Osteogenic Differentiation Of hPDLSCs Research Articles

Gastrodin attenuates lipopolysaccharide-induced inflammation and oxidative stress, and promotes the osteogenic differentiation of human periodontal ligament stem cells through enhancing sirtuin3 expression.

Periodontitis is a common inflammatory gum disease that destroys the periodontal tissue. Gastrodin (GAS) is the predominant bioactive component of Gastrodia elata Blume and exhibits anti-inflammatory, anti-apoptotic and antioxidant effects in various diseases, including bone-related diseases. The aim of the present study was to investigate whether GAS could protect lipopolysaccharide (LPS)-treated human periodontal ligament stem cells (hPDLSCs) against injury and inflammation, and to determine the potential underlying mechanisms. hPDLSCs were treated with LPS and GAS, alone or in combination, and cell viability, inflammation, oxidative stress levels and apoptosis were analyzed using a Cell Counting Kit-8 assay, ELISA assay, western blotting and flow cytometry, respectively. The osteogenic differentiation capacity was evaluated using an alkaline phosphatase (ALP) assay and Alizarin Red S staining. Sirtuin 3 (SIRT3) was silenced in cells treated with LPS and GAS to verify the involvement of SIRT3 in the effects of GAS. The results demonstrated that LPS-induced decrease in cell viability was rescued by treatment with 1, 10 or 50 µM GAS. The LPS-induced production of proinflammatory cytokines and increased level of oxidative stress were also inhibited following treatment with 50 µM GAS. Furthermore, GAS significantly promoted ALP activity, increased the number of mineralized nodules and increased the expression of proteins involved in osteogenic differentiation, including ALP, Runx2, osteocalcin and osteopontin, after osteoinduction, which were all downregulated following LPS stimulation. In addition, GAS prevented LPS-induced cell apoptosis and restored the imbalance of anti-apoptotic and proapoptotic proteins in hPDLSCs. In addition, SIRT3 knockdown significantly inhibited the protective effect of GAS on LPS-induced hPDLSC injury. In summary, the findings form the present study suggested that GAS may protect hPDLSCs from LPS-induced inflammation, apoptosis and oxidative stress, as well as promote their osteogenic differentiation. The effect of GAS on the osteogenic differentiation of hPDLSCs may therefore depend on the upregulated expression of SIRT3.

Daidzein promotes the proliferation and osteogenic differentiation of periodontal ligament stem cell.

Daidzein is a kind of isoflavone compound with many biological functions. However, the specific mechanism regarding the treatment of periodontitis with daidzein is still unclear. To investigate the effect of daidzein on the proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and its mechanism. Human periodontal ligament stem cells from clinical samples were isolated in vitro and identified by flow cytometry. hPDLSCs were treated with different concentrations of daidzein. Cell proliferation ability and viability were measured by MTT assay and cell colony formation assay. Osteogenic differentiation and calcification of hPDLSCs were observed by alkaline phosphatase (ALP) staining and alizarin red staining. Western blot was used to detect the expression of c-myc, CyclinD1, osteogenic differentiation-related proteins, and Wnt/β-catenin signaling pathway proteins in hPDLSCs. human periodontal ligament stem cells were positive for surface antigens CD146, STRO-1, and CD90 expression, but negative for CD45 expression, indicating the successful isolation of hPDLSCs. In addition, daidzein could significantly promote the proliferation, cell viability, ALP activity, and osteogenic differentiation of hPDLSC. At the same time, daidzein could notably increase the expression levels of c-myc, CyclinD1, osteogenic differentiation-related proteins, and Wnt/β-catenin signaling pathway proteins, while an inhibitor of Wnt/ β-catenin pathway, XAV-939, could reverse the effect caused by daidzein. Daidzein promotes the proliferation and osteogenic differentiation of hPDLSCs by activating Wnt/β-catenin signaling pathway.

ILK regulates osteogenic differentiation of Human Periodontal Ligament Stem Cells through YAP-mediated Mechanical Memory.

Mechanical memory meant the mechanical properties of the matrix could influence the cell fate even after the matrix was changed and has been justified in many kinds of cells. To utilize the phenomenon to improve periodontal tissue engineering, we studied whether mechanical memory existed in human periodontal ligament stem cells and testified if ILK plays a role in this process. The substrate of different stiffness was fabricated by gelatin methacrylate hydrogel. Two groups of hPDLSCs with stiff (St) and soft (So) matrix, respectively, were cultivated. Then, half of the cells exchanged their matrix stiffness in the fourth passage and therefore So, St, So-St, and St-So were formed. Morphology of hPDLSCs and intracellular location of YAP was observed via fluorescence staining, osteogenic differentiation of hPDLSCs was assessed by real-time PCR, ALP staining, and Western blot. Then, all these were reassessed after the ILK gene had been knocked down. The results showed that morphology and YAP location of hPDLSCs were different between matrix changed and unchanged groups; osteogenic genes expression, ALP staining, and Western blot also varied. After the ILK gene had been knocked down, the YAP location and osteogenic activity of hPDLSCs were significantly influenced. Thus, it could be concluded that mechanical memory exists in hPDLSCs; ILK is involved in this process.

Attenuation of Porphyromonas Gingival Lipopolysaccharide-Induced Periodontal Ligament Stem Cells Injury and Inflammation by Blocking Cell Pyroptosis

Periodontitis is a chronic inflammation of periodontal tissue, and programmed cell death plays an important role in chronic periodontitis induced by P. gingivalis. Studies have shown that the increased expression of pyroptosis-related NLRP3 inflammasome and the pro-inflammatory cytokines IL-1β and IL-18 in gingivitis, invasive periodontitis, and chronic periodontitis patients. The present study aimed to investigate whether the inhibition of pyroptosis could protect porphyromonas gingival lipopolysaccharide (pg-LPS)-induced human periodontal ligament stem cells (hPDLSCs) injury and inflammation. The hPDLSCs were treated with pg-LPS and ATP in the presence of caspase1/4 inhibitor VX765. The cell proliferation and survival were assessed by CCK-8, the osteogenic differentiation capacity was evaluated by Alkaline Phosphatase (ALP) assay and alizarin red staining. Then, cell apoptosis, cleavage of gasdermin D (GSDMD) and generation of inflammatory cytokines were estimated. Lastly, western blotting was used to detect the expression of potential target proteins. Results showed that the treatment of pg-LPS plus ATP significantly inhibited the proliferation, survival and osteogenic differentiation of hPDLSCs, while inducing cell apoptosis, pyroptosis and inflammation. However, the presence of VX765 partially recovered the cell proliferation, survival and osteogenic differentiation. At the same time, VX765 inhibited cell apoptosis, cleavage of GSDMD and generation of inflammatory cytokines. Besides, the expression of related proteins including Bax, Bcl-2, cleaved (c)-caspase3, c-caspase4, c-caspase1, Toll Like Receptor 4, High Mobility Group Box 1 (HMGB1) and NLRP3 was all rescued by VX765. In conclusion, our results revealed that the blocking of cell pyroptosis could protect hPDLSCs from pg-LPS-induced injury. Therefore, the application of pyroptosis inhibitor may be a valuable therapeutic approach for treating periodontitis.

Long non-coding RNA potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 regulates the proliferation and osteogenic differentiation of human periodontal ligament stem cells by targeting miR-24-3p.

This study aims to explore the effect and molecular mechanism of long non-coding RNA (lncRNA) potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1) on proliferation and osteogenic differentiation in human periodontal ligament stem cells (hPDLSCs). The hPDLSCs of normal periodontal tissues were isolated and cultured. The mineralized solution induced the osteoblast differentiation of hPDLSCs. The down-regulation of lncRNA KCNQ1OT1, the overexpression of anti-miR-24-3p on the proliferation and the levels of osteocalcin (OCN), osteopontin (OPN) and alkaline phosphatase (ALP) of hPDLSCs were investigated. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the levels of lncRNA KCNQ1OT1, miR-24-3p, OCN, OPN, and ALP. Methyl thiazolyl tetrazolium (MTT) method was used to detect cell viability and activity. Cell proliferation was evaluated by MTT. Western blot was used to detect protein expression. The targeted relationship between lncRNA KCNQ1OT1 and miR-24-3p was detected by double-luciferase experiment. The expression level of lncRNA KCNQ1OT1 increased, and that of miR-24-3p decreased during the osteogenesis of hPDLSCs (P<0.05). The down-regulation of lncRNA KCNQ1OT1 inhibited cell proliferation and reduced the mRNA and protein expression levels of OCN, OPN, and ALP (P<0.05). LncRNA KCNQ1OT1 targeted and regulated miR-24-3p. The overexpression of miR-24-3p inhibited cell proliferation and reduced the mRNA and protein expression levels of OCN, OPN, and ALP (P<0.05). Inhibition of miR-24-3p reversed the effect of the down-regulation of lncRNA KCNQ1OT1 on cell proliferation and mRNA and protein expression levels of OCN, OPN, and ALP (P<0.05). Down-regulation of lncRNA KCNQ1OT1 inhibited the proliferation and osteogenic differentiation of hPDLSCs by targeting the up-regulated expression of miR-24-3p.

MiR-200a-3p represses osteogenesis of human periodontal ligament stem cells by targeting ZEB2 and activating the NF-κB pathway

Objectives Human periodontal ligament stem cells (hPDLSCs) bear multilineage differentiation potential and represent the cytological basis of periodontal tissue regeneration. microRNA (miR) is accepted as a critical regulator of cell differentiation. This study explored the molecular mechanism of miR-200a-3p in osteogenesis of hPDLSCs. Material and methods hPDLSCs were cultured and identified in vitro. miR-200a-3p expression during osteogenic differentiation of hPDLSCs was detected. hPDLSCs were transfected with miR-200a-3p mimic or miR-200a-3p inhibitor. Alkaline phosphatase (ALP) activity, calcified nodules and osteogenesis-related genes of hPDLSCs were measured. The binding relationship between miR-200a-3p and ZEB2 was predicted and verified. hPDLSCs were infected with sh-ZEB2, and then the osteogenic capacity was examined. miR-200a-3p inhibitor-transfected hPDLSCs were infected with sh-ZEB2. The key proteins of the NF-κB pathway were measured. Results miR-200a-3p expression was downregulated during osteogenic differentiation of hPDLSCs. Upregulation of miR-200a-3p reduced ALP activity, calcified nodules and osteogenesis-related genes of hPDLSCs, while downregulation of miR-200a-3p facilitated the osteogenesis of hPDLSCs. miR-200a-3p targeted ZEB2. ZEB2 silencing repressed osteogenesis of hPDLSCs. ZEB2 silencing attenuated the promoting effect of miR-200a-3p inhibitor on osteogenesis of hPDLSCs. miR-200a-3p activated the NF-κB pathway by targeting ZEB2. Conclusion miR-200a-3p repressed osteogenesis of hPDLSCs by targeting ZEB2 and activating the NF-κB pathway. This study may offer insights for periodontal tissue regeneration engineering.

Thrombin-activated platelet-rich plasma enhances osteogenic differentiation of human periodontal ligament stem cells by activating SIRT1-mediated autophagy

BackgroundPlatelet-rich plasma (PRP) has the potential to be used for bone regeneration. However, its effect on osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and its effect on cell autophagy of hPDLSCs remain unknown. In this study, we investigated the effects of PRP on cell viability and osteogenic differentiation of hPDLSCs and the underlying molecular mechanisms.MethodshPDLSCs were isolated and identified by morphology and flow cytometry analysis. Next, thrombin-activated PRP was used to stimulate hPDLSCs. The MTT assay was used to analyze cell viability. Osteogenic differentiation was investigated using alkaline phosphatase (ALP) activity assay, alizarin red S (ARS) staining, and gene expression analysis of osteogenic markers. Expression of the autophagic proteins was determined using western blotting.ResultsThrombin-activated PRP significantly enhanced cell viability, ALP activity, osteogenic-related mRNA levels and alizarin red-mineralization activity in hPDLSCs in a dose-dependent manner. Furthermore, activated PRP dose-dependently increased LC3-II/I ratio and the expression of SIRT1 and Beclin-1. PRP treatment also enhanced the autophagic flux. It was also demonstrated that the inhibition of SIRT1 using sirtinol or suppression of autophagy by 3-methyladenine (3-MA) abrogated PRP-induced viability and osteogenic differentiation of hPDLSCs.ConclusionOur study suggested that thrombin-activated PRP accelerated the viability and osteogenic differentiation of hPDLSCs via SIRT1-mediated autophagy induction.

Betulinic acid promotes the osteogenic differentiation of human periodontal ligament stem cells by upregulating EGR1

Periodontitis is one of the most common chronic inflammations of the oral cavity, which eventually leads to tooth loss. Betulinic acid (BetA) is an organic acid that has anti-inflammatory effects and is derived from fruits and plants, but its effect on the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) is still unclear. This study aimed to explore the effect of BetA on the osteogenic differentiation of hPDLSCs and its mechanism. Our results revealed that BetA not only promoted the viability of hPDLSCs but also induced their osteogenic differentiation in a dose-dependent manner. In addition, RNA sequencing was used to screen the differentially expressed genes (DEGs) after hPDLSCs were treated with BetA, and 127 upregulated and 138 downregulated genes were identified. Gene Ontology enrichment analysis showed that DEGs were mainly involved in the response to lithium ions and the positive regulation of macrophage-derived foam cell differentiation. The Kyoto Encyclopedia of Genes and Genomes analysis results revealed that DEGs were enriched in the nuclear factor-κB and interleukin-17 signaling pathways. More importantly, we confirmed that early growth response gene 1 (EGR1), one of the three DEGs involved in bone formation, significantly promoted the expression of osteogenic markers and the mineralization of hPDLSCs. Knockdown of EGR1 obviously limited the effect of BetA on the osteogenic differentiation of hPDLSCs. In conclusion, BetA promoted the osteogenic differentiation of hPDLSCs through upregulating EGR1, and BetA might be a promising candidate in the clinical application of periodontal tissue regeneration.

LGALS3BP/Gal-3 promotes osteogenic differentiation of human periodontal ligament stem cells

ObjectiveTo identify the role of LGALS3BP/Gal-3 in the process of human periodontal ligament stem cells (hPDLSCs) differentiating into osteoblasts. MethodsIP-WB experiments were carried out to examine the binding of LGALS3BP and Gal-3. Western blot was performed to detect the expressions of LGALS3BP and Gal-3 in hPDLSCs with or without osteogenic differentiation inducement. The expressions of differentiation-related Oct4, Sox2 and Runx2 were also detected by western blot. Alkaline Phosphatase (ALP) Assay Kit was used to measure ALP activity in hPDLSCs. The mineralization ability of hPDLSCs was observed by staining with Alizarin Red S solution. ResultsLGALS3BP bound with Gal-3 in hPDLSCs, and the expression of LGALS3BP and Gal-3 was improved after osteogenic differentiation of hPDLSCs. Recombinant GAL-3 promoted the expression of differentiation-related proteins Oct4 and Sox2 and Runx2 in osteogenic differentiation-induced hPDLSCs. Recombinant GAL-3 also promoted the differentiation of osteogenesis-induced hPDLSCs. Furthermore, LGALS3BP had a facilitating effect on differentiation-related protein expression, while it could be reversed by shGal-3. LGALS3BP also promoted osteogenic capacity of hPDLSCs, and shGal-3 could reverse this effect. ConclusionLGALS3BP binds to Gal-3, producing a promoting effect on the osteogenic differentiation of human periodontal ligament stem cells.

Comparison of in vitro biocompatibility and antibacterial activity of two calcium silicate-based materials

This study is aimed at comparing and evaluating the biocompatibility and antibacterial activities of mineral trioxide aggregate (MTA) and iRoot BP Plus as novel retro-filling materials. Discs of both materials were prepared and incubated for 72 h to obtain material extracts in medium. Flow cytometry and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay were used to assess the rate of apoptosis and proliferation of human periodontal ligament stem cells (hPDLSCs) when exposed to eluates of both materials. The expression levels of alkaline phosphatase, collagen type I, osteocalcin, Runt-related transcription factor-2, and Osterix were tested for evaluating the osteogenic differentiation of hPDLSCs. The antibacterial activities of both materials were compared by the direct contact test. The hPDLSCs stimulated by MTA or iRoot BP Plus eluates showed significantly higher cell viability than that of the control group with no eluates. No significant differences were observed among the percentages of necrotic and apoptotic cells stimulated by MTA and iRoot BP Plus eluates and the control group. The expression of all osteogenic differentiation markers of hPDLSCs in both experimental groups were significantly higher than those of the control group, while the increment values in MTA group were significantly higher than those of the iRoot BP Plus group. The antibacterial activity against Enterococcus faecalis showed no significant difference between MTA and iRoot BP Plus. Therefore, both materials may be suitable for retro-filling applications.

PELP1 promotes the expression of RUNX2 via the ERK pathway during the osteogenic differentiation of human periodontal ligament stem cells

ObjectiveThe aim of this study was to determine the physiological function and mechanism of proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) at the molecular level in vitro. DesignDuring the osteogenic differentiation of hPDLSCs, the change of PELP1 and the osteogenic commitment markers runt-related transcription factor 2(RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN) were monitored by quantitative real-time PCR (qRT-PCR) and western blots. To elucidate how PELP1 regulates RUNX2, the expression of RUNX2, the phosphorylation of extracellular regulated protein kinases (ERK) and subcellular location of PELP1 were detected under conditions that PELP1 was either knockdown by specific siRNA or overexpressed. A pharmacological inhibitor of ERK, U0126 was used while PELP1 was overexpressed, and the expression of RUNX2 was monitored by qRT-PCR. ResultsPELP1 was upregulated during the osteogenic differentiation of hPDLSCs. Knockdown of PELP1 suppressed the expression of RUNX2, whereas overexpression of PELP1 increased RUNX2 expression. Moreover, PELP1 knockdown resulted in reduced ERK phosphorylation and RUNX2 expression, and PELP1 overexpression induced RUNX2 expression was inhibited by U0126 in the hPDLSCs. ConclusionsPELP1 regulates the expression of RUNX2 during the osteogenic differentiation of hPDLSCs and that the ERK pathway is involved in this process.

PWAR6 interacts with miR‑106a‑5p to regulate the osteogenic differentiation of human periodontal ligament stem cells.

Human periodontal ligament stem cells (hPDLSCs) associated with bone regeneration serve an important role in the treatment of periodontal disease. Long non-coding RNAs are involved in the osteogenesis of multiple stem cells and can act as a sponge of microRNAs (miRs). The present study aimed to investigate the interaction between Prader Willi/Angelman region RNA 6 (PWAR6) and miR-106a-5p, as well as their influences on the osteogenic differentiation of hPDLSCs. hPDLSCs were isolated and cultured in osteogenic medium (OM) or growth medium (GM) for 7 days prior to transfection with PWAR6 overexpression vector, short hairpin RNA PWAR6 or miR-106a-5p mimic. The expression levels of runt-related transcription factor 2, osteocalcin and bone morphogenetic protein 2 (BMP2) were detected by western blotting and reverse transcription-quantitative PCR (RT-qPCR), and the expression levels of PWAR6, miR-106a-5p and alkaline phosphatase (ALP) were determined by RT-qPCR. ALP activity assays and Alizarin red staining were performed to detect osteogenesis and mineralization, respectively. Luciferase activities of wild-type and mutant PWAR6 and BMP2 were assessed by conducting a dual-luciferase reporter assay. The results indicated that PWAR6 expression was upregulated in OM-incubated hPDLSCs compared with GM-incubated hPDLSCs, and PWAR6 overexpression increased the osteogenic differentiation and mineralization of hPDLSCs compared with the corresponding control group. By contrast, miR-106a-5p expression was downregulated in OM-incubated hPDLSCs compared with GM-incubated hPDLSCs. PWAR6 acted as a sponge of miR-106a-5p and PWAR6 overexpression promoted the osteogenesis of miR-106a-5p mimic-transfected hPDLSCs. BMP2 was predicted as a target gene of miR-106a-5p. Collectively, the results indicated that PWAR6 displayed a positive influence on the osteogenic differentiation of hPDLSCs. The results of the present study demonstrated that the PWAR6/miR-106a-5p interaction network may serve as a potential regulatory mechanism underlying hPDLSCs osteogenesis.

Effect of vitamin D3 on the osteogenic differentiation of human periodontal ligament stromal cells under inflammatory conditions.

ObjectivesVitamin D3 is known to activate osteogenic differentiation of human periodontal ligament stromal cells (hPDLSCs). Recently, inflammatory stimuli were shown to inhibit the transcriptional activity of hPDLSCs, but their effect on vitamin D3‐induced osteogenic differentiation is not known. The present study aimed to investigate whether the effects of 1,25‐dihydroxvitamin D3 (1,25(OH)2D3) and 25‐hydroxvitamin D3 (25(OH)D3) on the osteogenic differentiation of hPDLSCs are also altered under inflammatory conditions. Furthermore, the expression of osteogenesis‐related factors by hPDLSCs under osteogenic conditions was assessed in the presence of inflammatory stimuli.Materials and MethodsPrimary hPDLSCs of six donors were cultured in osteogenic induction medium containing either 1,25(OH)2D3 (0‐10 nM) or 25(OH)D3 (0‐100 nM) in the presence and absence of Porphyromonas gingivalis lipopolysaccharide (LPS) or Pam3CSK4 for 7, 14 and 21 days. Osteogenic differentiation of hPDLSCs was evaluated by analysis of mineralization as assessed by Alizarin Red S staining and gene expression levels of osteogenesis‐related factors osteocalcin, osteopontin and runt‐related transcription factor 2 (RUNX2) were analysed with qPCR.ResultsTreatment with 1,25(OH)2D3 significantly enhanced the osteogenic differentiation of hPDLSCs and their expression of osteocalcin and osteopontin. The 1,25(OH)2D3‐triggered expression of osteogenesis‐related factors was significantly lower in the presence of Pam3CSK4, but not P. gingivalis LPS. None of the inflammatory stimuli had significant effects on the 1,25(OH)2D3‐induced osteogenic differentiation. 25(OH)D3 neither affected gene expression levels nor osteogenic differentiation of hPDLSCs cultured in osteogenic induction medium.ConclusionThe results of this study indicate that inflammatory stimuli also diminish the 1,25(OH)2D3‐induced expression of osteogenesis‐related factors in hPDLSCs under osteogenic conditions, while having no effect on the osteogenic differentiation.

Dynamic proteomic profiling of human periodontal ligament stem cells during osteogenic differentiation

BackgroundHuman periodontal ligament stem cells (hPDLSCs) are ideal seed cells for periodontal regeneration. A greater understanding of the dynamic protein profiles during osteogenic differentiation contributed to the improvement of periodontal regeneration tissue engineering.MethodsTandem Mass Tag quantitative proteomics was utilized to reveal the temporal protein expression pattern during osteogenic differentiation of hPDLSCs on days 0, 3, 7 and 14. Differentially expressed proteins (DEPs) were clustered and functional annotated by Gene Ontology (GO) terms. Pathway enrichment analysis was performed based on the Kyoto Encyclopedia of Genes and Genomes database, followed by the predicted activation using Ingenuity Pathway Analysis software. Interaction networks of redox-sensitive signalling pathways and oxidative phosphorylation (OXPHOS) were conducted and the hub protein SOD2 was validated with western blotting.ResultsA total of 1024 DEPs were identified and clustered in 5 distinctive clusters representing dynamic tendencies. The GO enrichment results indicated that proteins with different tendencies show different functions. Pathway enrichment analysis found that OXPHOS was significantly involved, which further predicted continuous activation. Redox-sensitive signalling pathways with dynamic activation status showed associations with OXPHOS to various degrees, especially the sirtuin signalling pathway. SOD2, an important component of the sirtuin pathway, displays a persistent increase during osteogenesis. Data are available via ProteomeXchange with identifier PXD020908.ConclusionThis is the first in-depth dynamic proteomic analysis of osteogenic differentiation of hPDLSCs. It demonstrated a dynamic regulatory mechanism of hPDLSC osteogenesis and might provide a new perspective for research on periodontal regeneration.Graphical abstract

Asarylaldehyde enhances osteogenic differentiation of human periodontal ligament stem cells through the ERK/p38 MAPK signaling pathway

Periodontitis is an inflammatory disease that affects tooth-supporting tissues. Chronic inflammation can progress to periodontitis, which results in loss of alveolar bone. Asarylaldehyde is a potential substance for bone metabolism present in natural compounds. Here, we propose the application of asarylaldehyde in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) to prevent bone loss. We investigated the effect of asarylaldehyde on hPDLSCs together with bone differentiation media in vitro. The osteogenic differentiation effect was observed after treatment of hPDLSCs with several concentrations of asarylaldehyde. After 21 days, osteogenic cells were identified by mineralization. We also observed that asarylaldehyde increased the mRNA expression of osteoblast-specific markers in hPDLSCs. Interestingly, asarylaldehyde regulated the levels of alkaline phosphatase (ALP) transcriptional activity through the p38/extracellular-signal-regulated kinase (ERK) signaling pathway. Notably, asarylaldehyde induced hPDLSCs to promote osteogenic differentiation. These results suggest that asarylaldehyde plays a key role in the osteogenic differentiation of hPDLSCs. Asarylaldehyde may be a good candidate for the application of natural compounds in future in periodontal regeneration.

MiR-23b mediates TNF-α-Inhibited Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Targeting Runx2.

Periodontitis is the most prevalent oral infection disease, which causes the destruction of periodontal supporting tissues and eventual tooth loss. This study aimed to investigate the molecular mechanism of miRNA-23b (miR-23b) in regulating the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in an inflammatory environment. Results revealed that tumor necrosis factor-α (TNF‐α), a notoriously inflammatory cytokine, remarkably attenuated the osteogenic differentiation of hPDLSCs, which were partially rescued by SKL2001 (Wnt/β-catenin agonist). We further explored the underlying roles of miRNAs involved in TNF-α-inhibited osteogenesis of hPDLSCs. The miR-23b significantly increased with TNF-α stimulation, which was abolished by SKL2001. Similar to the effect of TNF-α, miR-23b agonist (agomir-23b) dramatically reduced the expression of runt-related transcription factor 2 (Runx2) and suppressed the osteogenic differentiation of hPDLSCs. The inhibition of miR-23b significantly increased Runx2, which is the major transcription factor during osteogenesis, thereby indicating that miR-23b was an endogenous regulator of Runx2 in hPDLSCs. Bioinformatic analysis and dual luciferase reporter assays confirmed that Runx2 was a target gene of miR-23b. Furthermore, the gain function assay of Runx2 revealed that the Runx2 overexpression efficiently reversed the suppression of the osteogenic differentiation of hPDLSCs with miR-23b agonist, suggesting that the suppressing effect of miR-23b on osteogenesis was mediated by Runx2 inhibition. Our study clarified that miR-23b mediated the TNF-α-inhibited osteogenic differentiation of hPDLSCs by targeting Runx2. Therefore, the expanded function of miR-23b in the osteogenesis of hPDLSCs under inflammatory conditions. This study might provide new insights and a novel therapeutic target for periodontitis.

MiR-375 promotes human periodontal ligament stem cells proliferation and osteogenic differentiation by targeting transducer of ERBB2, 2

BackgroundMiRNAs have been demonstrated to be important regulators during osteogenic differentiation in multiple types of stem cells. In the study, the interaction between miR-375 and TOB2 was analyzed to identify their functions on the proliferation and osteogenic differentiation of hPDLSCs. MethodshPDLSCs were isolated from human first premolars, and hPDLSCs stably expressing and silenced miR-375 were constructed using miR-375-ago and miR-375-antago, respectively. miR-375 and RUNX2 mRNA expression levels in hPDLSCs during osteogenic differentiation were investigated using qRT-PCR. The impact of miR-375 expression on hPDLSCs proliferation and osteogenic differentiation was determined using MTT assay, ALP assay, and alizarin red S staining. The protein expression levels of COL1A1, RUNX2 and OCN were detected using Western blot. The targeting of TOB2 by miR-375 was validated using dual luciferase reporter assay. ResultsThe expression levels of miR-375 were increased in hPDLSCs during osteogenic differentiation in a time-dependant manner, and was positively correlated with RUNX2 mRNA expression. miR-375 facilitated the proliferation and osteogenic differentiation of hPDLSCs, and promoted the protein expression levels of COL1A1, RUNX2 and OCN. Moreover, TOB2 protein expression was reduced in hPDLSCs during osteogenic differentiation in a time-dependant manner, and miR-375 directly targeted TOB2 expression. In addition, targeting TOB2 expression in hPDLSCs could rescue the suppression of cell proliferation and osteogenic differentiation by miR-375-antago. ConclusionIn summary, miR-375 promotes proliferation and osteogenic differentiation of hPDLSCs by targeting TOB2, which reveals a new regulatory mechanism underlying osteogenic differentiation of hPDLSCs by miR-375/TOB2 axis.

Resveratrol rescues TNF‑α‑induced inhibition of osteogenesis in human periodontal ligament stem cells via the ERK1/2 pathway.

Periodontitis is a common inflammatory disorder affecting the tissues surrounding the teeth, which can lead to the destruction of periodontal tissue and tooth loss. Resveratrol, a natural phytoalexin, exerts multiple biological effects. For example, its anti-inflammatory activity has been widely studied for the treatment of inflammatory bowel disease for a number of years. However, its effect on bone repair and new bone formation in an inflammatory microenvironment is not well understood. Accordingly, the effect of resveratrol on inflammation-affected human periodontal ligament stem cells (hPDLSCs) requires further investigation. In the present study, the effect of tumor necrosis factor-α (TNF-α), resveratrol, or the combination of both on the osteogenic differentiation of hPDLSCs, as well as the underlying mechanisms involved, were investigated. Cell Counting Kit-8 assay, alkaline phosphatase staining, Alizarin red staining, Oil Red O staining, reverse transcription-quantitative PCR and western blotting were used in the present study. It was demonstrated that resveratrol enhanced hPDLSC osteogenesis and reversed the inhibitory effects of TNF-α on this process. Further mechanistic studies indicated that resveratrol exerted anti-inflammatory activity by activating the ERK1/2 pathway, decreasing the secretion of interleukin (IL)-6 and IL-8 induced by TNF-α, and enhancing hPDLSCs osteogenesis. The present study suggested that resveratrol may be a novel and promising therapeutic choice for periodontitis.

Sulfonated chitosan oligosaccharide alleviates the inhibitory effect of basic fibroblast growth factor on osteogenic differentiation of human periodontal ligament stem cells.

Periodontal ligament stem cells (PDLSCs) play an essential role in periodontal tissue repair. Basic fibroblast growth factor (bFGF) has been used in the clinical treatment of periodontal disease. However, studies have shown that bFGF inhibits the osteogenic differentiation of PDLSCs, which is not conducive to alveolar bone repair. Sulfonated chitosan oligosaccharide (SCOS), a heparan-like compound, can maintain the conformation of bFGF and promote its proliferation activity. This study investigated the effects of bFGF in combination with SCOS on the osteogenic differentiation of hPDLSCs. hPDLSCs were isolated from healthy human periodontal ligament and identified by flow cytometry and immunofluorescence. The affinity between SCOS and bFGF was analyzed by surface plasmon resonance. Changes in osteogenic differentiation by combination of bFGF with SCOS were analyzed by alkaline phosphatase activity assay, Sirius Red staining, and Alizarin Red staining. Expression of genes and proteins was investigated by western blotting and reverse transcription-quantitative PCR. Extracted hPDLSCs were mesenchymal stem cells with pluripotent differentiation potential. SCOS exhibited an affinity for bFGF. bFGF (20ng/mL) promoted the proliferation of hPDLSCs, but inhibited their osteogenic differentiation. SCOS alleviated the inhibitory effect of bFGF on the osteogenic differentiation of hPDLSCs. SCOS can reduce the inhibitory effect of bFGF on the osteogenic differentiation of hPDLSCs. This study provides evidence for the clinical use of bFGF to repair periodontal tissue.

Effects of EZH2 inhibitor DZNeP on osteogenic differentiation of periodontal ligament stem cells

Objective To observe the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and to investigate the epigenetic regulation of EZH2 inhibitor DZNeP on osteogenic differentiation of hPDLSCs. Methods The hPDLSCs were isolated and cultured, and their proliferation under different concentrations of DZNeP (0, 1, 2, 5 and 10 μmol/L) was detected by MTT. The effects of DZNeP on osteogenic differentiation of hPDLSCs were observed by alkaline phosphatase (ALP) staining and alizarin red staining. The effect of DZNeP on the trimethylation of histone H3K27 in hPDLSCs was detected by immunofluorescence staining. Results Compared with the control group, the proliferation of hPDLSCs after 1, 2, 5 and 10 μmol/L DZNeP treatment for 48 h was significantly decreased, respectively (all P<0.05), and it was concentration-dependent. The result of ALP staining and alizarin red staining showed that DZNeP could promote the expression of early osteogenic markers ALP and the formation of advanced calcified nodules of hPDLSCs. The immunofluorescence staining result showed that the trimethylation fluorescence intensity of histone H3K27 was significantly decreased in the DZNeP group compared with the control group. Conclusions As an EZH2 inhibitor, DZNeP can inhibit the proliferation of hPDLSCs and promote the differentiation of hPDLSCs into osteoblasts in vitro, suggesting that DZNeP can be used as a potential small molecule drug for the treatment of periodontitis. Key words: Epigenetic regulation; EZH2; DZNeP; Human periodontal ligament stem cells; Osteogenic differentiation