogenesis, alkaline phosphatase activity, and cell-synthesized minerals were determined. Bone and vascular regenerations were investigated in rat critical-sized cranial defects in vivo.
hPDLSC-hUVEC-CPC-hPL group had 2-fold greater angiogenic expressions and cell-synthesized mineral synthesis than hPDLSC-hUVEC-CPC group (p < 0.05). Microcapillary-like structures were formed on scaffolds in vitro. hPDLSC-hUVEC-CPC-hPL group had more vessels than hPDLSC-hUVEC-CPC group (p < 0.05). In cranial defects in rats, hPDLSC-hUVEC-CPC-hPL group regenerated new bone amount that was 2.1 folds and 4.0 folds, respectively, that of hPDLSC-hUVEC-CPC group and CPC control (p < 0.05). New blood vessel density of hPDLSC-hUVEC-CPC-hPL group was 2 folds and 7.9 folds, respectively, that of hPDLSC-hUVEC-CPC group and CPC control (p < 0.05).
The hPL pre-culture method is promising to enhance bone regeneration via prevascularized CPC. Novel hPDLSC-hUVEC-CPC-hPL prevascularized construct increased new bone formation and blood vessel density by 4-8 folds over CPC control.
Novel hPDLSC-hUVEC-hPL-CPC prevascularized construct greatly increased bone and vascular regeneration in vivo and hence is promising for a wide range of craniofacial applications.

Exosomes derived from stem cells are a potential cell-free tool for tissue regeneration with therapeutic potential. However, its application in cementum repair is unclear. This study aimed to investigate the effect of human periodontal ligament stem cell-derived exosomes on the biological activity of cementoblasts, the main effector cells in cementum synthesis.
OCCM-30 cementoblasts were cultured with various human periodontal ligament stem cell-derived exosome concentrations. OCCM-30 cells proliferation, migration, and cementogenic mineralization were examined, along with the gene and protein expression of factors associated with cementoblastic mineralization.
Exosomal promoted the migration, proliferation, and mineralization of OCCM-30 cells. The exosome-treated group significantly increased the expression of cementogenic-related genes and proteins. Furthermore, the expression of p-PI3K and p-AKT was enhanced by exosome administration. Treatment with a PI3K/AKT inhibitor markedly attenuated the gene and protein expression of cementoblastic factors, and this effect was partially reversed by exosome administration.
Human periodontal ligament stem cell-derived exosomes can promote the activity of cementoblasts via the PI3K/AKT signaling pathway, providing a scientific basis for promoting the repair process in orthodontically induced inflammatory root resorption.

Periodontitis is a chronic inflammatory disease caused by Porphyromonas gingivalis and other bacteria, and human periodontal ligament stem cells (hPDLSCs) are a promising candidate for the treatment of periodontal supporting tissue defects. This study aimed to investigate the effect of 1α,25-dihydroxyvitamin D3 [1,25(OH)2VitD3] on osteogenic differentiation of hPDLSCs in an in vitro periodontitis model and whether it can improve inflammatory status. hPDLSCs were in vitro isolated and identified. After treatment with 1,25(OH)2VitD3 and ultrapure pure Porphyromonas gingivalis lipopolysaccharide (LPS-G), the viability of hPDLSCs was detected using Cell Counting Kit-8, the expressions of osteogenic markers and inflammatory genes using Western blotting and quantitative reverse transcription PCR (qRT-PCR), the levels of inflammatory factors in cells using enzyme linked immunosorbent assay (ELISA), and the fluorescence signal intensity of osteoblastic markers and inflammatory genes in cells using immunofluorescence assay. It was found that 1,25(OH)2VitD3 reversed the inhibition of hPDLSCs proliferation by LPS-G; LPS-G exhibited inhibitory effect on ALP, Runx2, and OPN expressions, and such inhibitory effect was significantly weakened when co-acting with 1,25(OH)2VitD3. Meanwhile, LPS-G upregulated the expressions of inflammatory genes IL-1β and Casp1, whereas 1,25(OH)2VitD3 antagonized such an effect and improved the inflammatory status. In conclusion, 1,25(OH)2VitD3 can reverse the inhibitory effect of LPS-G on hPDLSCs proliferation and osteogenic differentiation and suppress LPS-G-induced upregulation of inflammatory gene expressions.

Autologous tooth transplantation is the transplantation of an erupted, partially erupted, or unerupted tooth from one site to another in an individual. This is expected to maintain alveolar bone volume through physiological stimulation of the periodontal ligament (PDL). Tooth transplantation can be used for the closure of oroantral communication. As a simple, useful, and minimally invasive method, it should be considered a surgical option when a donor tooth is available in indicated cases. Herein, the authors report the case of a 20-year-old female patient whose left permanent maxillary first molar had to be extracted due to a longitudinal fracture and radicular cyst in the maxillary sinus floor. After the extraction, tooth 28 was exposed through an osteotomy and positioned in the gap. Nineteen years later, the autologous grafted tooth 28 could no longer be retained due to massive external resorption and was replaced by an implant. Human PDL stem cells can differentiate into bone-, fiber-, and cementum-forming cells and have the potential to build a PDL complex. Therefore, care must be taken to avoid damaging the PDL of the donor tooth during extraction. Autotransplanted teeth are expected to retain the alveolar bone volume. This case demonstrates the use of a transplanted tooth 28 for the treatment of a maxillary defect caused by the extraction of tooth 26 and removal of a radicular cyst. External resorption and regeneration of the bone of the maxillary sinus floor around the transplanted tooth occurred after 19 years.