Osterix Research Articles

Total flavonoids of Rhizoma Drynariae enhances CD31hiEmcnhi vessel formation and subsequent bone regeneration in rat models of distraction osteogenesis byactivating PDGF‑BB/VEGF/RUNX2/OSX signaling axis.

Total flavonoids of Rhizoma Drynariae (TFRD), extracted from the kidney-tonifying Traditional Chinese medicine Rhizoma Drynariae, can be effective in treating osteoporosis, bone fractures and defects. However, the pharmacological effects of TFRD on the specific vessel subtype CD31hiEmcnhi during distraction osteogenesis (DO) remains unclear. The present study aimed to investigate the effects of TFRD on CD31hiEmcnhi vessels in a rat model of DO. In the present study, tibial DO models were established using 60 rats with a distraction rate of 0.2 mm per day for 20 days. Co-immunofluorescence staining of CD31 and endomucin (Emcn) was conducted to determine CD31hiEmcnhi vessels. Radiographic, angiographic and histological analyses were performed to assess bone and vessel formation. Tube formation, alkaline phosphatase (ALP) and Von Kossa staining assays were performed to test angiogenesis of endothelial precursor cells (EPCs) and osteogenesis of bone marrow-derived mesenchymal stem cells (BMSCs). Additionally, expression levels of platelet-derived growth factor (PDGF)-BB, VEGF, runt-related transcription factor 2 (RUNX2) and Osterix (OSX) were determined by western blotting and reverse transcription-quantitative PCR. The in vivo assays demonstrated that TFRD markedly promoted CD31hiEmcnhi vessel formation during DO, whereas PDGF-BB neutralizing antibody suppressed vessel formation. Furthermore, the ALP, Von Kossa staining and tube formation assays indicated that TFRD notably elevated the angiogenic capacity of EPCs and osteogenic capacity of BMSCs under stress conditions, which was significantly suppressed by blocking PDGF-BB. The protein and mRNA levels of PDGF-BB, VEGF, RUNX2 and OSX were upregulated by TFRD, but downregulated by blocking PDGF-BB. Thus, TFRD could facilitate CD31hiEmcnhi vessel formation and subsequently enhance angiogenic-osteogenic coupling to regenerate bone defects during DO via the PDGF-BB/VEGF/RUNX2/OSX signaling axis, which indicated that CD31hiEmcnhi vessels could be a potential novel therapeutic target for DO, and TFRD may represent a promising drug for promoting bone regeneration in DO by increasing CD31hiEmcnhi vessels.

Effect of compression on mandibular fracture haematoma-derived cells

Mechanical stress induces a variety of biochemical and morphological reactions in bone cell biology. This study aimed to investigate appropriate pressures of osteogenesis on the biological responses of 3-dimensional cultured human mandibular fracture haematoma-derived cells by compressive loading. Six patients with mandibular fractures who underwent open reduction and internal fixation were included in the study. During the operation, fracture haematomas that formed fibrin clots were manually removed before irrigation. First, pressures were applied to human mandibular fracture haematoma-derived cell-seeded collagen sponges. The sponges were subjected to mechanical compression using loading equipment applied at no compression, 0.5, or 1 mm. Compressive loading was applied to the samples prior to compression for 0, 6, 12, or 24 hours. Collagen sponge samples were collected for quantification of mRNA using several parameters including alkaline phosphatase (ALP), osteopontin (OPN), osterix (OSX), runt-related gene 2 (RUNX2), protein level, and immunocytochemistry (anti-sclerostin). Among these the 0.5 mm compression group compared with the control and 1.0 mm compression groups upregulated mRNA expression of OPN and OSX after 24 hours. Additionally, compared with the control group, a significantly higher OSX gene expression was observed in both the 0.5 mm and 1.0 mm groups after 6, 12, and 24 hours of compression (p < 0.05). However, no significant differences were observed regarding ALP and RUNX2 expression. These results indicated increased stimulation of osteogenesis of the mandibular fracture-line gap in the 0.5 mm compression group compared with the control and 1.0 mm compression groups.

Sodium Selenite Promotes Osteoblast Differentiation via The WNT/ß-Catenin Signaling Pathway.

ObjectivesOsteoporosis is regarded as a silent disorder affecting bone slowly, leading to an increased risk of fractures. Lately, selenium has been found to be associated with the acquisition and maintenance of bone health by affecting the bone remodeling process. However, the mechanism of action of selenium on bone is poorly understood. Here, the objective of this study is to examine the protective effects and mechanism of sodium selenite on the differentiation process of osteoblasts as well as under oxidative stress-induced conditions by evaluating the expression of osteoblast differentiation markers in the sodium selenite and/or hydrogen peroxide (H2O2)-treated MC3T3-E1 cell line.Materials and Methods In this experimental study, we confirmed the inducible osteogenic effect of sodium selenite on MC3T3-E1 cells. Moreover, we investigated the recovery of expression levels of osteogenic markers of sodium selenite in H2O2-treated MC3T3-E1 cells. Results It was observed that sodium selenite could promote alkaline phosphatase (ALP) activity and collagen synthesis in pre-osteoblasts. Also, sodium selenite enhanced the mRNA expression levels of osteogenic transcriptional factors, like osterix (OSX) and runt-related transcription factor 2 (Runx2). In addition, the terminal differentiation markers, such as osteocalcin (OCN) and collagen 1α (Col1α) were also increased after the treatment of sodium selenite. Also treatment of sodium selenite recused the H2O2-induced inhibition of osteoblastic differentiation of pre-osteoblasts cells via the WNT signaling pathway, implicating its antioxidant activity. Furthermore, sodium selenite restored the H2O2 repressed β-catenin stability and axin-2 reporter activity in MC3T3-E1 cells. ConclusionIt may be concluded that sodium selenite can stimulate bone formation and rescue the oxidative repression of osteogenesis by activating WNT signaling pathways. Further detailed studies on the role of selenium and its ability to stimulate bone formation via the WNT signaling pathway may project it as a potential therapeutic intervention for osteoporosis.

Super-Enhancer-Associated Long Non-Coding RNA LINC01485 Promotes Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells by Regulating MiR-619-5p/RUNX2 Axis

ObjectiveTo investigate the mechanisms of super-enhancer-associated LINC01485/miR-619-5p/RUNX2 signaling axis involvement in osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs).MethodsOsteogenic differentiation of hBMSCs was induced in vitro. The expression levels of LINC01485 and miR-619-5p during osteogenesis were measured using quantitative real-time polymerase chain reaction (qRT-PCR). Osteogenic differentiation was examined by qRT-PCR, western blot, alkaline phosphatase (ALP) staining, ALP activity measurement, and Alizarin Red S (ARS) staining assays. Thereafter, the effects of LINC01485 and miR-619-5p on osteogenic differentiation of hBMSCs were evaluated by performing loss- and gain-of-function experiments. Subsequently, a fluorescence in situ hybridization (FISH) assay was employed to determine the cellular localization of LINC01485. Bioinformatics analysis, RNA antisense purification (RAP) assay, and dual-luciferase reporter assays were conducted to analyze the interactions of LINC01485, miR-619-5p, and RUNX2. Rescue experiments were performed to further delineate the role of the competitive endogenous RNA (ceRNA) signaling axis consisting of LINC01485/miR-619-5p/RUNX2 in osteogenic differentiation of hBMSCs.ResultsThe expression of LINC01485 was up-regulated during osteogenic differentiation of hBMSCs. The overexpression of LINC01485 promoted osteogenic differentiation of hBMSCs by up-regulating the expression of osteogenesis-related genes [e.g., runt-related transcription factor 2 (RUNX2), osterix (OSX), collagen type 1 alpha 1 (COL1A1), osteocalcin (OCN), and osteopontin (OPN)], and increasing the activity of ALP. ALP staining and ARS staining were also found to be increased upon overexpression of LINC01485. The opposing results were obtained upon LINC01485 interference in hBMSCs. miR-619-5p was found to inhibit osteogenic differentiation. FISH assay displayed that LINC01485 was mainly localized in the cytoplasm. RAP assay results showed that LINC01485 bound to miR-619-5p, and dual-luciferase reporter assay verified that LINC01485 bound to miR-619-5p, while miR-619-5p and RUNX2 bound to each other. Rescue experiments illustrated that LINC01485 could promote osteogenesis by increasing RUNX2 expression by sponging miR-619-5p.ConclusionLINC01485 could influence RUNX2 expression by acting as a ceRNA of miR-619-5p, thereby promoting osteogenic differentiation of hBMSCs. The LINC01485/miR-619-5p/RUNX2 axis might comprise a novel target in the bone tissue engineering field.

Insights Into Pulp Biomineralization in Human Teeth

IntroductionMineralized pulp (MP) compromises tooth function and its causation is unknown. The hypothesis of this study is that pulp mineralization is associated with pulpal tissue adaptation, increased mineral densities, and decreased permeabilities of tubular dentin and cementum. Methods will include correlative spatial mapping of physicochemical and biochemical characteristics of pulp, and contextualize these properties within the dentin-pulp complex (DPC) to reveal the inherent vunerabilities of pulp.MethodsSpecimens (N = 25) were scanned using micro X-ray computed tomography (micro-XCT) to visualize MP and measure mineral density (MD). Elemental spatial maps of MP were acquired using synchrotron X-ray fluorescence microprobe (μXRF) and energy dispersive X-ray spectroscopy (EDX). Extracted pulp tissues were sectioned for immunolabelling and the sections were imaged using a light microscope. Microscale morphologies and nanoscale ultrastructures of MP were imaged using scanning electron (SEM) and scanning transmission electron microscopy (STEM) techniques.ResultsHeterogeneous distribution of MD from 200 to 2,200 mg/cc, and an average MD of 892 (±407) mg/cc were observed. Highly mineralized pulp with increased number of occluded tubules, reduced pore diameter in cementum, and decreased connectivity in lateral channels were observed. H&amp;amp;E, trichrome, and von Kossa staining showed lower cell and collagen densities, and mineralized regions in pulp. The biomolecules osteopontin (OPN), osteocalcin (OCN), osterix (OSX), and bone sialoprotein (BSP) were immunolocalized around PGP 9.5 positive neurovascular bundles in MP. SEM and STEM revealed a wide range of nano/micro particulates in dentin tubules and spherulitic mineral aggregates in the collagen with intrafibrillar mineral surrounding neurovascular bundles. EDX and μXRF showed elevated counts of Ca, P, Mg, and Zn inside pulp and at the dentin-pulp interface (DPI) in the DPC.ConclusionColocalization of physical and chemical, and biomolecular compositions in MP suggest primary and secondary biomineralization pathways in pulp and dentin at a tissue level, and altered fluid dynamics at an organ level. Elevated counts of Zn at the mineralizing front in MP indicated its role in pulp biomineralization. These observations underpin the inherent mechano- and chemo-responsiveness of the neurovascular DPC and help elucidate the clinical subtleties related to pulpitis, dentin-bridge, and pulp stone formation.

Role of LINC01133 in Osteogenic Differentiation of Dental Pulp Stem Cells by Targeting miR-199b-5p.

Recently, increasing attention has been paid to the function of long non-coding RNAs (lncRNAs) in osteogenic differentiation (OD) of dental pulp stem cells (DPSCs). LINC01133 was reported to have a close relationship with tumorigenesis for multiple cancers, but no study has yet explored the role of LINC01133 in modulating OD of DPSCs. Alizarin red S (ARS) staining and alkaline phosphatase (ALP) staining were perfomed to assess the OD potential of DPSCs. Osteogenic markers including runt-related transcription factor 2 (RUNX2), osterix (OSX) and ALP expression levels in DPSCs were monitored by qRT-PCR and Western blot before and after cell transfection. Luciferase reporter gene assay detected the relationship between LINC01133 and miR-199b-5p. The expression of LINC01133 was low, while miR-199b-5p was increasingly expressed during OD of DPSCs. Overexpression of LINC01133 in DPSCs resulted in decreased expression of RUNX2, OSX, ALP, DSPP and DMP1, whose expression was reversed in DPSCs after transfections of miR-199b-5p overexpression. Co-transfection of pcDNA3.1-LINC01133 and miR-199b-5p mimic led to elevated expression of RUNX2, OSX, ALP, DSPP and DMP1 compared with pcDNA3.1-LINC01133 transfection alone. LINC01133 served as a sponge of miR-199b-5p. AKT3 was verified as a downstream effector of miR-199b-5p in DPSCs. LINC01133 inhibits the OD of DPSCs by upregulating AKT3 via sponging miR-199b-5p, which may act as a potential diagnostic biomarker for dentin regeneration in the dental pulp.

High-mobility group box chromosomal protein-1 deletion alleviates osteoporosis in OVX rat model via suppressing the osteoclastogenesis and inflammation

BackgroundOsteoporosis is a skeletal metabolic disease that constitutes a great threaten to human health. However, there is currently no gold standard for its treatment. High-mobility group box chromosomal protein-1 (HMGB-1) has been reported to play an important role in various orthopedic diseases. Till now, its role in osteoporosis remains elusive.MethodsRats underwent ovariectomy (OVX) were used to construct a postmenopausal model of osteoporosis. Then, rats were divided into sham groups without OVX surgery, OVX model group, HMGB-1 knockdown (HMGB-1 KD) OVX model groups. The expression of HMGB1 was evaluated by qRT-PCR and western blotting. Subsequently, the changes of trabeculae were evaluated by micro-computed tomography (CT) assay. Skeletal necrosis and metabolism were further analyzed by hematoxylin–eosin (HE) staining, Alcian blue staining and Masson’s trichrome staining. The contents of serum alkaline phosphatase (ALP) and osteocalcin were detected by ELISA assay. Expression of osteoclast-associated receptor (OSCAR) and tartrate-resistant acid phosphatase (TRAP) were determined to investigate the effects of HMGB-1 loss on osteoclastogenesis.ResultsSingle HMGB-1 deletion exerted no significant effect on rat trabeculae, serum ALP and osteocalcin. Noticeably, HMGB1 knockdown dramatically ameliorated OVX-induced changes in above indexes. Trabeculae structures of OVX rats were sparse with disorder arrangement, which were greatly recovered after HMGB-1 deletion. Enhanced osteoclastogenesis was observed in OVX rats by increasing number of TRAP + cells and expression of TRAP and OSCAR, and loss of HMGB1 ameliorated osteoclastogenesis in OVA rats. Moreover, HMGB-1 deletion antagonized OVX-evoked downregulation of osteoblast activity markers osterix (OSX), collagen type I alpha 1(COL1A1) and distal-less homeobox 2 (DLX2) protein. Furthermore, loss of HMGB-1 attenuated fluctuation of inflammatory factors in OVX rats. Additionally, HMGB-1 deficiency inhibited OVX-evoked activation of the Toll-like receptor (TLR) 4/NF-κB signaling pathway. Moreover, reactivating the TLR4 signaling further aggravated OVX-induced osteoporosis, which was reversed by HMGB1 knockdown.ConclusionHMGB-1 deletion alleviated OVX-triggered osteoporosis by suppressing osteoclastogenesis and inflammatory disorder via the inhibition of the TLR4 signaling. Therefore, HMGB-1 may be a promising therapeutic target for osteoporosis.

Effect of Cold Atmospheric Plasma (CAP) on Osteogenic Differentiation Potential of Human Osteoblasts.

Bone regeneration after oral and maxillofacial surgery is a long-term process, which involves various mechanisms. Recently, cold atmospheric plasma (CAP) has become known to accelerate wound healing and have an antimicrobial effect. Since the use of CAP in dentistry is not yet established, the aim of the present study was to investigate the effect of CAP on human calvaria osteoblasts (HCO). HCO were treated with CAP for different durations of time and distances to the cells. Cell proliferation was determined by MTT assay and cell toxicity by LDH assay. Additionally, RT-qPCR was used to investigate effects on osteogenic markers, such as alkaline phosphatase (ALP), bone morphogenic protein (BMP)2, collagen (COL)1A1, osteonectin (SPARC), osteoprotegerin (OPG), osterix (OSX), receptor activator of NF-κB (RANK), RANK Ligand (RANKL), and Runt-related transcription factor (RUNX)2. There were small differences in cell proliferation and LDH release regarding treatment duration and distance to the cells. However, an increase in the expression of RANK and RANKL was observed at longer treatment times. Additionally, CAP caused a significant increase in mRNA expression of genes relevant to osteogenesis. In conclusion, CAP has a stimulating effect on osteoblasts and may thus represent a potential therapeutic approach in the regeneration of hard tissue defects.

Hirudin promotes proliferation and osteogenic differentiation of HBMSCs via activation of cyclic guanosine monophosphate (cGMP)/protein kinase-G (PKG) signaling pathway

ABSTRACT Osteoporosis is a public health problem resulting in higher susceptibility to bone fracture. Hirudin is known as a direct thrombin inhibitor, which is isolated from the salivary gland of the medicinal leech. The present study aimed to evaluate the effect of Hirudin on the proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (HBMSCs). In our study, the effect of Hirudin on the proliferation of HBMSCs was evaluated with the CCK-8 and MTT assays. The capacity of osteogenic differentiation and mineralization of HBMSCs was evaluated with ALP and alizarin red staining, respectively. cGMP content was determined by ELISA. Western blotting and qRT-PCR were used to investigate the effect of Hirudin on the expression of osteoblast-specific markers, including Runx2, osterix (OSX), osteocalcin (OCN), and collagen1 (Col1). In our study, Hirudin treatment promoted cell viability. Moreover, Hirudin treatment increased ALP activity of HBMSCs and red coloration of alizarin. Interestingly, cGMP inhibitor partly reversed the effect of Hirudin on the proliferation, differentiation and mineralization of HBMSCs. In conclusion, Hirudin promoted the proliferation, differentiation and mineralization of HBMSCs via activation of cGMP signaling pathway. Hence, Hirudin contributed to bone remodeling and might represent as an effective agent for the treatment of osteoporosis.

The role of ZBTB16 in odontogenic differentiation of dental pulp stem cells

ObjectiveOdontogenic differentiation of dental pulp stem cells (DPSCs) is highly controlled by the activation of several transcription factors. The zinc finger and BTB domain-containing 16 (ZBTB16) gene encodes a BTB/POZ domain and zinc finger containing transcription factors and is involved in several biological processes, but little is known about its role in odontogenic differentiation. The main goal of the current study was to determine the role of ZBTB16 in odontogenic differentiation of DPSCs. DesignZBTB16, runt-related transcription factor 2 (RUNX2), and osterix (OSX) were silenced via small-hairpin RNA (shRNA) lentivirus. The odontoblastic differentiation of DPSCs was detected by alkaline phosphatase (ALP) staining, activity measurement, and alizarin red S staining in vitro. The gene and protein expression levels were assessed by RT-qPCR and western blotting. Further, an ectopic implantation experiment was performed to explore the role of ZBTB16 in mineralization regulation in vivo followed by histological examination. ResultsThe silencing of ZBTB16 attenuated ALP activity and mineralized nodules formation by DPSCs. In addition, knockdown of ZBTB16 impaired the expression of markers involved in odontogenic differentiation, including dentin sialophosphoprotein, dentin matrix acidic phosphoprotein 1, and collagen 1 in vitro and vivo. Silencing the OSX gene suppressed ZBTB16 expression and, in turn, OSX expression decreased after ZBTB16 knockdown. However, shRUNX2 did not suppress ZBTB16 expression and shZBTB16 did not affect RUNX2 expression. ConclusionsZBTB16 may play an important role in modulating the odontoblastic differentiation of DPSCs and act as a regulator of OSX in a possible feed-back cycle independent of RUNX2.

Immunocytochemistry and western blot test for the in-situ detection of biomarkers of osteogenesis

There are two ways that bones can form: intramembranous and endochondral ossification. Specific osteogenic markers, such as insulin-like growth factor (IGF-1), osteocalcin (OCN), Runt-related transcription factor 2 (RUNX2), and osterix (OSX), accompany osteoblast differentiation from an undifferentiated state to a functional state. IGF-1 hormones are the main regulators in growth, differentiation, and apoptosis in cells and tissues mediated by IGF-1 receptors (IGF-1R). Biomolecular technology aims to study nucleic acids and their regulation and expression of proteins. Techniques that can be used when analyzing proteins include the Bradford protein assay, immunocytochemistry, immunohistochemistry, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and the western blot test. To better understand the biomarkers of osteogenesis, the use of in-situ detection is suggested, such as immunocytochemistry and the western blot test. For this review, the author adhered to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) standards. The research examined 50 articles; only 4 articles were selected for this review. In the immunocytochemical test, IGF-1 expression was found in the nucleus and IGF-1R expression in the cell membrane, and it was found that RUNX2, OSN, and OCN are important for osteogenesis. During the western blot test, it was found that the addition of IGF-1 in dental stem cells could increase the expression of RUNX2, OSX, and OCN proteins. Based on this information, it appears that both immunocytochemistry and western blot tests can detect the main biomarkers of osteogenesis.

MicroRNA-34a and microRNA-146a target CELF3 and suppress the osteogenic differentiation of periodontal ligament stem cells under cyclic mechanical stretch

Background/purposeDuring orthodontic tooth movement, mechanical forces induce the osteogenic differentiation of periodontal ligament stem cells (PDLSCs), which contributes to alveolar bone remodeling. MicroRNAs (miRNAs) are involved in regulating PDLSC osteogenic differentiation. Therefore, we intended to explore the role of miR-34a and miR-146a in osteogenic differentiation of PDLSCs under cyclic stretch. Materials and methodsPhenotypic identification of PDLSCs was determined by flow cytometry analysis. PDLSCs were incubated with osteogenic differentiation medium for 3 weeks and the osteogenic differentiation capability was detected by Alizarin Red staining. To mimic the orthodontic forces, cyclic mechanical stretch was applied to PDLSCs. Alkaline phosphatase (ALP) activity assay and ALP staining were used for evaluating the ALP activity. The expression of osteogenesis markers in PDLSCs was assessed by western blotting and qRT-PCR. The binding between miR-34a (or miR-146a) and CUGBP Elav-like family member 3 (CELF3) was validated by luciferase reporter assay. ResultsCyclic stretch elevated ALP activity and the expression of osteogenesis markers, osteopontin (OPN), runt-related transcription factor 2 (RUNX2), type I collagen (COL1), ALP, osteocalcin (OCN) and osterix (OSX), in PDLSCs. MiR-146a and miR-34a were downregulated in PDLSCs under cyclic stretch. Either overexpressing miR-146a and miR-34a reduced ALP activity and the expression of osteogenesis markers. CELF3 was a target of both miR-146a and miR-34a. CELF3 silencing attenuated while CELF3 overexpression enhanced ALP activity and the expression of osteogenesis markers. ConclusionMiR-34a and miR-146a repress cyclic stretch-induced osteogenic differentiation of PDLSCs via regulating the expression of CELF3.

Diabetes impairs fracture healing through disruption of cilia formation in osteoblasts.

Diabetes-associated fracture risk and impaired fracture healing represents a serious health threat. It is well known that type 1 diabetes mellitus (T1DM) impairs fracture healing due to its effect on osteoblasts and their progenitor cells. Previous studies have showed that primary cilia and intraflagellar transport protein 80 (IFT80) are critical for bone formation. However, whether TIDM impairs fracture healing due to influencing ciliary gene expression and cilia formation is unknown. Here, we investigated the effect of T1DM on primary cilia in a streptozotocin induced diabetes mouse model and examined the impact of cilia on fracture healing in osteoblasts by deletion of IFT80 in osteoblast linage using osterix (OSX)-cre (OSXcretTAIFT80f/f). The results showed that diabetes inhibited ciliary gene expression and primary cilia formation to an extent that was similar to normoglycemic mice with IFT80 deletion. Moreover, diabetic mice and normoglycemic mice with cilia loss in osteoblasts (OSXcretTAIFT80f/f) both exhibited delayed fracture healing with significantly reduced bone density and mechanical strength as well as with reduced expression of osteoblast markers, decreased angiogenesis and proliferation of bone lining cells at the fracture sites. In vitro studies showed that advanced glycation end products (AGEs) downregulated IFT80 expression in osteoblast progenitors. Moreover, AGEs and IFT80 deletion significantly reduced cilia number and length which inhibited differentiation of primary osteoblast precursors. Thus, this study for the first time report that primary cilia are essential for bone regeneration during fracture healing and loss of cilia caused by diabetes in osteoblasts resulted in defective diabetic fracture healing.

REV-ERBs negatively regulate mineralization of the cementoblasts

ObjectiveThe role of circadian clock in cementogenesis is unclear. This study examines the role of REV-ERBs, one of circadian clock proteins, in proliferation, migration and mineralization of cementoblasts to fill the gap in knowledge. MethodsExpression pattern of REV-ERBα in cementoblasts was investigated in vivo and in vitro. CCK-8 assay, scratch wound healing assay, alkaline phosphatase (ALP) and alizarin red S (ARS) staining were performed to evaluate the effects of REV-ERBs activation by SR9009 on proliferation, migration and mineralization of OCCM-30, an immortalized cementoblast cell line. Furthermore, mineralization related markers including osterix (OSX), ALP, bone sialoprotein (BSP) and osteocalcin (OCN) were evaluated. ResultsStrong expression of REV-ERBα was found in cellular cementum around tooth apex. Rev-erbα mRNA oscillated periodically in OCCM-30 and declined after mineralization induction. REV-ERBs activation by SR9009 inhibited proliferation but promoted migration of OCCM-30 in vitro. Results of ALP and ARS staining suggested that REV-ERBs activation negatively regulated mineralization of OCCM-30. Mechanically, REV-ERBs activation attenuated the expression of OSX and its downstream targets including ALP, BSP and OCN. ConclusionsREV-ERBs are involved in cementogenesis and negatively regulate mineralization of cementoblasts via inhibiting OSX expression. Our study provides a potential target regarding periodontal and cementum regeneration.

Mechanosensitive Piezo1 in Periodontal Ligament Cells Promotes Alveolar Bone Remodeling During Orthodontic Tooth Movement.

Orthodontic tooth movement (OTM) is a process depending on the remodeling of periodontal tissues surrounding the roots. Orthodontic forces trigger the conversion of mechanical stimuli into intercellular chemical signals within periodontal ligament (PDL) cells, activating alveolar bone remodeling, and thereby, initiating OTM. Recently, the mechanosensitive ion channel Piezo1 has been found to play pivotal roles in the different types of human cells by transforming external physical stimuli into intercellular chemical signals. However, the function of Piezo1 during the mechanotransduction process of PDL cells has rarely been reported. Herein, we established a rat OTM model to study the potential role of Piezo1 during the mechanotransduction process of PDL cells and investigate its effects on the tension side of alveolar bone remodeling. A total of 60 male Sprague-Dawley rats were randomly assigned into three groups: the OTM + inhibitor (INH) group, the OTM group, and the control (CON) group. Nickel-titanium orthodontic springs were applied to trigger tooth movement. Mice were sacrificed on days 0, 3, 7, and 14 after orthodontic movement for the radiographic, histological, immunohistochemical, and molecular biological analyses. Our results revealed that the Piezo1 channel was activated by orthodontic force and mainly expressed in the PDL cells during the whole tooth movement period. The activation of the Piezo1 channel was essential for maintaining the rate of orthodontic tooth movement and facilitation of new alveolar bone formation on the tension side. Reduced osteogenesis-associated transcription factors such as Runt-related transcription factor 2 (RUNX2), Osterix (OSX), and receptor activator of nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio were examined when the function of Piezo1 was inhibited. In summary, Piezo1 plays a critical role in mediating both the osteogenesis and osteoclastic activities on the tension side during OTM.

Osteopontin induces osteogenic differentiation by human periodontal ligament cells via calcium binding domain-ALK-1 interaction.

Recently we have generated recombinant human osteopontin (rhOPN) using a plant platform (Nicotiana benthamiana) and demonstrated, when coated on culture plates, its osteogenic induction capacity of human periodontal ligament (PDL) cells. The aim of this study is to elucidate the molecular mechanism underlying the rhOPN-induced osteogenic differentiation of human PDL cells. Full length rhOPN (FL-OPN) and three constructs of OPN containing integrin binding domain (N142), calcium binding domain (C122) and mutated calcium-binding domain (C122δ) were generated from N. benthamiana. Human PDL cells were isolated from extracted third molars and cultured on FL-OPN, N142, C122, or C122δ-coated surfaces. Real-time PCR and Western blot analyses were used to determine mRNA and protein expression. In vitro calcification was determined by Alizarin red staining. A chemical inhibitor and RNAi silencing were used to elucidate signaling pathways. In silico analyses were performed to predict the protein-protein interaction. In vivo analysis was performed using a rat calvaria defect model. Human PDL cells seeded on FL-OPN and C122-coated surfaces significantly increased both mRNA and protein expression of osterix (OSX) and enhanced in vitro calcification. Soluble FL-OPN as well as a surface coated with N142 did not affect OSX expression. Inhibition of activin receptor-like kinase (ALK-1) abolished the induction of osterix expression. In silico analysis suggested a possible interaction between the calcium binding domain (CaBD) of OPN and ALK-1 receptor. C122, but not C122δ coated surfaces, induced the expression of p-Smad-1 and this induction was inhibited by an ALK-1 inhibitor and RNAi against ALK-1. In vivo data showed that 3D porous scaffold containing C-122 enhanced new bone formation as compared to scaffold alone. The results suggest that next to full length OPN, the CaBD of OPN, if coated to a surface, induces osteogenic differentiation via interaction with ALK-1 receptor.

Effects of idiopathic hypoparathyroidism on tooth eruption of rats

Objective: To explore the effects of reduced parathyroid function in early growth and development on tooth eruption and enamel development by establishing an animal model of idiopathic hypoparathyroidism (IHP) and to explore the mechanism of IHP affecting tooth eruption with a view to provide experimental basis for early diagnosis and clinical treatment of IHP. Methods: Forty-eight SD rats at postnatal day 7 were randomly and equally divided into sham operation group and IHP group. The bilateral parathyroidectomy (PTX) was performed by using carbon nanoparticles technique to establish an IHP rat model, while no parathyroids were removed in the sham operation group using the same technique. Serum was extracted after surgery, serum calcium, serum phosphorus, and serum parathyroid hormone (PTH) concentrations were detected in order to verify the success of the modeling. At postnatal day 14, day 25 and day 38 (P14, P25 and P38) the rats were sacrificed to collect the mandible samples (six from each group) and to analyze the volume of enamel, the height of the tooth eruption and the bone microarchitecture parameters of the root-oriented alveolar bone of mandibular third molar quantitatively by micro-CT scanning. Histological sections were prepared. The distribution and expression levels of osteoblast differentiation markers runt-related transcription factor 2 (RUNX2) and osterix (OSX) in the alveolar bone around the third molar were detected by immunohistochemical staining and the osteoclast activity was detected by tartrate-resistant acid phosphatase (TRAP) staining. After each of the third molars was isolated, the microhardness of the enamel was measure by using a microhardness tester and the enamel microstructure was photographed by using scanning electron microscope. Primary dental follicle stem cells were isolated from other six mandibulars from each group at P14 and cultured in vitro. The cell proliferation activity was tested by cell colony forming units detection. After induction of dental follicle stem cells into osteogenic differentiation, the degree of mineralization was detected by using alkaline phosphatase staining and alizarin red staining. The mRNA of mandibular tissues and dental follicle cells were extracted, the expression of genes related to osteoblasts and osteoclast differentiations and parathyroid hormone receptor 1 (PTH1R) were detected by real-time quantitative PCR. Results: Bilateral parathyroidectomy was successfully performed on rats with the help of carbon nanoparticles under stereomicroscope. After surgery, the serum calcium concentration reduced, the serum phosphorus concentration increased and the serum PTH concentration distinctly reduced (P<0.01). The volume of enamel [(4.58±0.24) mm3] and the microhardness [(167.76±21.86) MPa] in IHP group were significantly lower than that in sham operation group [(5.22±0.46) mm3, P<0.05; (223.92±10.94) MPa, P<0.01, respectively]. The eruption height of the mandibular third molar in the IHP group was respectively lower than that in the sham operation group (P<0.05). The bone volume over total volume and trabecular number of the root-oriented alveolar bone of the mandibular third molars in the IHP group were respectively lower than that in sham operation group (P<0.05). The expression levels of RUNX2 and OSX proteins in the root-oriented alveolar bone of the mandibular third molars in the IHP group respectively reduced, compared to that in sham operation group (P<0.05). Furthermore, the number of osteoclasts (3.86±1.07) in crown-oriented alveolar bone in the IHP group was respectively lower than that in sham operation group (6.43±1.27) (P<0.01). The proliferative activity of dental follicle stem cells in the IHP group respectively decreased (P<0.01). After the induction of osteogenic differentiation, the mineralization ability of dental follicle stem cells in the IHP group was weakened. In the mandibular tissues of IHP group, the expression levels of osteogenesis related genes such as RUNX2 and OSX and the the expression of PTH1R significantly reduced (P<0.05). The receptor activator of nuclear factor-κB ligand/osteoprotegerin (RANKL/OPG) ratio reduced significantly (P<0.01) compared to those of sham operation group. Also in the dental follicle cells of IHP group, the expression levels of osteogenesis related genes such as RUNX2 and OSX, the RANKL/OPG ratio and the expression of PTH1R significantly decreased simultaneously compared to that in sham operation group (P<0.01). Conclusions: Under the condition of idiopathic hypoparathyroidism, the weakening of PTH/PTH1R signaling may reduce the proliferative activity of dental follicle stem cells, inhibit their regulation for osteoblast and osteoclast differentiations and functions, thereby interfere the bone remodeling of alveolar bone around the tooth germ during tooth eruption, which eventually leads to delayed tooth eruption.

Improving osteogenic activity of Y-TZP (Yttria-stabilized tetragonal zirconia polycrystal) surfaces by grafting of silanes with different end groups

Poor osteogenesis caused by limited bioactivity is one of the challenges affecting the use of zirconia-based materials as implants. In this study, four representative silanes with different end groups [γ-methacryloxypropyltrimethoxysilane (γ-MPS, with a vinyl end group), methyltrimethoxysilane (MTMS, methyl end), 3-mercaptopropyltrimethoxysilane (MPTS, mercapto end), and 3-aminopropyltriethoxysilane (APTES, amino end)] were grafted on a Y-TZP surface with the purpose of improving its osteogenic activity. Thermodynamic and analytical chemistry results confirmed that the terminal alkoxy groups of the silanes could condense with the hydroxyl groups on the zirconia surface to form covalent Zr–O–Si bonds; in particular, mercapto- terminated silanes were more effective at combining with zirconia than hydrophobic vinyl- and methyl-terminated silanes. Y-TZP modified with mercapto- and amino-terminated silanes showed improved hydrophilicity, which in turn promoted cell adhesion and growth. In vitro cell experiments showed that mercapto- and amino-terminated silanes promoted osteogenic differentiation and mineralization by increasing osteocalcin (OCN) and osterix (OSX) expression levels, while vinyl- and methyl-terminal silanes exhibited inhibitory effects on the collagen type 1 (COL-1), Runt-related transcription factor 2 (Runx2), and OCN expressions. Therefore, grafting mercapto- and amino-terminal silanes is a simple and effective strategy for enhancing the osteogenic potential of Y-TZP implants.

Anthocyanin-enriched polyphenols from Hibiscus syriacus L. (Malvaceae) exert anti-osteoporosis effects by inhibiting GSK-3β and subsequently activating β-catenin

BackgroundThe bark and petal of Hibiscus syriacus L. (Malvaceae) have been used to relieve pain in traditional Korean medicine. Recently, we identified anthocyanin-enriched polyphenols from the petal of H. syriacus L. (AHs) and determined its anti-melanogenic, anti-inflammatory, and anti-oxidative properties. Nevertheless, the osteogenic potential of AHs remains unknown. PurposeThis study was aimed to investigating the effect of AHs on osteoblast differentiation and osteogenesis in osteoblastic cell lines and zebrafish larvae. Furthermore, we investigated whether AHs ameliorates prednisolone (PDS)-induced osteoporosis. Study design and methodsCell viability was assessed by cellular morphology, MTT assay, and flow cytometry analysis, and osteoblast differentiation was measured alizarin red staining, alkaline phosphatase (ALP) activity, and osteoblast-specific marker expression. Osteogenic and anti-osteoporotic effects of AHs were determined in zebrafish larvae. ResultsAHs enhanced calcification and ALP activity concomitant with the increased expression of osterix (OSX), runt-related transcription factor 2 (RUNX2), and ALP in MC3T3-E1 preosteoblast and MG-63 osteosarcoma cells. Additionally, AHs accelerated vertebral formation and mineralization in zebrafish larvae, concurrent with the increased expression of OSX, RUNX2a, and ALP. Furthermore, PDS-induced loss of osteogenic activity and vertebral formation were restored by treatment with AHs, accompanied by a significant recovery of calcification, ALP activity, and osteogenic marker expression. Molecular docking studies showed that 16 components in AHs fit to glucagon synthase kinase-3β (GSK-3β); particularly, isovitexin-4′-O-glucoside most strongly binds to the peptide backbone of GSK-3β at GLY47(O), GLY47(N), and ASN361(O), with a binding score of -7.3. Subsequently, AHs phosphorylated GSK-3β at SER9 (an inactive form) and released β-catenin into the nucleus. Pretreatment with FH535, a Wnt/β-catenin inhibitor, significantly inhibited AH-induced vertebral formation in zebrafish larvae. ConclusionAHs stimulate osteogenic activities through the inhibition of GSK-3β and subsequent activation of β-catenin, leading to anti-osteoporosis effects.

Fisetin promotes osteoblast differentiation and osteogenesis through GSK-3β phosphorylation at Ser9 and consequent β-catenin activation, inhibiting osteoporosis

Fisetin is a bioactive flavonol that inhibits osteoclastogenesis and promotes osteoblastogenesis. However, the osteogenic activity of fisetin needs to be comprehensively elucidated. In the present study, we observed that fisetin significantly increased alkaline phosphatase (ALP) activity and bone mineralization in MC3T3-E1 preosteoblasts, accompanied by a significant increase in runt-related transcription factor 2 (RUNX2), ALP, collagen type Ⅰ alpha 1 (Col1α1), osterix (OSX), osteocalcin (OCN), and bone morphogenetic protein 4 (BMP4) expression. Furthermore, fisetin promoted vertebral formation in zebrafish larvae, with the highest fisetin concentration comparable with that observed in β-glycerophosphate treatment. Fisetin also inhibited prednisolone (PDS)-induced anti-osteoblastic genes, including nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), tartrate-resistant acid phosphatase-6 (ACP6), dendritic cell-specific transmembrane protein (DC-STAMP), and cathepsin K (CTSK). Fisetin potently mitigated the PDS-induced inhibition of ALP activity and bone mineralization, as well as vertebral resorption in zebrafish larvae. Moreover, we confirmed that fisetin-induced osteogenic effect was activated through phosphorylation of glycogen synthase kinase-3β (GSK-3β) at Ser9, consequently releasing β-catenin from the destructive complex to promote its nuclear translocation. β-Catenin inhibition by FH535 and the stabilization of GSK-3β by DOI hydrochloride remarkably inhibited fisetin-induced osteogenic activities, indicating that the GSK-3β/β-catenin signaling pathway plays a vital role in fisetin-induced osteogenesis. Collectively, our findings suggest that fisetin stimulates osteogenic activity and could be used as an effective strategy to prevent bone resorption.