Osteogenic Differentiation Of hPDLSCs Research Articles

Low-energy red light-emitting diode irradiation enhances osteogenic differentiation of periodontal ligament stem cells by regulating miR-146a-5p.

The study aimed to investigate the role of miR-146a-5p in osteogenesis of hPDLSCs irradiated with low-energy red LEDs. After irradiation with 5 J/cm2 red LED, miR-146a-5p expression was detected by real-time quantitative polymerase chain reaction (RT-qPCR), and osteogenic markers expression was determined by RT-qPCR and Western blotting. Alkaline phosphatase (ALP) activity was assessed by ALP staining, and mineralization was assessed by Alizarin Red staining, respectively. Lentiviral vectors were designed to regulate miR-146a-5p expression. Dual-luciferase reporter assay was performed to confirm the targeted relationship between miR-146a-5p and MAPK1. Short hairpin RNA (shRNA) was used to regulate MAPK1 expression. RT-qPCR and western blotting revealed that 5 J/cm2 irradiation elevated the levels of the osteogenic markers osterix (OSX) and bone sialoprotein (BSP) in hPDLSCs. miR-146a-5p is downregulated in hPDLSCs under the low-energy red LED light irradiation. miR-146a-5p underexpression markedly promoted the osteogenic potential of hPDLSCs. miR-146a-5p targeted MAPK1. 5 J/cm2 red LED irradiation rescued the inhibitory effects of upregulated miR-146a-5p on osteogenic differentiation, and the positive influence of red LED irradiation could be reversed by downregulated MAPK1. These findings confirm that miR-146a-5p is involved in the effect of LED irradiation on the osteogenic differentiation of hPDLSCs by targeting MAPK1. Red LED irradiation may be a potential clinical adjunct therapy for periodontal regeneration.

Compression Promotes the Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Regulating METTL14-mediated IGF1.

Orthodontic treatment involves the application of mechanical force to induce periodontal tissue remodeling and ultimately promote tooth movement. It is essential to study the response mechanisms of human periodontal ligament stem cells (hPDLSCs) to improve orthodontic treatment. In this study, hPDLSCs treated with compressive force were used to simulate orthodontic treatment. Cell viability and cell death were assessed using the CCK-8 assay and TUNEL staining. Alkaline phosphatase (ALP) and alizarin red staining were performed to evaluate osteogenic differentiation. The binding relationship between IGF1 and METTL14 was assessed using RIP and dual-luciferase reporter assays. The compressive force treatment promoted the viability and osteogenic differentiation of hPDLSCs. Additionally, m6A and METTL14 levels in hPDLSCs increased after compressive force treatment, whereas METTL14 knockdown decreased cell viability and inhibited the osteogenic differentiation of hPDLSCs treated with compressive force. Furthermore, the upregulation of METTL14 increased m6A levels, mRNA stability, and IGF1 expression. RIP and dual-luciferase reporter assays confirmed the interaction between METTL14 and IGF1. Furthermore, rescue experiments demonstrated that IGF1 overexpression reversed the effects of METTL14 knockdown in hPDLSCs treated with compressive force. In conclusion, this study demonstrated that compressive force promotes cell viability and osteogenic differentiation of hPDLSCs by regulating IGF1 levels mediated by METTL14.

MiR-508-5p suppresses osteogenic differentiation of human periodontal ligament stem cells via targeting sex-determining region Y-related HMG-box 11

Background/PurposeThe local inflammatory microenvironment created by periodontitis negatively impacts periodontal tissue regeneration, necessitating the development of methods to enhance the regenerative capacity of stem cells. This study explored the regulatory role and underlying mechanism of miR-508-5p in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). Materials and methodsThe regulatory roles of miR-508–5p in osteogenic differentiation of hPDLSCs were investigated through its inhibition or overexpression. Expression of the sex-determining region Y-related HMG-box 11 (SOX11) and osteogenic markers was analyzed using Western blot and real-time PCR. Osteogenesis was measured using alizarin red S (ARS) staining and alkaline phosphatase (ALP) staining. A dual luciferase reporter assay was performed to confirm SOX11 as a target of miR-508-5p. ResultsDuring the osteogenic differentiation of hPDLSCs, miR-508-5p expression level gradually decreased, while that of SOX11 increased. miR-508-5p inhibition significantly promoted osteogenesis in hPDLSCs, while overexpression inhibited the process. SOX11 overexpression reversed the suppressive effects of miR-508-5p on the osteogenic differentiation of hPDLSCs. miR-508–5p downregulation significantly increased SOX11; a dual luciferase reporter assay provided evidence for their direct targeting. ConclusionmiR-508-5p downregulation promotes the osteogenic differentiation of hPDLSCs by targeting SOX11.

TAZ reverses the inhibitory effects of LPS on the osteogenic differentiation of human periodontal ligament stem cells through the NF-κB signaling pathway

BackgroundHuman 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.MethodsLPS 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.ResultsLPS 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.ConclusionOverexpressing 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.

FBLN5 was Regulated by PRDM9, and Promoted Senescence and Osteogenic Differentiation of Human Periodontal Ligament Stem Cells.

Periodontal ligament stem cells (PDLSCs) are ideal seed cells for periodontal tissue regeneration. Our previous studies have indicated that the histone methyltransferase PRDM9 plays an important role in human periodontal ligament stem cells (hPDLSCs). Whether FBLN5, which is a downstream gene of PRDM9, also has a potential impact on hPDLSCs is still unclear. Senescence was assessed using β-galactosidase and Enzyme-linked immunosorbent assay (ELISA). Osteogenic differentiation potential of hPDLSCs was measured through Alkaline phosphatase (ALP) activity assay and Alizarin red detection, while gene expression levels were evaluated using western blot and RT-qPCR analysis. FBLN5 overexpression promoted the osteogenic differentiation and senescence of hPDLSCs. FBLN5 knockdown inhibited the osteogenic differentiation and senescence of hPDLSCs. Knockdown of PRDM9 decreased the expression of FBLN5 in hPDLSCs and inhibited senescence of hPDLSCs. Additionally, both FBLN5 and PRDM9 promoted the expression of phosphorylated p38 MAPK, Erk1/2 and JNK. The p38 MAPK pathway inhibitor SB203580 and the Erk1/2 pathway inhibitor PD98059 have the same effects on inhibiting the osteogenic differentiation and senescence of hPDLSCs. The JNK pathway inhibitor SP600125 reduced the senescence of hPDLSCs. FBLN5 promoted senescence and osteogenic differentiation of hPDLSCs via activation of the MAPK signaling pathway. FBLN5 was positively targeted by PRDM9, which also activated the MAPK signaling pathway.

Triptolide mitigates the inhibition of osteogenesis induced by TNF-α in human periodontal ligament stem cells via the p-IκBα/NF-κB signaling pathway: an in-vitro study

BackgroundTriptolide is a widely utilized natural anti-inflammatory drug in clinical practice. Aim of this study was to evaluate effects of triptolide on hPDLSCs osteogenesis in an inflammatory setting and to investigate underlying mechanisms.MethodsUsing the tissue block method to obtain hPDLSCs from extracted premolar or third molar. Flow cytometry, osteogenic and adipogenic induction were carried out in order to characterise the features of the cells acquired. hPDLSC proliferative activity was assessed by CCK-8 assay to determine the effect of TNF-α and/or triptolide. The impact of triptolide on the osteogenic differentiation of hPDLSCs was investigated by ALP staining and quantification. Osteogenesis-associated genes and proteins expression level were assessed through PCR and Western blotting assay. Finally, BAY-117,082 was used to study the NF-κB pathway.ResultsIn the group treated with TNF-α, there was an elevation in inflammation levels while osteogenic ability and the expression of both osteogenesis-associated genes and proteins decreased. In the group co-treated with TNF-α and triptolide, inflammation levels were reduced and osteogenic ability as well as the expression of both osteogenesis-associated genes and proteins were enhanced. At the end of the experiment, both triptolide and BAY-117,082 exerted similar inhibitory effects on the NF-κB pathway.ConclusionThe osteogenic inhibition of hPDLSCs by TNF-α can be alleviated through triptolide, with the involvement of the p-IκBα/NF-κB pathway in this mechanism.

CoCl2-Induced hypoxia promotes hPDLSCs osteogenic differentiation through AKT/mTOR/4EBP-1/HIF-1α signaling and facilitates the repair of alveolar bone defects.

Bone defects are characterized by a hypoxic environment, which affects bone tissue repair. However, the role of hypoxia in the repair of alveolar bone defects remains unclear. Human periodontal ligament stem cells (hPDLSCs) are high-quality seed cells for repairing alveolar bone defects, whose behavior changes under hypoxia. However, their mechanism of action is not known and needs to be elucidated. We hypothesized that hypoxia might be beneficial to alveolar bone defect repair and the osteogenic differentiation of hPDLSCs. To test this hypothesis, cobalt chloride (CoCl2) was used to create a hypoxic environment, both in vitro and in vivo. In vitro study, the best osteogenic effect was observed after 48 h of hypoxia in hPDLSCs, and the AKT/mammalian target of rapamycin/eukaryotic translation initiation factor 4e-binding protein 1 (AKT/mTOR/4EBP-1) signaling pathway was significantly upregulated. Inhibition of the AKT/mTOR/4EBP-1 signaling pathway decreased the osteogenic ability of hPDLSCs under hypoxia and hypoxia-inducible factor 1 alpha (HIF-1α) expression. The inhibition of HIF-1α also decreased the osteogenic capacity of hPDLSCs under hypoxia without significantly affecting the level of phosphorylation of AKT/mTOR/4EBP-1. In vitro study, Micro-CT and tissue staining results show better bone regeneration in hypoxic group than control group. These results suggested that hypoxia promoted alveolar bone defect repair and osteogenic differentiation of hPDLSCs, probably through AKT/mTOR/4EBP-1/HIF-1α signaling. These findings provided important insights into the regulatory mechanism of hypoxia in hPDLSCs and elucidated the effect of hypoxia on the healing of alveolar bone defects. This study highlighted the importance of physiological oxygen conditions for tissue engineering.

Effects of sitagliptin activation of the stromal cell-derived factor-1/CXC chemokine receptor 4 signaling pathway on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells induced by lipopolysaccharide.

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. 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. 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). 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.

Exploring the Role of Wnt Ligands in Osteogenic Differentiation of Human Periodontal Ligament Stem Cells.

This study aimed to investigate the functions of 19types of Wnt ligands during the process of osteogenic differentiation inhuman periodontal ligament stem cells (hPDLSCs), with particular attention to WNT3A and WNT4. The expression levels of 19 types ofWnt ligands were examined using real-time quantitative polymerase chain reaction (real-time qPCR) during hPDLSCs osteogenic differentiation at 7, 10, and 14days. Knockdown of WNT3A and WNT4 expression was achieved using adenovirus vectors, and conditioned medium derived from WNT3A and WNT4 overexpression plasmids was employed to investigate their roles in hPDLSCs osteogenesis. Osteogenic-specific genes were analyzed using real-time qPCR. Alkaline phosphatase (ALP) and alizarin red S activities and staining were employed to assess hPDLSCs' osteogenic differentiation ability. During hPDLSCs osteogenic differentiation, the expression of 19types of Wnt ligands varied, with WNT3A and WNT4 showing significant upregulation. Inhibiting WNT3A and WNT4 expression hindered hPDLSCs' osteogenic capacity. Conditioned medium of WNT3A promoted early osteogenic differentiation, while WNT4 facilitated late osteogenesis slightly. Wnt ligands, particularly WNT3A and WNT4, play an important role in hPDLSCs' osteogenic differentiation, highlighting their potential as promoters of osteogenesis. Given the challenging nature of alveolar bone regeneration, therapeutic strategies that target WNT3A and WNT4 signaling pathways offer promising opportunities. Additionally, innovative gene therapy approaches aimed at regulating of WNT3A and WNT4 expression hold potential for improving alveolar bone regeneration outcomes.

Identifying genes for regulating osteogenic differentiation of human periodontal ligament stem cells in inflammatory environments by bioinformatics analysis.

Periodontitis is an immuno-inflammatory disease caused by dental plaque biofilms and inflammations. The regeneration of bone tissue in inflammatory environment is of great significance for the treatment of periodontal disease, but the specific molecular mechanism of bone formation in periodontitis still needs further exploration. The objective of this study was to identify key osteogenesis-related genes (ORGs) in periodontitis. We used two datasets from the Gene Expression Omnibus (GEO) database to find differentially expressed mRNAs and miRNAs, further performed Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Then we predicted the downstream genes of the differentially expressed miRNAs (DEMs) by the TargetScan database and established a miRNA-mRNA regulatory network. Finally, the osteogenic mechanism of periodontitis was explored through quantitative real-time PCR (qRT-PCR) by inducing inflammatory environment and osteogenic differentiation of hPDLSCs. Through differential expression analysis and prediction of downstream target genes of DEMs, we created a miRNA-mRNA regulatory network consisting of 29 DEMs and 11 differentially expressed osteogenesis-related genes (DEORGs). In addition, the qRT-PCR results demonstrated that BTBD3, PLAT, AKAP12, SGK1, and GLCE expression levels were significantly upregulated, while those of TIMP3, ZCCHC14, LIN7A, DNAH6, NNT, and ITGA6 were downregulated under the dual effects of inflammatory stimulation and osteogenic induction. DEORGs might be important factors in the osteogenic phase of periodontitis, and the miRNA-mRNA network may shed light on the clarification of the role and mechanism of osteogenesis in periodontitis and contribute to the development of novel therapeutic strategies.

LncRNA urothelial cancer associated 1 promotes the osteogenic differentiation of human periodontal ligament stem cells by regulating the miR-96–5p/Osx axis

ObjectivesTo investigate the expression of long non-coding RNA (lncRNA) urothelial cancer associated 1 (UCA1) in human periodontal ligament stem cells (hPDLSCs), its effect on osteogenic differentiation of hPDLSCs and its mechanism. DesignThe expression of osteogenic genes Osx, Runx2, Ocn and Opn was explored by qPCR. Protein expression in hPDLSCs was estimated by Western blot. The osteogenic differentiation of hPDLSCs was detected by Alizarin red staining assays. The interaction between UCA1 and miR-96–5p was explored by RNA pulldown assay and dual luciferase assay. The interaction between miR-96–5p and Osx 3′-UTR was measured by dual luciferase assay. ResultsThe expression of UCA1 and miR-96–5p was negatively correlated in hPDLSCs. During the osteogenic differentiation of hPDLSCs, the expression of UCA1 was increased, while the expression of miR-96–5p was decreased. Knockdown of UCA1 in hPDLSCs inhibited osteogenic differentiation but induced upregulation of miR-96–5p expression, and vice versa. In addition, miR-96–5p partially reversed the positive effect of UCA1 on osteogenic differentiation of hPDLSCs. Notably, UCA1 was identified as a miR-96–5p sponge, and miR-96–5p targeted Osx. ConclusionsOur results demonstrated that the novel UCA1/miR-96–5p/Osx pathway regulates osteogenic differentiation of hPDLSCs and sheds new insights and targets for periodontitis therapeutics.

Resveratrol modulates the inflammatory response in hPDLSCs via the NRF2/HO-1 and NF-κB pathways and promotes osteogenic differentiation.

The purpose of this study was to investigate resveratrol's specific role as an anti-inflammatory and osteogenic differentiation of hPDLSCs in periodontitis and to reveal the mechanisms involved. Numerous studies have shown that inhibiting the inflammatory response of periodontal tissues and promoting the regeneration of alveolar bone are crucial treatments for periodontitis. Resveratrol has been found to have certain anti-inflammatory property. However, the anti-inflammatory mechanism and osteogenic effect of resveratrol in periodontitis are poorly understood. We constructed an invitro periodontitis model by LPS stimulation of hPDLSCs and performed WB, RT-qPCR, and immunofluorescence to analyze inflammatory factors and related pathways. In addition, we explored the osteogenic ability of resveratrol in invitro models. In vitro, resveratrol ameliorated the inflammatory response associated with activation of the NF-κB pathway through activation of the NRF2/HO-1 pathway, characterized by inhibition of p65/p50 nuclear translocation and the proinflammatory cytokines interleukin-1β levels. Resveratrol also has a positive effect on osteogenic differentiation. Observations suggest that resveratrol modulates the inflammatory response in hPDLSCs via the NRF2/HO-1 and NF-κB pathways and promotes osteogenic differentiation.

AFF4 regulates osteogenic potential of human periodontal ligament stem cells via mTOR-ULK1-autophagy axis.

Scaffold protein AF4/FMR2 family member 4 (AFF4) has been found to play a role in osteogenic commitment of stem cells. However, function of AFF4 in human periodontal ligament stem cells (hPDLSCs) has not been studied yet. This present study aims to investigate the biological effect of AFF4 on osteogenic differentiation of hPDLSCs and potential mechanistic pathway. First, AFF4 expression profile was evaluated in conditions of periodontitis and osteogenic differentiation of hPDLSCs by immunohistochemical staining, western blot and qRT-PCR. Next, si-RNA mediated knockdown and lentiviral transduction mediated overexpression of AFF4 were adopted to explore impact of AFF4 on osteogenic capacity of hPDLSCs. Then, possible mechanistic pathway was identified. At last, pharmacological agonist of autophagy, rapamycin, was utilized to affirm the role of autophagy in AFF4-regulated osteogenesis of hPDLSCs. First, AFF4 expressions were significantly lower in inflamed periodontal tissues and lipopolysaccharides-treated hPDLSCs than controls, and were up-regulated during osteogenic differentiation of hPDLSCs. Next, osteogenic potential of hPDLSCs was impaired by AFF4 knockdown and potentiated by AFF4 overexpression. Moreover, AFF4 was found to positively regulate autophagic activity in hPDLSCs. At last, rapamycin treatment was shown to be able to partly restore AFF4 knockdown-suppressed osteogenic differentiation. Our study demonstrates that AFF4 regulates osteogenic potential of hPDLSCs via targeting autophagic activity. The involvement of AFF4 in periodontal homeostasis was identified for the first time.

Roles of human periodontal ligament stem cells in osteogenesis and inflammation in periodontitis models: Effect of 1α,25-dihydroxyvitamin D3

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.

Effect of psoralen on the regulation of osteogenic differentiation induced by periodontal stem cell-derived exosomes

Periodontitis is a chronic inflammatory disease that is the main cause of tooth loss in adults, and the key to periodontitis treatment is the repair and regenerate of periodontal bone tissue. Psoralen is the main component of the Psoralea corylifolia Linn, which shows antibacterial, anti-inflammatoryand osteogenic activities. It promotes the differentiation of periodontal ligament stem cells toward osteogenesis. Exosomes secreted by stem cells play important roles in information transmission during the osteogenic differentiation process. The aim of this paper was to investigate the role of psoralen in regulating osteogenic miRNA information in periodontal stem cells and in periodontal stem cells exosomes and the specific mechanism of its action. Experimental results show that exosomes of human periodontal ligament stem cell origin treated with psoralen (hPDLSCs + Pso-Exos) were not significantly different from untreated exosomes (hPDLSC-Exos) in terms of size and morphology. Thirty-five differentially expressed miRNAs were found to be upregulated and 58 differentially expressed miRNAs were found to be downregulated in the hPDLSCs + Pso-Exos compared to the hPDLSC-Exos (P < 0.05). hsa-miR-125b-5p was associated with osteogenic differentiation. Among them, hsa-miR-125b-5p was associated with osteogenic differentiation. After hsa-miR-125b-5p was inhibited, the osteogenesis level of hPDLSCs was enhanced. In summary, the osteogenic differentiation of hPDLSCs was promoted by psoralen through the downregulation of hsa-miR-125b-5p gene expression in hPDLSCs, and the expression of the hsa-miR-125b-5p gene was also downregulated in exosomes. This finding provides a new therapeutic idea for using psoralen to promote periodontal tissue regeneration.

Long non‑coding RNA SNHG5 promotes osteogenic differentiation of human periodontal ligament stem cells via mediating miR‑23b‑3p/Runx2 axis.

The treatment of bone loss due to periodontitis has posed a great challenge for physicians for decades. Therefore, it is of extraordinary significance to identify an effective regeneration scheme for alveolar bone. This study aimed to investigate long non-coding RNA (lncRNA) small nucleolar RNA host gene 5 (SNHG5) whether sponges microRNA-23b-3p (miR-23b-3p) to achieve the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). Results revealed that the expression of SNHG5 was upregulated whereas that of miR-23b-3p was downregulated in osteogenic hPDLSCs. Alizarin red staining assays and qRT-PCR demonstrated that SNHG5 silencing or miR-23b-3p overexpression inhibits hPDLSCs osteogenic differentiation and vice versa. In addition, miR-23b-3p partially abolished the promotive effect of SNHG5 on osteogenic differentiation of hPDLSCs. Dual luciferase report and RNA pulldown assay verified that miR-23b-3p is a regulatory target of SNHG5 and that Runx2 is a gene target of miR-23b-3p. In brief, the results demonstrate that SNHG5 promotes the osteogenic differentiation of hPDLSCs by regulating the miR-23b-3p/Runx2 axis. Our study provides novel mechanistic insights into the critical role of lncRNA SNHG5 as a miR-23b-3p sponge to regulate Runx2 expression in hPDLSCs and may serve as a potential therapeutics target for periodontitis.

Novel injectable calcium phosphate scaffold with human periodontal ligament stem cell encapsulation in microbeads for bone regeneration

Objectives: 1) Develop a novel construct of human periodontal ligament stem cells (hPDLSCs) encapsulated in degradable alginate microbeads (DAMB) with human platelet lysate (hPL) and injectable calcium phosphate cement (ICPC); 2) Investigate the proliferation and osteogenic differentiation of hPDLSCs in ICPC with hPL as a xeno-free supplement and animal serum replacement for bone tissue engineering applications.Methods: hPDLSCs were encapsulated in alginate-fibrin microbeads (DAMB + fibrin), alginate-hPL degradable microbeads (DAMB + hPL), or alginate-fibrin-hPL microbeads (DAMB + fibrin + hPL). The proliferation and osteogenic differentiation of hPDLSCs were investigated in culturing with the ICPC scaffold.Results: Flexural strength of ICPC was 8.4 ± 0.91 MPa, and elastic modulus was 1.56 ± 0.1 GPa, exceeding those of cancellous bone. hPDLSCs had higher viability in DAMB + fibrin + hPL group than in DAMB + fibrin. ALP was 69.97 ± 16.96 mU/mg for ICPC + DAMB + fibrin + hPL group, higher than 30.68 ± 2.86 mU/mg of ICPC + DAMB + fibrin (p &amp;lt; 0.05) and 4.12 ± 1.65 mU/mg of control (p &amp;lt; 0.01). At 7 days, osteogenic gene expressions (ALP, RUNX2, COL1, and OPN) in ICPC + DAMB + fibrin + hPL and ICPC + DAMB + fibrin were 4–11 folds that of control. At 21 days, the hPDLSC-synthesized bone mineral amounts in ICPC + DAMB + fibrin + hPL and ICPC + DAMB + fibrin were 13.2 folds and 11.1 folds that of control group, respectively.Conclusion: The novel injectable CPC scaffold encapsulating hPDLSCs and hPL is promising to protect and deliver hPDLSCs. The hPL-based medium significantly enhanced the osteogenic differentiation of hPDLSCs in ICPC + DAMB + fibrin + hPL construct, suggesting a promising xeno-free approach for bone tissue regeneration applications.

Lactate inhibits osteogenic differentiation of human periodontal ligament stem cells via autophagy through the MCT1-mTOR signaling pathway.

Periodontal ligament stem cells (PDLSCs) play a crucial role in periodontal bone regeneration. Lactate used to be considered a waste product of glucose metabolism. In recent years, a few pieces of evidence revealed its roles in regulating the osteogenic differentiation of stem cells, but the standpoints were controversial. This study aims to investigate the effects and the mechanisms of lactate on the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs). The hPDLSCs were treated with different concentrations of lactic acid and lactate to differentiate the effects of the acidic PH and ion lactate. Proliferation and cytotoxicity were measured by Cell Counting Kit-8 (CCK8) assay and Live/Dead assay. The osteogenic differentiation of hPDLSCs was analyzed by alizarin red staining, alkaline phosphatase (ALP) staining, and then osteogenic proteins and genes were measured by western blot and reverse transcription-quantitative PCR (qRT-PCR). To investigate the potential signaling pathways, MCT1 inhibitor, G-protein inhibitors, and rapamycin were used, and then autophagy-related proteins and osteogenic proteins were measured by western blot. The inhibition of lactic acid on the osteogenic differentiation of hPDLSCs was more significant than lactate at the same concentration. Lactate inhibited the expression of ALP which can be rescued by Gα inhibitor. Alizarin red staining, the protein expression levels of osteocalcin (OCN), osteoprotegerin (OPN), osterix (OSX), and beclin1, LC3-II/LC3-I were decreased by lactate and partly rescued by MCT1 inhibitor. Rapamycin restored the protein expression levels of beclin1, LC3-II/LC3-I and OCN, OPN, OSX under the high lactate conditions. Lactate inhibits the expression of ALP via Gα subunit signaling, and inhibits mineralized nodules formation and the expression of osteogenic-related proteins via reducing autophagy through the MCT1-mTOR signaling pathway.

Low-intensity pulsed ultrasound enhances immunomodulation and facilitates osteogenesis of human periodontal ligament stem cells by inhibiting the NF-κB pathway.

Human periodontal ligament stem cells (hPDLSCs) are vital in cellular regeneration and tissue repair due to their multilineage differentiation potential. Low intensity pulsed ultrasound (LIPUS) has been applied for treating bone and cartilage defects. This study explored the role of LIPUS in the immunomodulation and osteogenesis of hPDLSCs. hPDLSCs were cultured in vitro, and the effect of different intensities of LIPUS (30, 60, and 90 mW/cm2) on hPDLSC viability was measured. hPDLSCs irradiated by LIPUS and stimulated by lipopolysaccharide (LPS) and LIPUS (90 mW/cm2) were co-cultured with peripheral blood mononuclear cells (PBMCs). Levels of immunomodulatory factors in hPDLSCs and inflammatory factors in PBMCs were estimated, along with determination of osteogenesis-related gene expression in LIPUS-irradiated hPDLSCs. The mineralized nodules and alkaline phosphatase (ALP) activity of hPDLSCs and levels of IκBα, p-IκBα, and p65 subunits of NF-κB were determined. hPDLSC viability was increased as LIPUS intensity increased. Immunomodulatory factors were elevated in LIPUS-irradiated hPDLSCs, and inflammatory factors were reduced in PBMCs. Osteogenesis-related genes, mineralized nodules, and ALP activity were promoted in LIPUS-irradiated hPDLSCs. The cytoplasm of hPDLSCs showed increased IκBα and p65 and decreased p-IκBα at increased LIPUS intensity. After LPS and LIPUS treatment, the inhibitory effect of LIPUS irradiation on the NF-κB pathway was partially reversed, and the immunoregulation and osteogenic differentiation of hPDLSCs were decreased. LIPUS irradiation enhanced immunomodulation and osteogenic differentiation abilities of hPDLSCs by inhibiting the NF-κB pathway, and the effect is dose-dependent. This study may offer novel insights relevant to periodontal tissue engineering.Graphical

Effects of nuclear factor-κB signaling pathway on periodontal ligament stem cells under lipopolysaccharide-induced inflammation

ABSTRACT Lipopolysaccharide (LPS) induces inflammatory stress and apoptosis. This study focused on the effect of nuclear factor kappa B (NF-κB) signaling pathway on proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) after LPS induction and its mechanism. We first isolated hPDLSCs from human tooth root samples in vitro. Then, flow cytometry detected positive expression of cell surface antigens CD146 and STRO-1 and negative expression of CD45, suggesting the hPDLSCs were successfully isolated. LPS significantly induced increased apoptosis and diminished proliferation of hPDLSCs. The NF-κB pathway agonist phorbol 12-myristate 13-acetate (PMA) or p65 overexpression inhibited the proliferation of LPS-treated hPDLSCs and promoted apoptosis. PMA also promoted LPS-induced up-regulation of the expression of inflammatory factors TNF-α and IL-6 and down-regulation of the expression of anti-inflammatory factor IL-10. Additionally, LPS was confirmed to lead to a reduction of alkaline phosphatase (ALP) activity, calcium nodules, and expression of osteogenic markers Runt-related transcription factor 2 (Runx2) and osteopontin. This reduction could be promoted by PMA. Western blotting further indicated that PMA could promote LPS-induced decrease of expression of p65 (cytoplasm), and total cellular proteins IKKα and IKKβ in hPDLSCs, while protein expression of p-IκBα (cytoplasm) and p65 (nucleus), and p-IκBα/IκBα ratio was elevated. By contrast, inhibition of the NF-κB pathway (PDTC) or small-interfering RNA targeting NF-κB/p65 (p65 siRNA) showed the opposite results. In conclusion, activation of NF-κB signaling in LPS-induced inflammatory environment can inhibit the proliferation and osteogenic differentiation of hPDLSCs. This study provides a theory foundation for the clinical treatment of periodontitis.