UNASSIGNED: The present study aimed to explore the function of human bone marrow mesenchymal stem cells (hBMMSCs)-derived exosomal long noncoding RNA histocompatibility leukocyte antigen complex P5 (HCP5) in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) to improve chronic periodontitis (CP).
UNASSIGNED: Exosomes were extracted from hBMMSCs. Alizarin red S staining was used to detect mineralised nodules. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to measure HCP5 and miR-24-3p expression. The mRNA and protein levels of alkaline phosphatase (ALP), osteocalcin, osterix, runt-related transcription factor 2, bone morphogenetic protein 2, osteopontin, fibronectin, collagen 1, heme oxygenase 1 (HO1), P38, and ETS transcription factor ELK1 (ELK1) were detected using RT-qPCR and Western blot. Enzyme-linked immunosorbent assay (ELISA) kits were used to determine the HO1 and carbon monoxide concentrations. Heme, biliverdin, and Fe2+ levels were determined using detection kits. Micro-computed tomography, hematoxylin and eosin staining, ALP staining, tartrate-resistant acid phosphatase staining, ELISA, and RT-qPCR were conducted to evaluate the effect of HCP5 on CP mice. Dual luciferase, RNA immunoprecipitation, and RNA pulldown experiments were performed to identify the interactions among HCP5, miR-24-3p, and HO1.
UNASSIGNED: The osteogenic ability of hPDLSCs significantly increased when co-cultured with hBMMSCs or hBMMSCs exosomes. Overexpression of HCP5 and HO1 in hBMMSCs exosomes promoted the osteogenic differentiation of hPDLSCs, and knockdown of HCP5 repressed the osteogenic differentiation of hPDLSCs. HCP5 knockdown enhanced the inflammatory response and repressed osteogenesis in CP mice. MiR-24-3p overexpression diminished the stimulatory effect of HCP5 on the osteogenic ability of hPDLSCs. Mechanistically, HCP5 acted as a sponge for miR-24-3p and regulated HO1 expression, and HO1 activated the P38/ELK1 pathway.
UNASSIGNED: HBMMSCs-derived exosomal HCP5 promotes the osteogenic differentiation of hPDLSCs and alleviates CP by regulating the miR-24-3p/HO1/P38/ELK1 signalling pathway.

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.

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。.

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.

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.

It has been reported that protein arginine methyltransferase 5 (PRMT5) serves a significant role in osteogenic differentiation and inflammatory response. Nevertheless, its role in periodontitis as well as its underlying mechanism remain to be elucidated. The aim of the present study was to explore the role of PRMT5 in periodontitis and whether PRMT5 could reduce liposaccharide (LPS)-induced inflammation of human periodontal ligament stem cells (hPDLSCs) and promote osteogenic differentiation through STAT3/NF-κB signaling. In the current study, the expression levels of PRMT5 were determined in LPS-induced hPDLSCs by reverse transcription-quantitative PCR and western blot analysis. ELISA and western blot analysis were employed to assess the secretion and expression levels of inflammatory factors, respectively. The osteogenic differentiation and mineralization potential of hPDLSCs were evaluated using alkaline phosphatase (ALP) activity assay, Alizarin red staining and western blot analysis. Additionally, western blot analysis was applied to determine the expression levels of the STAT3/NF-κB signaling pathway-related proteins. The results showed that the expression levels of PRMT5 were significantly enhanced in LPS-induced hPDLSCs. Additionally, PRMT5 knockdown reduced the contents of IL-1β, IL-6, TNF-α, inducible nitric oxide synthase and cyclooxygenase-2. PRMT5 depletion also enhanced ALP activity, improved the mineralization ability and upregulated bone morphogenetic protein 2, osteocalcin and runt-related transcription factor 2 in LPS-induced hPDLSCs. Furthermore, PRMT5 knockdown inhibited inflammation and promoted the osteogenic differentiation of hPDLSCs via blocking the activation of the STAT3/NF-κB signaling pathway. In conclusion, PRMT5 inhibition suppressed LPS-induced inflammation and accelerated osteogenic differentiation in hPDLSCs via regulating STAT3/NF-κB signaling, thus providing a potential targeted therapy for the improvement of periodontitis.

UNASSIGNED: To investigate the roles of Notoginsenoside R1 (NG-R1) on the proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and explore its possible mechanism.
UNASSIGNED: hPDLSCs were isolated and, then characterized by flow cytometry. Cell-counting kit-8 (CCK-8) and colony assays were used to validate the effect of different NG-R1 concentrations on hPDLSCs proliferation and the optimal concentration was determined. Quantitative detection of alkaline phosphatase (ALP) activity at optimal concentration and the mineralization of the cells was investigated by Alizarin Red S staining. qRT-PCR and Western blot were utilized to examine the factors expression levels of ALP, Runx Family Transcription Factor 2 (RUNX2), Collagen I (Col-1) and catenin beta 1 (CTNNB1; β-catenin). In addition, the tankyrase inhibitor XAV-939 was used to explore NG-R1\'s role in canonical Wnt signaling.
UNASSIGNED: hPDLSCs were positive for surface antigens CD90 while negative for CD34 and CD45, which indicated that we have successfully isolated the hPDLSCs. Furthermore, a concentration of 20μmol NG-R1 dramatically enhanced hPDLSCs proliferation, ALP activity, and mineral deposition. ALP, RUNX2, COL-1, and β-catenin expression were all rised in comparison to control group. After XAV-939 was added to disrupt the canonical Wnt signaling, the impact of NG-R1 appeared to be reversed.
UNASSIGNED: These findings suggest that NG-R1 can stimulate osteogenic differentiation of hPDLSCs, which is probably attributable to canonical Wnt signaling activation.

Periodontitis often causes damage to the periodontal tissue and affects the function of human periodontal ligament stem cells (hPDLSCs). Low-level laser therapy (LLLT) has been used for periodontal treatment and can upregulate the proliferation and osteogenesis of hPDLSCs. The purpose of this study was to investigate the effects of LLLT on the proliferation, osteogenic differentiation, inflammatory reaction, and oxidative stress of hPDLSCs in an inflammatory environment (pPDLSCs). We designed one control group and three testing groups (treated with Nd:YAG laser at 4, 8, and 16 J/cm2) of hPDLSCs from periodontitis patients who were diagnosed with stable phase periodontitis. Cell proliferation was measured by colony-forming unit fibroblast (CFU-F) assays and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The osteogenic capacity of the cells was determined by alkaline phosphatase (ALP) staining, ALP activity assays, Alizarin Red S staining and the mRNA transcript levels of runt-related transcription factor 2 (Runx2), ALP, and osteocalcin (OCN). The effects of LLLT on the secretion of tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β by PDLSCs were measured by enzyme-linked immunosorbent assay (ELISA). We also evaluated the oxidative stress of hPDLSCs and pPDLSCs by measuring the reactive oxygen species (ROS) level, malondialdehyde (MDA) level, and superoxide dismutase (SOD) activity after treatment with LLLT at 4, 8, and 16 J/cm2. Our results demonstrated that LLLT could modulate the osteogenic potential of pPDLSCs at 8 J/cm2. Inflammatory stimuli induced excess ROS release, and LLLT at 4-8 J/cm2 promoted oxidative stress levels in hPDLSCs but decreased the expression of inflammatory cytokines and ROS levels in pPDLSCs. Moreover, LLLT at 16 J/cm2 could significantly suppress proliferation and osteogenic differentiation and promote inflammatory cytokines and ROS levels in pPDLSCs. In conclusion, LLLT could regulate proliferation, osteogenesis, inflammatory reaction, and oxidative stress of human periodontal ligament stem cells under inflammatory conditions.

Periodontitis is a complex dental condition that has a number of different etiologies. Lin-28 homolog A (LIN28A) has been previously reported to regulate inflammation, where its expression levels have been indicated to be lower in periodontal tissues following periodontitis. However, there is a lack of evidence to indicate the precise role of LIN28A in periodontitis. In the present study, LIN28A and Runt-related transcription factor 2 (RUNX2) expression were measured in human periodontal biopsy tissues using reverse transcription-quantitative PCR (RT-qPCR). RT-qPCR and western blot analyses were also used to measure LIN28A and RUNX2 expression in human periodontal ligament stem cells (hPDLSCs) following lipopolysaccharide (LPS) induction. Following construction of the LIN28A overexpression plasmid, the expression of LIN28A, RUNX2, osteopontin, osterix and osteocalcin were detected using RT-qPCR and western blotting. Additionally, RT-qPCR was used for the detection of proinflammatory biomarkers (IL-8, IL-1β and IL-6) and alkaline phosphatase (ALP) expression. Protein expression of intranuclear and cytoplasmic NF-κB p65 and NF-κB p65 phosphorylation were assessed using western blot analysis. The expression of antioxidant factors including SOD and GSH were determined using corresponding commercial assay kits. ALP and the mineralization capacity of hPDLSCs were detected by ALP activity assay and Alizarin red staining. The expression of LIN28A was found to be decreased in periodontal biopsy tissues from periodontitis patients compared with normal tissues and LPS-induced hPDLSCs compared with untreated hPDLSCs, which was positively correlated with RUNX2 expression. LIN28A overexpression was revealed to attenuate inflammatory damage and oxidative stress whilst improving ALP active damage, restoring RUNX2 expression and osteoblastic mineralization in LPS-induced hPDLSCs. In conclusion, the present study suggests that LIN28A serves a key role as a mediator of osteoblast differentiation and mineralization. In addition, LIN28A was able to alleviate inflammatory injury and oxidative stress in LPS-induced hPDLSCs.