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

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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High Glucose Enhances the Odonto/Osteogenic Differentiation of Stem Cells from Apical Papilla via NF-KappaB Signaling Pathway

BioMed Research International ◽

10.1155/2019/5068258 ◽

2019 ◽

Vol 2019 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Yanping Wang ◽

Yanqiu Wang ◽

Yadie Lu ◽

Jinhua Yu

Keyword(s):

Stem Cells ◽

Western Blot ◽

Osteogenic Differentiation ◽

Real Time ◽

Signaling Pathway ◽

Mtt Assay ◽

High Glucose ◽

Rt Pcr ◽

Alizarin Red ◽

Apical Papilla

Objective. The transport and metabolism of glucose are important during mammalian development. High glucose can mediate the biological characteristics of mesenchymal stem cells (MSCs). However, the role of high glucose in the odonto/osteogenic differentiation of stem cells from apical papilla (SCAPs) is unclear. Materials and Methods. SCAPs were isolated and identified in vitro. Then, SCAPs were cultured in normal α-MEM and high glucose α-MEM separately. MTT assay was applied to observe the proliferation of SCAPs. ALP activity, alizarin red staining, real-time RT-PCR, and western blot were used to detect the odonto/osteogenic capacity of SCAPs as well as the participation of NF-κB pathway. Results. SCAPs in 25mmol/L glucose group expressed the maximum proteins of RUNX2 and ALP as compared with those in 5, 10, and 15 mmol/L groups. MTT assay showed that 25 mmol/L glucose suppressed the proliferation of SCAPs. ALP assay, alizarin red staining, real-time RT-PCR, and western blot showed 25 mmol/L high glucose can obviously enhance the odonto/osteogenic capacity of SCAPs. Moreover, the NF-κB pathway was activated in 25mmol/L glucose-treated SCAPs and the odonto/osteogenic differentiation was inhibited following the inhibition of NF-κB signaling pathway. Conclusions. High glucose can enhance the odonto/osteogenic capacity of SCAPs via NF-κB pathway.

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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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Intermittent Administration of Parathyroid Hormone Enhances Odonto/Osteogenic Differentiation of Stem Cells from the Apical Papilla via JNK and P38 MAPK Pathways

Stem Cells International ◽

10.1155/2020/5128128 ◽

2020 ◽

Vol 2020 ◽

pp. 1-13 ◽

Cited By ~ 1

Author(s):

Xiyao Pang ◽

Ying Zhuang ◽

Zehan Li ◽

Shuanglin Jing ◽

Qin Cai ◽

...

Keyword(s):

Stem Cells ◽

Parathyroid Hormone ◽

Osteogenic Differentiation ◽

P38 Mapk ◽

Cell Counting ◽

Mrna Levels ◽

Alizarin Red ◽

Alp Activity ◽

Mapk Pathways ◽

Apical Papilla

Objective. Parathyroid hormone (PTH) is considered to be essential during the tooth development. Stem cells from the apical papilla (SCAPs) are responsible for dentine formation. However, the interaction between PTH and SCAPs remains unclear. This study was aimed at investigating the effects of PTH on odonto/osteogenic differentiation capacity of SCAPs and elucidating the underlying molecular mechanisms. Materials and Methods. Here, SCAPs were isolated and identified in vitro. Effects of PTH on the proliferation of SCAPs were determined by Cell Counting Kit-8 (CCK-8), flow cytometry (FCM), and EdU. Alkaline phosphatase (ALP) activity, alizarin red staining, Western blot, and RT-PCR were carried out to detect the odonto/osteogenic differentiation of PTH-treated SCAPs as well as the participation of the MAPK signaling pathway. Results. An ALP activity assay determined that 10-8 mol/L PTH was the optimal concentration for the induction of SCAPs with no significant influence on the proliferation of SCAPs as indicated by CCK-8, FCM, and EdU. The expression of odonto/osteogenic markers was significantly upregulated in mRNA levels and protein levels. Moreover, intermittent treatment of PTH also increased phosphorylation of JNK and P38, and the differentiation was suppressed following the inhibition of JNK and P38 MAPK pathways. Conclusion. PTH can regulate the odonto/osteogenic differentiation of SCAPs via JNK and P38 MAPK pathways.

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Epigallocatechin-3-Gallate Promotes Osteo-/Odontogenic Differentiation of Stem Cells from the Apical Papilla through Activating the BMP–Smad Signaling Pathway

Molecules ◽

10.3390/molecules26061580 ◽

2021 ◽

Vol 26 (6) ◽

pp. 1580

Author(s):

Zeni Liu ◽

Yuxiu Lin ◽

Xiaolin Fang ◽

Jingwen Yang ◽

Zhi Chen

Keyword(s):

Stem Cells ◽

Alkaline Phosphatase ◽

Signaling Pathway ◽

Matrix Protein ◽

Type I ◽

Alizarin Red ◽

Smad Signaling ◽

Odontogenic Differentiation ◽

Smad Signaling Pathway ◽

Apical Papilla

Stem cells from apical papilla (SCAPs) are desirable sources of dentin regeneration. Epigallocatechin-3-gallate (EGCG), a natural component of green tea, shows potential in promoting the osteogenic differentiation of bone mesenchymal stem cells. However, whether EGCG regulates the odontogenic differentiation of SCAPs and how this occurs remain unknown. SCAPs from immature human third molars (16–20 years, n = 5) were treated with a medium containing different concentrations of EGCG or bone morphogenic protein 2 (BMP2), with or without LDN193189 (an inhibitor of the canonical BMP pathway). Cell proliferation and migration were analyzed using a CCK-8 assay and wound-healing assay, respectively. Osteo-/odontogenic differentiation was evaluated via alkaline phosphatase staining, alizarin red S staining, and the expression of osteo-/odontogenic markers using qPCR and Western blotting. We found that EGCG (1 or 10 μM) promoted the proliferation of SCAPs, increased alkaline phosphatase activity and mineral deposition, and upregulated the expression of osteo-/odontogenic markers including dentin sialophosphoprotein (Dspp), dentin matrix protein-1 (Dmp-1), bone sialoprotein (Bsp), and Type I collagen (Col1), along with the elevated expression of BMP2 and phosphorylation level of Smad1/5/9 (p < 0.01). EGCG at concentrations below 10 μM had no significant influence on cell migration. Moreover, EGCG-induced osteo-/odontogenic differentiation was significantly attenuated via LDN193189 treatment (p < 0.01). Furthermore, EGCG showed the ability to promote mineralization comparable with that of recombinant BMP2. Our study demonstrated that EGCG promotes the osteo-/odontogenic differentiation of SCAPs through the BMP–Smad signaling pathway.

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Downregulation of the LncRNA MEG3 Promotes Osteogenic Differentiation of BMSCs and Bone Repairing by Activating Wnt/β-Catenin Signaling Pathway

Journal of Clinical Medicine ◽

10.3390/jcm11020395 ◽

2022 ◽

Vol 11 (2) ◽

pp. 395

Author(s):

Juan Liu ◽

Xin Qi ◽

Xiao-Hong Wang ◽

Hong-Sheng Miao ◽

Zi-Chao Xue ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Osteogenic Differentiation ◽

Signaling Pathway ◽

Rat Model ◽

Cell Counting ◽

Alizarin Red ◽

Skull Defects ◽

Endogenous Expression ◽

Alkaline Phosphatase Staining ◽

Bone Repairing

Background: Previous studies have demonstrated that long non-coding RNA maternally expressed gene 3 (MEG3) emerged as a key regulator in development and tumorigenesis. This study aims to investigate the function and mechanism of MEG3 in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and explores the use of MEG3 in skull defects bone repairing. Methods: Endogenous expression of MEG3 during BMSCs osteogenic differentiation was detected by quantitative real-time polymerase chain reaction (qPCR). MEG3 was knockdown in BMSCs by lentiviral transduction. The proliferation, osteogenic-related genes and proteins expression of MEG3 knockdown BMSCs were assessed by Cell Counting Kit-8 (CCK-8) assay, qPCR, alizarin red and alkaline phosphatase staining. Western blot was used to detect β-catenin expression in MEG3 knockdown BMSCs. Dickkopf 1 (DKK1) was used to block wnt/β-catenin pathway. The osteogenic-related genes and proteins expression of MEG3 knockdown BMSCs after wnt/β-catenin inhibition were assessed by qPCR, alizarin red and alkaline phosphatase staining. MEG3 knockdown BMSCs scaffold with PHMG were implanted in a critical-sized skull defects of rat model. Micro-computed tomography(micro-CT), hematoxylin and eosin staining and immunohistochemistry were performed to evaluate the bone repairing. Results: Endogenous expression of MEG3 was increased during osteogenic differentiation of BMSCs. Downregulation of MEG3 could promote osteogenic differentiation of BMSCs in vitro. Notably, a further mechanism study revealed that MEG3 knockdown could activate Wnt/β-catenin signaling pathway in BMSCs. Wnt/β-catenin inhibition would impair MEG3-induced osteogenic differentiation of BMSCs. By using poly (3-hydroxybutyrate-co-3-hydroxyhexanoate, PHBHHx)-mesoporous bioactive glass (PHMG) scaffold with MEG3 knockdown BMSCs, we found that downregulation of MEG3 in BMSCs could accelerate bone repairing in a critical-sized skull defects rat model. Conclusions: Our study reveals the important role of MEG3 during osteogenic differentiation and bone regeneration. Thus, MEG3 engineered BMSCs may be effective potential therapeutic targets for skull defects.

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Establishing a deeper understanding of the osteogenic differentiation of monolayer cultured human pluripotent stem cells using novel and detailed analyses

Stem Cell Research & Therapy ◽

10.1186/s13287-020-02085-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ping Zhou ◽

Jia-Min Shi ◽

Jing-E Song ◽

Yu Han ◽

Hong-Jiao Li ◽

...

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Pluripotent Stem Cells ◽

Embryonic Stem ◽

Telomerase Activity ◽

Human Pluripotent Stem Cells ◽

Cell Counting ◽

Cell Type ◽

Alizarin Red

Abstract Background Derivation of osteoblast-like cells from human pluripotent stem cells (hPSCs) is a popular topic in bone tissue engineering. Although many improvements have been achieved, the low induction efficiency because of spontaneous differentiation hampers their applications. To solve this problem, a detailed understanding of the osteogenic differentiation process of hPSCs is urgently needed. Methods Monolayer cultured human embryonic stem cells and human-induced pluripotent stem cells were differentiated in commonly applied serum-containing osteogenic medium for 35 days. In addition to traditional assays such as cell viability detection, reverse transcription-polymerase chain reaction, immunofluorescence, and alizarin red staining, we also applied studies of cell counting, cell telomerase activity, and flow cytometry as essential indicators to analyse the cell type changes in each week. Results The population of differentiated cells was quite heterogeneous throughout the 35 days of induction. Then, cell telomerase activity and cell cycle analyses have value in evaluating the cell type and tumourigenicity of the obtained cells. Finally, a dynamic map was made to integrate the analysis of these results during osteogenic differentiation of hPSCs, and the cell types at defined stages were concluded. Conclusions Our results lay the foundation to improve the in vitro osteogenic differentiation efficiency of hPSCs by supplementing with functional compounds at the desired stage, and then establishing a stepwise induction system in the future.

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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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An Inhibitory Role of Osthole in Rat MSCs Osteogenic Differentiation and Proliferation via Wnt/β-Catenin and Erk1/2-MAPK Pathways

Cellular Physiology and Biochemistry ◽

10.1159/000445590 ◽

2016 ◽

Vol 38 (6) ◽

pp. 2375-2388 ◽

Cited By ~ 12

Author(s):

Hongyang Hu ◽

Min Chen ◽

Guangzu Dai ◽

Guoqing Du ◽

Xuezong Wang ◽

...

Keyword(s):

Osteogenic Differentiation ◽

Signaling Pathways ◽

Western Blotting ◽

Underlying Mechanism ◽

Mapk Signaling ◽

Cell Counting ◽

Nuclear Antigen ◽

Dependent Manner ◽

Alizarin Red ◽

Mapk Signaling Pathways

Background/Aims: Bone marrow-derived mesenchymal stem cells (MSCs) are responsible for new bone formation during adulthood. Accumulating evidences showed that Osthole promotes the osteogenic differentiation in primary osteoblasts. The aim of this study was to investigate whether Osthole exhibits a potential to stimulate the osteogenic differentiation of MSCs and the underlying mechanism. Methods: MSCs were treated with a gradient concentration of Osthole (6.25 µM, 12.5 µM, and 25 µM). Cell proliferation was assessed by western blotting with the proliferating cell nuclear antigen (PCNA) and Cyclin D1 antibodies, fluorescence activated cell sorting (FACS), and cell counting kit 8 (CCK8). MSCs were cultured in osteogenesis-induced medium for one or two weeks. The osteogenic differentiation of MSCs was estimated by Alkaline Phosphatase (ALP) staining, Alizarin red staining, Calcium influx, and quantitative PCR (qPCR). The underlying mechanism of Osthole-induced osteogenesis was further evaluated by western blotting with antibodies in Wnt/β-catenin, PI3K/Akt, BMPs/smad1/5/8, and MAPK signaling pathways. Results: Osthole inhibited proliferation of rat MSCs in a dose-dependent manner. Osthole suppressed osteogenic differentiation of rat MSCs by down-regulating the activities of Wnt/β-catenin and Erk1/2-MAPK signaling. Conclusions: Osthole inhibits the proliferation and osteogenic differentiation of rat MSCs, which might be mediated through blocking the Wnt/β-catenin and Erk1/2-MAPK signaling pathways.

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The Role of Pannexin3-Modified Human Dental Pulp-Derived Mesenchymal Stromal Cells in Repairing Rat Cranial Critical-Sized Bone Defects

Cellular Physiology and Biochemistry ◽

10.1159/000486023 ◽

2017 ◽

Vol 44 (6) ◽

pp. 2174-2188 ◽

Cited By ~ 3

Author(s):

Fangfang Song ◽

Hualing Sun ◽

Liyuan Huang ◽

Dongjie Fu ◽

Cui Huang

Keyword(s):

Osteogenic Differentiation ◽

Signaling Pathway ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Dental Pulp ◽

Underlying Mechanism ◽

Bone Defects ◽

Dependent Manner ◽

Alizarin Red ◽

Human Dental Pulp

Background/Aims: Human dental pulp-derived mesenchymal stromal cells (hDPSCs) are promising seed cells for tissue engineering due to their easy accessibility and multi-lineage differentiation. Pannexin3 (Panx3) plays crucial roles during bone development and differentiation. The aim of the present study was to investigate the effect of Panx3 on osteogenesis of hDPSCs and the underlying mechanism. Methods: Utilizing qRT-PCR, Western blot, and immunohistochemistry, we explored the change of Panx3 during osteogenic differentiation of hDPSCs. Next, hDPSCs with loss (Panx3 knockdown) and gain (Panx3 overexpression) of Panx3 function were developed to investigate the effects of Panx3 on osteogenic differentiation of hDPSC and the underlying mechanism. Finally, a commercial β-TCP scaffold carrying Panx3-modified hDPSCs was utilized to evaluate bone defect repair. Results: Panx3 was upregulated during osteogenic differentiation in a time-dependent manner. Panx3 overexpression promoted osteogenic differentiation of hDPSCs, whereas depletion of Panx3 resulted in a decline of differentiation, evidenced by upregulated expression of mineralization-related markers, increased alkaline phosphatase (ALP) activity, and enhanced ALP and Alizarin red staining. Panx3 was found to interact with the Wnt/β-catenin signaling pathway, forming a negative feedback loop. However, Wnt/β-catenin did not contribute to enhancement of osteogenic differentiation as observed in Panx3 overexpression. Moreover, Panx3 promoted osteogenic differentiation of hDPSCs via increasing ERK signaling pathway. Micro-CT and histological staining results showed that Panx3-modified hDPSCs significantly improved ossification of critical-sized bone defects. Conclusion: These findings suggest that Panx3 is a crucial modulator of hDPSCs differentiation.

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Bioinformatics analysis and identification of circular RNAs promoting the osteogenic differentiation of human bone marrow mesenchymal stem cells on titanium treated by surface mechanical attrition

PeerJ ◽

10.7717/peerj.9292 ◽

2020 ◽

Vol 8 ◽

pp. e9292

Author(s):

Shanshan Zhu ◽

Yuhe Zhu ◽

Zhenbo Wang ◽

Chen Liang ◽

Nanjue Cao ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Human Bone ◽

Cell Counting ◽

Circular Rnas ◽

Control Group ◽

Alizarin Red ◽

Qrt Pcr ◽

Mechanical Attrition

Background To analyze and identify the circular RNAs (circRNAs) involved in promoting the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs) on titanium by surface mechanical attrition treatment (SMAT). Methods The experimental material was SMAT titanium and the control material was annealed titanium. Cell Counting Kits-8 (CCK-8) was used to detect the proliferation of hBMSCs, and alkaline phosphatase (ALP) activity and alizarin red staining were used to detect the osteogenic differentiation of hBMSCs on the sample surfaces. The bioinformatics prediction software miwalk3.0 was used to construct competing endogenous RNA (ceRNA) networks by predicting circRNAs with osteogenesis-related messenger RNAs (mRNAs) and microRNAs (miRNAs). The circRNAs located at the key positions in the networks were selected and analyzed by quantitative real-time PCR (QRT-PCR). Results Compared with annealed titanium, SMAT titanium could promote the proliferation and osteogenic differentiation of hBMSCs. The total number of predicted circRNAs was 51. Among these, 30 circRNAs and 8 miRNAs constituted 6 ceRNA networks. Circ-LTBP2 was selected for verification. QRT-PCR results showed that the expression levels of hsa_circ_0032599, hsa_circ_0032600 and hsa_circ_0032601 were upregulated in the experimental group compared with those in the control group; the differential expression of hsa_circ_0032600 was the most obvious and statistically significant, with a fold change (FC) = 4.25 ± 1.60, p-values (p) < 0.05.

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