microRNA-214-3p Suppresses Ankylosing Spondylitis Fibroblast Osteogenesis via BMP–TGFβ Axis and BMP2

Recent investigations suggest microRNAs (miRs) exert functions in fibroblast osteogenesis in ankylosing spondylitis (AS), an inflammatory rheumatic disease. But the mechanism of miR-214-3p in osteogenic differentiation in AS is not clearly understood yet. In this study, fibroblasts were obtained from the capsular ligament of patients with AS and femoral neck fracture and cultured for osteogenic induction and identified. The roles of miR-214-3p and bone morphogenic protein 2 (BMP2) in AS fibroblast osteogenesis were assessed via gain- and loss-of-function, alizarin red S staining, and alkaline phosphatase (ALP) detection. Levels of miR-214-3p, BMP2, osteogenic differentiation-related proteins, and BMP–TGFβ axis-related proteins were further measured. Consequently, miR-214-3p was downregulated in AS fibroblasts, with enhanced ALP activity and calcium nodules, which were reversed by miR-214-3p overexpression. BMP2 was a target gene of miR-214-3p and promoted AS fibroblast osteogenesis by activating BMP–TGFβ axis, while miR-214-3p inhibited AS fibroblast osteogenesis by targeting BMP2. Together, miR-214-3p could prevent AS fibroblast osteogenic differentiation by targeting BMP2 and blocking BMP–TGFβ axis. This study may offer a novel insight for AS treatment.

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Impaired Osteogenic Differentiation Of Mesenchymal Stem Cells Derived From Bone Marrow Of Patients With Low-Risk Myelodysplastic Syndromes

Blood ◽

10.1182/blood.v122.21.1579.1579 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1579-1579

Author(s):

Chunkang Chang ◽

Chengming Fei ◽

Youshan Zhao ◽

Juan Guo ◽

Xiao Li

Keyword(s):

Alkaline Phosphatase ◽

Osteogenic Differentiation ◽

Lower Risk ◽

Conflicts Of Interest ◽

Osteoblastic Differentiation ◽

Differentiation Markers ◽

Alizarin Red ◽

Hematopoietic Microenvironment ◽

Alkaline Phosphatase Staining ◽

Osteogenic Induction

Abstract Background The pathogenesis of MDS has not been completely understood, and insufficiency of the hematopoietic microenvironment can be an important factor. MSCs and osteoblasts are key components of the hematopoietic microenvironment. Studying osteoblastic differentiation of MSCs quantitatively may help to understand the pathogenesis of MDS. Methods 38 patients with MDS and 15 normal donors were investigated in this study. Osteoblastic differentiation assays were performed in 16 MDS cases and 8 controls. The expression of osteogenic differentiation markers were measured by real-time PCR. Alkaline phosphatase staining was performed with Alkaline Phosphatase staining kit after 3,7,14 days of incubation. ALP activity was assessed at 3, 7, and 10 days after osteogenic differentiation. Mineralization analysis was performed at 7, 14 and 21 days of osteogenic induction. The areas of mineralization were measured by Image-Pro Plus 6.0 software. Results Both MDS-MSCs and normal cells displayed same fibroblast-like morphology and similar antigen expression. The expression level of RUNX2 was significantly decreased in MSCs from MDS, compaired with normal controls, especially in lower-risk MDS. After osteogenic induction, lower-risk MDS showed lower alkaline phosphatase activity, less intense alizarin red S staining, and lower gene expression of osteogenic differentiation markers, however, higher-risk MDS was normal. Conclusions We concluded that impaired osteogenic differentiation of MSCs was seen mainly in patients with lower-risk MDS. It may contribute to the ineffective hamatopoiesis of MDS. Disclosures: No relevant conflicts of interest to declare.

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Effect of miR-192-5p on Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in Idiopathic Scoliosis by Regulating Wnt/β-catenin Signaling Pathway via RSPO1

10.21203/rs.3.rs-961628/v1 ◽

2021 ◽

Author(s):

Yifan Yang ◽

Jing Xu ◽

Qingxin Su ◽

Yiran Wu ◽

Qizheng Li ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Alkaline Phosphatase ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Signaling Pathway ◽

Alizarin Red ◽

Alkaline Phosphatase Staining ◽

Marrow Mesenchymal Stem Cells ◽

Related Proteins

Abstract BackgroundIdiopathic scoliosis (IS) is the most common structural scoliosis, which seriously affects not only patient’s physical and mental health but also quality of patient’s life. Abnormal osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is one of the causes of IS. However, the regulation mechanism of osteogenic differentiation of BMSCs in patients with IS remains to be further studied.MethodsSerum samples of 135 patients with IS were collected, and the expression of miRNA were detected by RT-qPCR. BMSCs from patients with IS were collected and the expression of miR-192-5p in BMSCs from IS patients and normal BMSCs was detected by RT-qPCR. Double luciferase reporter genes assay was used to verify the targeting relationship between miR-192-5p and RSPO1. The levels of RSPO1, osteogenic related proteins (OC, OPN and RUNX2) and Wnt/β-catenin signaling pathway related proteins (WNT3A and β-catenin) were detected by Western blotting. Alkaline phosphatase staining and alizarin red staining were used to evaluate the osteogenesis of BMSCs.ResultsmiR-192-5p was significantly up-regulated in serum and BMSCs of patients with IS. Alkaline phosphatase staining and alizarin red staining showed that miR-192-5p inhibitor promoted the osteogenic differentiation of BMSCs from IS patients. miR-192-5p targeted down-regulated the expression of RSPO1 in BMSCs from IS patients. In addition, overexpression of RSPO1 activated Wnt/β-catenin signaling pathway in BMSCs from IS patients. Furthermore, miR-192-5p/RSPO1 axis regulated levels of osteogenic related proteins (OC, OPN and RUNX2) in BMSCs from IS patients through Wnt/β-catenin signaling pathway, and affected the osteogenic differentiation of BMSCs.ConclusionmiR-192-5p, which was highly expressed in patients with IS, inhibited Wnt/β-catenin signaling pathway by down-regulating RSPO1 protein and then reduced the osteogenic differentiation ability of BMSCs.

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Stretch-Induced Lncrna SNHG8 Inhibits Osteogenic Differentiation by Regulating EZH2 in Hpdlscs

10.21203/rs.3.rs-362066/v1 ◽

2021 ◽

Author(s):

Zijie Zhang ◽

Qin He ◽

Xiaolu Zhao ◽

Xiaoyu Li ◽

Fulan Wei

Keyword(s):

Alkaline Phosphatase ◽

Western Blot ◽

Osteogenic Differentiation ◽

High Throughput ◽

High Throughput Sequencing ◽

Tooth Movement ◽

Alizarin Red ◽

Qrt Pcr ◽

Osteogenic Induction

Abstract Background: Periodontal ligament stem cells (PDLSCs) are important for the remodeling of the alveolar bone while tooth moving. However, the effect of long non-coding RNA (lncRNA) on osteogenic differentiation of PDLSCs under mechanical force remains unclear.Methods: In this study, we compared stretched and non-stretched PDLSCs by high-throughput sequencing. The verification and selection of lncRNAs were achieved by quantitative reverse transcription polymerase chain reaction (qRT-PCR). PDLSCs osteogenic differentiation potentials were assessed by alkaline phosphatase (ALP) staining, Alizarin Red staining, qRT-PCR, and western blot. The application of mechanical force used Flexcell-FX-6000-Tension System in vitro, and constructing rats’ tooth movement model in vivo. To verify the osteogenic regulation ability of small nucleolar RNA host gene 8 (SNHG8), PDLSCs were stretched or applied osteogenic induction after been infected by lentivirus. RNA fluorescence in situ hybridization, isolation of nuclear and cytoplasmic RNA, qRT-PCR and western blot were performed to locate SNHG8. Western blot and qRT-PCR to find the relationship between enhancer of zeste homolog 2 (EZH2) and SNHG8.Results: Our results demonstrated that among lncRNAs altered screened by high-throughput sequencing, the expression level of SNHG8 steadily decreased after being stretched. Analysis of mRNA expression and protein levels revealed an upregulation of ALP and RUNX2, ALP and Alizarin Red staining showed more obvious alkaline phosphatase and more mineralized nodules in SNHG8 knockdown PDLSCs. In vivo experiments showed lower expression of the homologous gene of SNHG8 after tooth movement, and better ability of ectopic osteogenesis after knockdown SNHG8. The verification of SNHG8’s nuclear location led us to infer that SNHG8 may interact with EZH2. The qRT-PCR and western blot results disclosed EZH2 expression reduced along with the knockdown of SNHG8. Furthermore, knockdown of EZH2 lead to PDLSCs’ osteogenic differentiation ability increasing under osteogenic induction according to the mRNA level of ALP and RUNX2 accompanied by ALP and Alizarin Red staining results.Conclusion: In general, our study confirmed that mechanically sensitive lncRNA SNHG8 can influence the osteogenic differentiation of PDLSCs through epigenetic pathways without directly encoding protein, which provides solid evidence for the regulation by non-coding genes.

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Mechanical Sensing Element PDLIM5 Promotes Osteogenesis of Human Fibroblasts by Affecting the Activity of Microfilaments

Biomolecules ◽

10.3390/biom11050759 ◽

2021 ◽

Vol 11 (5) ◽

pp. 759

Author(s):

Xiaolan Huang ◽

Rongmei Qu ◽

Yan Peng ◽

Yuchao Yang ◽

Tingyu Fan ◽

...

Keyword(s):

Osteogenic Differentiation ◽

Morphological Changes ◽

Human Fibroblasts ◽

Inhibitory Effect ◽

Induction Medium ◽

Sensing Element ◽

Differentiation Potential ◽

Osteogenic Induction Medium ◽

Osteogenic Induction ◽

Related Proteins

Human skin fibroblasts (HSFs) approximate the multidirectional differentiation potential of mesenchymal stem cells, so they are often used in differentiation, cell cultures, and injury repair. They are an important seed source in the field of bone tissue engineering. However, there are a few studies describing the mechanism of osteogenic differentiation of HSFs. Here, osteogenic induction medium was used to induce fibroblasts to differentiate into osteoblasts, and the role of the mechanical sensitive element PDLIM5 in microfilament-mediated osteogenic differentiation of human fibroblasts was evaluated. The depolymerization of microfilaments inhibited the expression of osteogenesis-related proteins and alkaline phosphatase activity of HSFs, while the polymerization of microfilaments enhanced the osteogenic differentiation of HSFs. The evaluation of potential protein molecules affecting changes in microfilaments showed that during the osteogenic differentiation of HSFs, the expression of PDLIM5 increased with increasing induction time, and decreased under the state of microfilament depolymerization. Lentivirus-mediated PDLIM5 knockdown by shRNA weakened the osteogenic differentiation ability of HSFs and inhibited the expression and morphological changes of microfilament protein. The inhibitory effect of knocking down PDLIM5 on HSF osteogenic differentiation was reversed by a microfilament stabilizer. Taken together, these data suggest that PDLIM5 can mediate the osteogenic differentiation of fibroblasts by affecting the formation and polymerization of microfilaments.

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The inhibitory effects of vancomycin on rat bone marrow–derived mesenchymal stem cell differentiation

Journal of Neurosurgery Spine ◽

10.3171/2020.10.spine201511 ◽

2021 ◽

pp. 1-5

Author(s):

Kari Hanson ◽

Carly Isder ◽

Kristen Shogren ◽

Anthony L. Mikula ◽

Lichun Lu ◽

...

Keyword(s):

Bone Marrow ◽

Alkaline Phosphatase ◽

Osteogenic Differentiation ◽

Phosphatase Activity ◽

Alkaline Phosphatase Activity ◽

High Dose ◽

Inhibitory Effects ◽

Alizarin Red ◽

Osteogenic Factors

OBJECTIVE The use of intrawound vancomycin powder in spine surgery has been shown to decrease the rate of surgical site infections; however, the optimal dose is unknown. High-dose vancomycin inhibits osteoblast proliferation in vitro and may decrease the rate of solid arthrodesis. Bone marrow–derived mesenchymal stem cells (BMSCs) are multipotent cells that are a source of osteogenesis in spine fusions. The purpose of this study was to determine the effects of vancomycin on rat BMSC viability and differentiation in vitro. METHODS BMSCs were isolated from the femurs of immature female rats, cultured, and then split into two equal groups; half were treated to stimulate osteoblastic differentiation and half were not. Osteogenesis was stimulated by the addition of 50 µg/mL l-ascorbic acid, 10 mM β-glycerol phosphate, and 0.1 µM dexamethasone. Vancomycin was added to cell culture medium at concentrations of 0, 0.04, 0.4, or 4 mg/mL. Early differentiation was determined by alkaline phosphatase activity (4 days posttreatment) and late differentiation by alizarin red staining for mineralization (9 days posttreatment). Cell viability was determined at both the early and late time points by measurement of formazan colorimetric product. RESULTS Viability within the first 4 days decreased with high-dose vancomycin treatment, with cells receiving 4 mg/mL vancomycin having 40%–60% viability compared to the control. A gradual decrease in alizarin red staining and nodule formation was observed with increasing vancomycin doses. In the presence of the osteogenic factors, vancomycin did not have deleterious effects on alkaline phosphatase activity, whereas a trend toward reduced activity was seen in the absence of osteogenic factors when compared to osteogenically treated cells. CONCLUSIONS Vancomycin reduced BMSC viability and impaired late osteogenic differentiation with high-dose treatment. Therefore, the inhibitory effects of high-dose vancomycin on spinal fusion may result from both reduced BMSC viability and some impairment of osteogenic differentiation.

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Optimizing Osteogenic Differentiation of Ovine Adipose-Derived Stem Cells by Osteogenic Induction Medium and FGFb, BMP2, or NELL1 In Vitro

Stem Cells International ◽

10.1155/2018/9781393 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 5

Author(s):

Emil Østergaard Nielsen ◽

Li Chen ◽

Jonas Overgaard Hansen ◽

Matilda Degn ◽

Søren Overgaard ◽

...

Keyword(s):

Osteogenic Differentiation ◽

Stromal Cells ◽

Basic Medium ◽

Induction Medium ◽

Control Analysis ◽

Alizarin Red ◽

Osteogenic Induction Medium ◽

Osteogenic Induction ◽

Fibroblast Growth Factor Basic

Although adipose-derived stromal cells (ADSCs) have been a major focus as an alternative to autologous bone graft in orthopedic surgery, bone formation potential of ADSCs is not well known and cytokines as osteogenic inducers on ADSCs are being investigated. This study aimed at isolating ADSCs from ovine adipose tissue (AT) and optimizing osteogenic differentiation of ovine ADSCs (oADSC) by culture medium and growth factors. Four AT samples were harvested from two female ovine (Texel/Gotland breed), and oADSCs were isolated and analyzed by flow cytometry for surface markers CD29, CD44, CD31, and CD45. Osteogenic differentiation was made in vitro by seeding oADSCs in osteogenic induction medium (OIM) containing fibroblast growth factor basic (FGFb), bone morphogenetic protein 2 (BMP2), or NEL-like molecule 1 (NELL1) in 4 different dosages (1, 10, 50, and 100 ng/ml, respectively). Basic medium (DMEM) was used as control. Analysis was made after 14 days by Alizarin red staining (ARS) and quantification. This study successfully harvested AT from ovine and verified isolated cells for minimal criteria for adipose stromal cells which suggests a feasible method for isolation of oADSCs. OIM showed significantly higher ARS to basic medium, and FGFb 10 ng/ml revealed significantly higher ARS to OIM alone after 14 days.

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Azithromycin Promotes the Osteogenic Differentiation of Human Periodontal Ligament Stem Cells after Stimulation with TNF-α

Stem Cells International ◽

10.1155/2018/7961962 ◽

2018 ◽

Vol 2018 ◽

pp. 1-11 ◽

Cited By ~ 1

Author(s):

Tingting Meng ◽

Ying Zhou ◽

Jingkun Li ◽

Meilin Hu ◽

Xiaomeng Li ◽

...

Keyword(s):

Stem Cells ◽

Alkaline Phosphatase ◽

Osteogenic Differentiation ◽

Alkaline Phosphatase Activity ◽

Periodontal Ligament ◽

Caspase 3 ◽

Caspase 8 ◽

Periodontal Ligament Stem Cells ◽

Alizarin Red ◽

Induced Apoptosis

Background and Objective. This study investigated the effects and underlying mechanisms of azithromycin (AZM) treatment on the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) after their stimulation with TNF-α in vitro. Methods. PDLSCs were isolated from periodontal ligaments from extracted teeth, and MTS assay was used to evaluate whether AZM and TNF-α had toxic effects on PDLSCs viability and proliferation. After stimulating PDLSCs with TNF-α and AZM, we analyzed alkaline phosphatase staining, alkaline phosphatase activity, and alizarin red staining to detect osteogenic differentiation. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis was performed to detect the mRNA expression of osteogenic-related genes, including RUNX2, OCN, and BSP. Western blotting was used to measure the NF-κB signaling pathway proteins p65, phosphorylated p65, IκB-α, phosphorylated IκB-α, and β-catenin as well as the apoptosis-related proteins caspase-8 and caspase-3. Annexin V assay was used to detect PDLSCs apoptosis. Results. TNF-α stimulation of PDLSCs decreased alkaline phosphatase and alizarin red staining, alkaline phosphatase activity, and mRNA expression of RUNX2, OCN, and BSP in osteogenic-conditioned medium. AZM enhanced the osteogenic differentiation of PDLSCs that were stimulated with TNF-α. Western blot analysis showed that β-catenin, phosphorated p65, and phosphorylated IκB-α protein expression decreased in PDLSCs treated with AZM. In addition, pretreatment of PDLSCs with AZM (10 μg/ml, 20 μg/ml) prevented TNF-α-induced apoptosis by decreasing caspase-8 and caspase-3 expression. Conclusions. Our results showed that AZM promotes PDLSCs osteogenic differentiation in an inflammatory microenvironment by inhibiting the WNT and NF-κB signaling pathways and by suppressing TNF-α-induced apoptosis. This suggests that AZM has potential as a clinical therapeutic for periodontitis.

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Yunnan Baiyao Conditioned Medium Promotes the Odonto/Osteogenic Capacity of Stem Cells from Apical Papilla via Nuclear Factor Kappa B Signaling Pathway

BioMed Research International ◽

10.1155/2019/9327386 ◽

2019 ◽

Vol 2019 ◽

pp. 1-11 ◽

Cited By ~ 1

Author(s):

Xiyao Pang ◽

Yanqiu Wang ◽

Jintao Wu ◽

Zhou Zhou ◽

Tao Xu ◽

...

Keyword(s):

Stem Cells ◽

Alkaline Phosphatase ◽

Osteogenic Differentiation ◽

Conditioned Medium ◽

Signaling Pathway ◽

Cell Counting ◽

Dependent Manner ◽

Alizarin Red ◽

Apical Papilla ◽

Yunnan Baiyao

Yunnan Baiyao is a traditional Chinese herbal remedy that has long been used for its characteristics of wound healing, bone regeneration, and anti-inflammation. However, the effects of Yunnan Baiyao on the odonto/osteogenic differentiation of stem cells from apical papilla (SCAPs) and the potential mechanisms remain unclear. The aim of this study was to investigate the odonto/osteogenic differentiation effects of Yunnan Baiyao on SCAPs and the underlying mechanisms involved. SCAPs were isolated and cocultured with Yunnan Baiyao conditioned media. The proliferation ability was determined by cell counting kit 8 and flow cytometry. The differentiation capacity and the involvement of NF-κB pathway were investigated by alkaline phosphatase assay, alizarin red staining, immunofluorescence assay, real-time RT-PCR, and western blot analyses. Yunnan Baiyao conditioned medium at the concentration of 50 μg/mL upregulated alkaline phosphatase activity, induced more mineralized nodules, and increased the expression of odonto/osteogenic genes/proteins (e.g., OCN/OCN, OPN/OPN, OSX/OSX, RUNX2/RUNX2, ALP/ALP, COL-I/COL-I, DMP1, DSP/DSPP) of SCAPs. In addition, the expression of cytoplasmic phos-IκBα, phos-P65, and nuclear P65 was significantly increased in Yunnan Baiyao conditioned medium treated SCAPs in a time-dependent manner. Conversely, the differentiation of Yunnan Baiyao conditioned medium treated SCAPs was obviously inhibited when these stem cells were cocultured with the specific NF-κB inhibitor BMS345541. Yunnan Baiyao can promote the odonto/osteogenic differentiation of SCAPs via the NF-κB signaling pathway.

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The Role and Mechanism of Exosomes from Umbilical Cord Mesenchymal Stem Cells in Inducing Osteogenesis and Preventing Osteoporosis

Cell Transplantation ◽

10.1177/09636897211057465 ◽

2021 ◽

Vol 30 ◽

pp. 096368972110574

Author(s):

Ge Yahao ◽

Wang Xinjia

Keyword(s):

Stem Cell ◽

Osteogenic Differentiation ◽

Umbilical Cord ◽

Cell Counting ◽

Control Group ◽

Human Umbilical Cord ◽

Promoting Effect ◽

Alizarin Red ◽

Osteogenic Induction

Mesenchymal stem cell (MSC) exosomes promote tissue regeneration and repair, and thus might be used to treat many diseases; however, the influence of microenvironmental conditions on exosomes remains unclear. The present study aimed to analyze the effect of osteogenic induction on the functions of human umbilical cord MSC (HucMSC)-derived exosomes. Exosomes from standardized stem cell culture (Exo1) and osteogenic differentiation-exosomes (Exo2) were co-cultured with osteoblasts, separately. Cell counting kit-8 assays, alkaline phosphatase and alizarin red staining were used to observe the exosomes’ effects on osteoblast proliferation and differentiation. The levels of osteogenic differentiation-related proteins were analyzed using western blotting. Estrogen-deficient osteoporosis model mice were established, and treated with the two exosome preparations. Micro-computed tomography and hematoxylin and eosin staining were performed after 6 weeks. MicroRNAs in Exo1 and Exo2 were sequenced and analyzed using bioinformatic analyses. Compared with Exo1 group, Exo2 had a stronger osteogenic differentiation promoting effect, but a weaker proliferation promoting effect. In ovariectomy-induced osteoporosis mice, both Exo1 and Exo2 improved the tibial density and reversed osteoporosis in vivo. High-throughput microRNA sequencing identified 221 differentially expressed microRNAs in HucMSC-derived exosomes upon osteogenic induction as compared with the untreated control group. Importantly, we found that 41 of these microRNAs are potentially critical for MSC-secreted exosomes during osteogenic induction. Mechanistically, exosomal miRNAs derived from osteogenic induced-HucMSCs are involved in bone development and differentiation, such as osteoclast differentiation and the MAPK signaling pathway. The expression of hsa-mir-2110 and hsa-mir-328-3p gradually increased with prolonged osteogenic differentiation and regulated target genes associated with bone differentiation, suggesting that they are probably the most important osteogenesis regulatory microRNAs in exosomes. In conclusion, we examined the contribution of osteogenic induction to the function of exosomes secreted by HucMSCs following osteogenic differentiation in vitro and in vivo, and reveal the underlying molecular mechanisms of exosome action during osteoporosis.

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TRIM16 Positively Regulates Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Modulating the Ubiquitination and Degradation of RUNX2

10.21203/rs.3.rs-38207/v1 ◽

2020 ◽

Author(s):

Yi Zhao ◽

Qiaoli Zhai ◽

Hong Liu ◽

Xun Xi ◽

Shuai Chen ◽

...

Keyword(s):

Stem Cells ◽

Alkaline Phosphatase ◽

Bone Regeneration ◽

Osteogenic Differentiation ◽

Periodontal Ligament ◽

Osteogenic Potential ◽

Common Disease ◽

Periodontal Ligament Stem Cells ◽

Alizarin Red ◽

Periodontal Tissues

Abstract BackgroundPeriodontal disease is a common disease that compromises the integrity of tooth-supporting tissues. Bone regeneration is the ultimate goal of periodontal therapies, in which osteogenic differentiation of human periodontal ligament stem cells plays a critical role. The tripartite motif (TRIM)16 is downregulated in periodontal tissues of patients with periodontitis and involved in osteogenic differentiation of human bone marrow mesenchymal stem cells(hBMSCs).However, the role of TRIM16 in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) is largely unknown.MethodshPDLSCs were isolated and identified by immunophenotype assays using flow cytometry. Overexpression plasmids and specific short-hairpin RNAs (shRNAs) were constructed to manipulate the expression of target molecules. Alkaline phosphatase (ALP) staining, alizarin red staining (ARS) and enzyme‐linked immunosorbent assays (ELISA) were used to evaluate osteogenic potential capacity. Reverse transcription quantitative PCR (RT-qPCR) and Western blot analysis were performed to determine the expression of osteogenic-related markers and activation of relevant signaling pathways. Co-immunoprecipitation assays were performed to confirm the interactions between proteins and the ubiquitination of RUNX2. A LC-MS/MS analysis was performed to explore the different expression proteins in present of TRIM16.ResultsTRIM16 significantly promoted alkaline phosphatase activity and mineralized nodule formation, and positively regulated the osteogenic differentiation of hPDLSCs by enhancing protein expression of RUNX2, COL1A1 and OCN. Mechanistically, TRIM16 serves as a pivotal factor that stabilizes RUNX2 protein levels by decreasing CHIP-mediated K48-linked ubiquitination degradation of the RUNX2 protein. Besides, TRIM16 significantly increased expression of COL1A1 via activation of p38MAPK/RUNX2.ConclusionThis study identified a novel mechanism of TRIM16 in regulating stability of the RUNX2 protein, which may promote the osteogenic differentiation of hPDLSCs. TRIM16 may be a potential target of stem cell based-bone regeneration for periodontal therapies.

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