mRNA Expression Of Osteogenic Markers Research Articles

Sustained BMP-2 delivery via alginate microbeads and polydopamine-coated 3D-Printed PCL/β-TCP scaffold enhances bone regeneration in long bone segmental defects

Background/ObjectiveRepair of long bone defects remains a major challenge in clinical practice, necessitating the use of bone grafts, growth factors, and mechanical stability. Hence, a combination therapy involving a 3D-printed polycaprolactone (PCL)/β-tricalcium phosphate (β-TCP) scaffold coated with polydopamine (PDA) and alginate microbeads (AM) for sustained delivery of bone morphogenetic protein-2 (BMP-2) was investigated to treat long bone segmental defects. MethodsSeveral in vitro analyses were performed to evaluate the scaffold osteogenic effects in vitro such as PDA surface modification, namely, hydrophilicity and cell adhesion; cytotoxicity and BMP-2 release kinetics using CCK-8 assay and ELISA, respectively; osteogenic differentiation in canine adipose-derived mesenchymal stem cells (Ad-MSCs); formation of mineralized nodules using ALP staining and ARS staining; and mRNA expression of osteogenic differentiation markers using RT-qPCR. Bone regeneration in femoral bone defects was evaluated in vivo using a rabbit femoral segmental bone defect model by performing radiography, micro-computed tomography, and histological observation (hematoxylin and eosin and Masson's trichrome staining). ResultsThe PDA-coated 3D-printed scaffold demonstrated increased hydrophilicity, cell adhesion, and cell proliferation compared with that of the control. BMP-2 release kinetics assessment showed that BMP-2 AM showed a reduced initial burst and continuous release for 28 days. In vitro co-culture with canine Ad-MSCs showed an increase in mineralization and mRNA expression of osteogenic markers in the BMP-2 AM group compared with that of the BMP-2-adsorbed scaffold group. In vivo bone regeneration evaluation 12 weeks after surgery showed that the BMP-2 AM/PDA group exhibited the highest bone volume in the scaffold, followed by the BMP-2/PDA group. High cortical bone connectivity was observed in the PDA-coated scaffold groups. ConclusionThese findings suggest that the combined use of PDA-coated 3D-printed bone scaffolds and BMP-2 AM can successfully induce bone regeneration even in load-bearing bone segmental defects. The translational potential of this articleA 3D-printed PCL/β-TCP scaffold was fabricated to mimic the cortical bone of the femur. Along with the application of PDA surface modification and sustained BMP-2 release via AM, the developed scaffold could provide suitable osteoconduction, osteoinduction, and osteogenesis in both in vitro settings and in vivo rabbit femoral segmental bone defect models. Therefore, our findings suggest a promising therapeutic option for treating challenging long bone segmental defects, with potential for future clinical application.

MgO-enhanced β-TCP promotes osteogenesis in both in vitro and in vivo rat models

Allogeneic bone grafts are used to treat bone defects in orthopedic surgery, but the osteogenic potential of artificial bones remains a challenge. In this study, we developed a β-tricalcium phosphate (β-TCP) formulation containing MgO, ZnO, SrO, and SiO2 and compared its bone-forming ability with that of β-TCP without biological elements. We prepared β-TCP discs with 60% porosity containing 1.0 wt% of these biological elements. β-TCP scaffolds were loaded with bone marrow-derived mesenchymal stem cells (BMSC) from 7-week-old male rats and cultured for 2 weeks. ALP activity and mRNA expression of osteogenic markers were evaluated. In addition, scaffolds were implanted subcutaneously in rats and analyzed after 7 weeks. In vitro, the MgO group showed lower Ca concentrations and higher osteogenic marker expression compared to controls. In vivo, the MgO group showed higher ALP activity compared to controls, and RT-qPCR analysis showed significant expression of BMP2 and VEGF. Histopathology, fluorescent immunostaining, and micro-CT also showed relatively better bone formation in the MgO group. β-TCP with MgO may enhance bone morphology in vitro and in vivo and improve the prognosis of patients with substantial and refractory bone defects.

Plasma exosomal miR-30a-5p inhibits osteogenic differentiation of bone marrow mesenchymal stem cells from a chronic unpredictable mild stress-induced depression rat model

With rising society stress, depression-induced osteoporosis is increasing. However, the mechanism involved is unclear. In this study, we explored the effect of plasma exosomal miRNAs on bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation in a chronic unpredictable mild stress (CUMS)-induced depression rat model. After 12 weeks of CUMS-induced depression, the pathological changes in the bone tissue and markers of osteogenic differentiation were tested by micro-computed tomography, hematoxylin-eosin staining, and quantitative real-time reverse transcription PCR (qRT-PCR). Plasma exosomes from rats were isolated and co-incubated with BMSCs for 14 d to detect the effect on osteogenic markers. Next-generation sequencing identified the miRNAs in the plasma exosomes, and the differential miRNAs were analyzed and verified by qRT-PCR. BMSCs were infected with lentivirus to upregulate miRNA-30a-5p and incubated in a medium that induced osteogenic differentiation for 14 d. The effect of miR-30a-5p on osteogenic differentiation was determined by qPCR and alizarin red staining. CUMS-induced depression rat model was established successfully, and exhibited reduced bone mass and damaged bone microstructure compared to that of the controls. The observed pathological changes suggested the occurrence of osteoporosis in the CUMS group, and the mRNA expression of osteogenic markers was also significantly reduced. Incubation of BMSCs with plasma exosomes from the CUMS group for 14 d resulted in a significant decrease in the expression of osteogenic markers. Twenty-five differentially expressed miRNAs in plasma exosomes were identified and upregulation of miR-30a-5p was observed to significantly inhibit the expression of osteogenic markers in BMSCs. Our findings contributed to a comprehensive understanding of the mechanism of osteoporosis caused by depression, and demonstrated the potential of miR-30a-5p as a novel biomarker or therapeutic target for the treatment of osteoporosis.

Ginsenoside Rg1 modulates PI3K/AKT pathway for enhanced osteogenesis via GPER

BackgroundOsteoporosis is a systemic skeletal disorder characterized by decreased bone density and the degradation of bone tissue microarchitecture. Ginsenoside Rg1, derived from Panax ginseng, has been a part of traditional Chinese medicine in China for centuries, particularly for treating osteoporosis. However, there remains limited research on the osteogenic potential of Rg1 within the glucocorticoid-induced osteoporosis (GIOP) model and its specific mechanisms. PurposeThe primary objective of this study is to investigate the osteogenic potential of Rg1 within the GIOP model and explore the signaling pathways associated with its in vivo and in vitro effects. MethodsCell proliferation, differentiation and mineralization were evaluated by the Cell counting kit 8(CCK8) assay, alkaline phosphatase (ALP) test and Alizarin Red S staining, respectively. The qPCR technique was used to determine the relative expression of mRNA and the western blot was used to determine the relative expression of protein. In vivo experiments, spinal vertebrae staining in zebrafish larvae was accomplished by alizarin red S staining. ResultsZebrafish larvae's hatching, survival, malformation, and heart rate were unaffected by 50 μM of Rg1 in vivo, while the MEC3T3-E1 cell line's proliferation was unaffected by 50 μM of Rg1 in vitro. Meanwhile, Rg1 was shown to improve osteogenic differentiation or bone formation as well as the level of mRNA expression of osteogenic markers in vivo and in vitro. Treatment with Rg1 significantly increased the expression of G protein-coupled estrogen receptor (GPER) and pAKT. In addition, the GPER inhibitor G15 could significantly reduce the mRNA and protein expression levels of GPER and phosphorylated AKT, LY294002, a PI3K/AKT pathway inhibitor, markedly suppresses the expression of phosphorylated AKT, yet shows no significant impact on GPER expression. Both G15 and LY294002 can significantly blocked the Rg1-mediated enhancement of osteogenesis capacity in the GIOP model. In contrast, when both the agonists G1 of GPER and LY294002 were added, G1 increased the relative expression of mRNA and protein of GPER, but not the expression of osteogenic capacity and osteogenic markers. ConclusionsThis study investigates the mineralization effects and mechanisms of Ginsenoside Rg1 both in vitro and in vivo. For the first time, we propose that Rg1 might regulate osteogenesis by modulating AKT phosphorylation through mediating GPER expression within the PI3K/AKT pathway in the GIOP model. This discovery introduces novel targets and avenues for osteoporosis treatment.

Dissecting specific Wnt components governing osteogenic differentiation potential by human periodontal ligament stem cells through interleukin-6

Periodontal ligament stem cells (PDLSCs) play a significant role on periodontal tissue and alveolar bone homeostasis. During inflammation, interleukin (IL)-6 serves as one of key cytokine players controlling tissue reaction as well as alveolar bone tissue remodeling. It is believed that periodontal tissue inflammation causes periodontium degradation, especially alveolar bone. However, in this study, we show that an inflammatory mediator, IL-6, may serve another direction on alveolar bone homeostasis during inflammatory condition. We found that, IL-6 at 10 and 20 ng/mL was not cytotoxic and dose-dependently exerted beneficial effects on osteogenic differentiation of human PDLSCs (hPDLSCs), as demonstrated by increased alkaline phosphatase activity, mRNA expression of osteogenic markers, and matrix mineralization. The presence of physiological and inflammatory level of IL-6, the osteogenic differentiation potential by hPDLSCs was enhanced by several possible mechanisms including transforming growth factor (TGF), Wnt, and Notch pathways. After in-depth and thorough exploration, we found that Wnt pathway serves as key regulator controlling osteogenic differentiation by hPDLSCs amid the IL-6 presentation. Surprisingly, apart from other mesenchymal stem cells, distinct Wnt components are employed by hPDLSCs, and both canonical and non-canonical Wnt pathways are triggered by different mechanisms. Further validation by gene silencing, treatment with recombinant Wnt ligands, and β-catenin stabilization/translocation confirmed that IL-6 governed the canonical Wnt/β-catenin pathway via either WNT2B or WNT10B and employed WNT5A to activate the non-canonical Wnt pathway. These findings fulfill the homeostasis pathway governing periodontal tissue and alveolar bone regeneration and may serve for further therapeutic regimen design for restoring the tissues.

Recombinant irisin enhances the extracellular matrix formation, remodeling potential, and differentiation of human periodontal ligament cells cultured in 3D.

Irisin is expressed in human periodontal ligament (hPDL), and its administration enhances growth, migration and matrix deposition in hPDL cells cultured in monolayers in vitro. To identify whether irisin affects the gene expression patterns directing the morphology, mechanical properties, extracellular matrix (ECM) formation, osteogenic activity and angiogenic potential in hPDL cell spheroids cultured in 3D. Spheroids of primary human hPDL cells were generated in a rotational 3D culture system and treated with or without irisin. The gene expression patterns were evaluated by Affymetrix microarrays. The morphology of the spheroids was characterized using histological staining. Mechanical properties were quantified by nanoindentation. The osteogenic and angiogenic potential of spheroids were assessed through immunofluorescence staining for collagen type I, periostin fibronectin and von Willebrand factor (vWF), and mRNA expression of osteogenic markers. The secretion of multiple myokines was evaluated using Luminex immunoassays. Approximately 1000 genes were differentially expressed between control and irisin-treated groups by Affymetrix. Several genes related to ECM organization were differentially expressed, and multiple deubiquitinating enzymes were upregulated in the irisin-exposed samples analyzed. These represent cellular and molecular mechanisms indicative of a role for irisin in tissue remodeling. Irisin induced a rim-like structure on the outer region of the hPDL spheroids, ECM-related protein expression and the stiffness of the spheroids were enhanced by irisin. The expression of osteogenic and angiogenetic markers was increased by irisin. Irisin altered the morphology in primary hPDL cell-derived spheroids, enhanced its ECM deposition, mechanical properties, differentiation and remodeling potential.

MiRNA-Based Early Healing Mechanism of Extraction Sockets: miR-190a-5p, a Potential Enhancer of Bone Healing.

Objective Tooth extraction causes a wound with hard and soft tissue defects in the alveolar ridge. Few studies have reported the function of microRNAs (miRNAs) in the healing of extraction sockets. This study used bioinformatics analysis to reveal the possible relevance and role of miRNAs during the early stages following tooth extraction. Materials and Methods Socket tissues from beagle dogs (Canis familiaris; two males and two females) were collected 1 and 12 hours after extraction of premolars on both sides of the mandible. miRNA expression was profiled through miRNA sequencing, and hub miRNAs showing characteristic expression patterns were selected and subjected to target enrichment analysis. Alkaline phosphatase (ALP) activity analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were performed to verify the effect of hub miRNA on osteoblast differentiation and bone regeneration in vivo. Results Five miRNAs were identified to have consistently high expression levels, with cfa-miR-451 showing the highest expression. Additionally, 20 hub miRNAs were selected as candidates expected to play an important role in the healing process. Pathways, such as the MAPK, axon guidance, TGF-β, and Wnt signaling, were significantly enriched. Among hub miRNAs, miR-190a-5p increased ALP activity and mRNA expression of osteogenic markers and increased new bone formation in vivo. Conclusions Our findings suggest that miRNAs may be involved in the earliest stages of socket healing after tooth extraction and can play an important role in moderating the entire socket healing mechanism in the extraction socket.

Osteogenic effects of microRNA-335-5p/lipidoid nanoparticles coated on titanium surface

ObjectiveIn this study, we aimed to investigate the therapeutic potential of miR-335-5p lipidoid nanocomplexes coated on Titanium (Ti) SLActive surface by lyophilization. DesignIn our model, we coated miR-335-5p/Lipidoid nanoparticles on titanium implant, seeded GFP-labelled mouse bone marrow stromal cells (BMSCs) onto the functionalized Ti implant surface, and analyzed the transfection efficiency, cell adhesion, proliferation, and osteogenic activity of the bone-implant interface. ResultsThe Ti SLActive surface displayed a suitable hydrophilicity ability and provided a large surface area for miRNA loading, enabling spatial retention of the miRNAs within the nanopores until cellular delivery. We demonstrated a high transfection efficiency of miR-335-5p lipidoid nanoparticles in BMSCs seeded onto the Ti SLActive surface, even after 14 days. Alkaline phosphatase (ALP) activity and cell vitality were significantly increased in BMSCs transfected with miR-335-5p at 7 and 14 days as opposed to cells transfected with negative controls. When miR-335-5p transfected BMSCs were induced to undergo osteogenic differentiation, we detected increased mRNA expression of osteogenic markers including Alkaline phosphatase (ALP), collagen I (COL1), osteocalcin (OCN) and bone sialoprotein (BSP) at 7 and 14 days as compared with negative controls. ConclusionMiR-335-5p lipidoid nanoparticles could be used as a new cost-effective methodology to increase the osteogenic capacity of biomedical Ti implants.

Induction of osteogenic differentiation of bone marrow stromal cells on 3D polyester-based scaffolds solely by subphysiological fluidic stimulation in a laminar flow bioreactor

The fatal determination of bone marrow mesenchymal stem/stromal cells (BMSC) is closely associated with mechano-environmental factors in addition to biochemical clues. The aim of this study was to induce osteogenesis in the absence of chemical stimuli using a custom-designed laminar flow bioreactor. BMSC were seeded onto synthetic microporous scaffolds and subjected to the subphysiological level of fluid flow for up to 21 days. During the perfusion, cell proliferation was significantly inhibited. There were also morphological changes, with F-actin polymerisation and upregulation of ROCK1. Notably, in BMSC subjected to flow, mRNA expression of osteogenic markers was significantly upregulated and RUNX2 was localised in the nuclei. Further, under perfusion, there was greater deposition of collagen type 1 and calcium onto the scaffolds. The results confirm that an appropriate level of fluid stimuli preconditions BMSC towards the osteoblastic lineage on 3D scaffolds in the absence of chemical stimulation, which highlights the utility of flow bioreactors in bone tissue engineering.

Low-magnitude vibration promotes osteogenesis of osteoblasts in ovariectomized osteoporotic rats via the estrogen receptor α

The purpose of this study was to investigate the effect of low-magnitude vibration on osteogenesis of osteoblasts in ovariectomized rats with osteoporosis via estrogen receptor α(ERα). The mRNA expression of osteogenic markers were examined with qRT-PCR, based on which the optimal vibration parameter for promoting osteogenesis was determined (45 Hz × 0.9 g, g = 9.8 m/s2). Then we loaded the optimal vibration parameter on the osteoblasts of ovariectomized rats with osteoporosis. The protein expression of osteogenic markers and ERα were detected with Western blot; the distribution of ERα was examined with immunofluorescence technique. Finally, through inhibiting the expression of ERα with estrogen receptor inhibitor ICI182780, the protein and mRNA expression of osteogenic markers were examined. First, the results showed that low-magnitude vibration could promote the expression of osteogenic markers and ERα in osteoblasts of ovariectomized rats with osteoporosis (P < 0.05), and make ERα transfer to the nucleus. On the other hand, the results also showed that after inhibiting the expression of ERα in osteoblasts of ovariectomized rats with osteoporosis, the protein and mRNA expression of osteogenic marker were decreased (P < 0.05). In our study, low-magnitude vibration played an important role in the osteogenesis of osteoblasts in ovariectomized rats with osteoporosis through increasing the expression and causing translocation of ERα. Furthermore, it provides a theoretical basis for the application of low-magnitude vibration in the prevention and treatment of postmenopausal osteoporosis.

Sulfurous thermal waters stimulate the osteogenic differentiation of human mesenchymal stromal cells – An in vitro study

Strategies aimed at delaying the onset of bone tissue degeneration and the resulting skeletal fragility are key to decrease the risk of bone fracture correlated to ageing. The therapeutic properties of sulfurous thermal waters (STWs), rich in hydrogen sulfide (H2S), have been claimed for centuries. However, the direct regulation of bone cells by STWs has not been investigated yet. Here we aimed at analyzing the effect of STWs on cultured human mesenchymal stromal cells (hMSCs) derived from bone tissue. Two concentrations of STWs from 2 health spa centers in Italy (here named STW-1 and STW-2) containing, respectively, high and moderate quantities of H2S, were added to the culture media. Cytotoxicity and osteogenic differentiation were evaluated. We provided first evidence that treatment of hMSCs with STWs results in a sharp increase in intracellular H2S content, coherent with the different concentrations of H2S, thereby reveling that STWs-released H2S is internalized by cells. STWs treatment significantly induced osteogenic differentiation of hMSCs. In particular, mineral apposition was increased with a similar pattern by the two STWs, while mRNA expression of osteogenic markers (BSP, OC, RUNX-2, OPN) was differently affected. Only STW-2 induced a significant, dose-dependent increase in these gene expression. These findings support the rationale for the use of STWs as a complementary treatment of bone wasting diseases.

MiR-16-5p regulates postmenopausal osteoporosis by directly targeting VEGFA.

In this study, we used bioinformatics tools, and experiments with patient tissues and human mesenchymal stem cells (hMSCs) to identify differentially regulated genes (DEGs) and microRNAs (miRNAs) that promote postmenopausal osteoporosis. By analyzing the GSE56815 dataset from the NCBI GEO database, we identified 638 DEGs, including 371 upregulated and 267 downregulated genes, in postmenopausal women with low bone density. Enrichment and protein-protein interaction network analyses showed that TP53, RPS27A, and VEGFA were the top three hub genes with the highest degree of betweenness and closeness centrality. TargetScanHuman and DIANA software analyses and dual luciferase reporter assays confirmed that miR-16a-5p directly targets the 3’UTR of VEGFA. Postmenopausal patients with osteoporosis showed higher miR-16-5p and lower VEGFA levels than those without osteoporosis (n=10 each). VEGFA levels were higher in miR-16-5p knockdown hMSCs and were reduced in miR-16-5p-overexpressing hMSCs. mRNA expression of osteogenic markers, ALP, OCN, and RUNX2, as well as calcium deposition based on Alizarin red staining, correlated inversely with miR-16-5p levels and correlated positively with VEGFA levels. These findings suggest that miR-16-5p suppresses osteogenesis by inhibiting VEGFA expression and is a promising target for postmenopausal osteoporosis therapy.

Loss of receptor tyrosine kinase-like orphan receptor 2 impairs the osteogenesis of mBMSCs by inhibiting signal transducer and activator of transcription 3

BackgroundReceptor tyrosine kinase-like orphan receptor 2 (Ror2) plays a key role in bone formation, but its signaling pathway is not completely understood. Signal transducer and activator of transcription 3 (Stat3) takes part in maintaining bone homeostasis. The aim of this study is to reveal the role and mechanism of Ror2 in the osteogenic differentiation from mouse bone marrow mesenchymal stem cells (mBMSCs) and to explore the effect of Stat3 on Ror2-mediated osteogenesis.MethodsRor2 CKO mice were generated via the Cre-loxp recombination system using Prrx1-Cre transgenic mice. Quantitative real-time PCR and western blot were performed to assess the expression of Stat3 and osteogenic markers in Ror2-knockdown mBMSCs (mBMSC-sh-Ror2). After being incubated in osteogenic induction medium for 3 weeks, Alizarin Red staining and western blot were used to examine the calcium deposit and osteogenic markers in Stat3 overexpression in mBMSC-sh-Ror2.ResultsLoss of Ror2 in mesenchymal or osteoblast progenitor cells led to a dwarfism phenotype in vivo. The mRNA expression of osteogenic markers (osteocalcin, osteopontin (OPN), and collagen I) in the ulna proximal epiphysis of Ror2 CKO mice was significantly decreased (P < 0.05). The mRNA and protein expression of Stat3 and osteogenic markers (Runx2, osterix, and OPN) decreased in mBMSC-sh-Ror2 cells (P < 0.05). The overexpression of Stat3 in mBMSC-sh-Ror2 cells rescued the calcium deposit and expression of Runx2, osterix, and OPN to a level comparable to normal mBMSCs.ConclusionsRor2 was essential for skeleton development by regulating mBMSCs’ osteogenesis and osteoblast differentiation. Loss of Ror2 may impair the osteogenesis of mBMSCs by inhibiting Stat3.

Repair processes of flat bones formed via intramembranous versus endochondral ossification

ObjectivesAlthough fractures occur in various bones, including long, short, and flat bones, fracture repair investigations focus on the diaphysis of the long bone. The cell composition, osteogenic capacity, and bone matrix differ among osteogenesis patterns. However, the differences in the bone repair process have not been studied. Here, we compared the bone repair processes in the parietal bone and scapula of adolescent mice. MethodsBone apertures were created in the parietal bone and scapula. Samples were collected at indicated times after surgery, and the repair process was analyzed using micro-computed tomography, histological, immunohistochemical, and mRNA expression analyses. ResultsIn both repair processes, cartilage formation was not detected on the periosteum side. The parietal bone aperture was gradually filled with newly formed bone produced from the edge of the aperture by day 14 but was not completely repaired even by day 49. In the scapula, a bony callus was detected on the periosteum at day 7, and the aperture was bridged by day 14. Subsequently, the bony callus was remodeled to the original bone architecture. Alkaline phosphatase activity and osteocalcin synthesis occurred earlier in the repair region of the scapular periosteum, compared with that in the parietal periosteum. The mRNA expression of osteogenic markers in the periosteum was markedly upregulated in the scapula versus the parietal bone. ConclusionOur study findings clarify the differences between parietal bone and scapula repair and suggest that the bone repair process differs among ossification patterns.

Dickkopf-1 may regulate bone coupling by attenuating wnt/β-catenin signaling in chronic apical periodontitis

ObjectiveAlveolar bone loss is a common outcome of chronic apical periodontitis. In this study, we investigated the involvement of the Dickkopf-1-Wnt/β-catenin signaling pathway in the attenuation of osteogenic differentiation induced by Escherichia coli lipopolysaccharide, and we evaluated the use of Dickkopf-1 inhibitor and Dickkopf-1 recombinant protein to reverse bone loss in different phases of osteogenic differentiation. MethodsMC3T3-E1 cells grown in osteogenic medium were treated with Escherichia coli lipopolysaccharide for 24h during osteogenic induction on days 0, 1, 7, 14 and 21. Dickkopf-1 siRNA was added on days 0 and 1, and Dickkopf-1 recombinant was added on days 7, 14, and 21. Quantitative real-time PCR, Western blotting and alkaline phosphatase activity assays were performed to measure osteogenic marker expression and Wnt/β-catenin signaling. A rat apical periodontitis model was used to further evaluate the function of Dickkopf-1 in relation to bone loss. ResultsMC3T3-E1 cells treated with Escherichia coli lipopolysaccharide showed decreased mRNA expression of osteogenic markers. Wnt/β-catenin signaling was also inhibited, and Dickkopf-1 showed corresponding variations as quantified by Western blotting. Using Dickkopf-1 inhibitor or Dickkopf-1 recombinant protein at different phases of osteogenic differentiation in vitro partially reversed the decrease in osteogenic marker expression. The rat apical periodontitis model indicated that the Dickkopf-1 inhibitor could restore bone loss in the periapical area in vivo. ConclusionsDickkopf-1 may play a key regulatory role in determining the outcome for bone in inflammatory environments, and modulating the Wnt/β-catenin signaling pathway via Dickkopf-1 inhibitor or recombinant protein may provide a potential therapeutic option to prevent bone destruction in endodontic disease.

Overexpression of MiR-335-5p Promotes Bone Formation and Regeneration in Mice.

MicroRNAs (miRNAs) and the Wnt signaling pathway play critical roles in regulating bone development and homeostasis. Our previous study revealed high expression of miR-335-5p in osteoblasts and hypertrophic chondrocytes in mouse embryos and the ability of miR-335-5p to promote osteogenic differentiation by downregulating Wnt antagonist Dickkopf-1 (DKK1). The purpose of this study was to investigate the effects of miR-335-5p constitutive overexpression on bone formation and regeneration in vivo. To that end, we generated a transgenic mouse line specifically overexpressing miR-335-5p in osteoblasts lineage by the osterix promoter and characterized its bone phenotype. Bone histomorphometry and μCT analysis revealed higher bone mass and increased parameters of bone formation in transgenic mice than in wild-type littermates. Increased bone mass in transgenic mice bones also correlated with enhanced expression of osteogenic differentiation markers. Upon osteogenic induction, bone marrow stromal cells (BMSCs) isolated from transgenic mice displayed higher mRNA expression of osteogenic markers than wild-type mice BMSCs cultures. Protein expression of Runx2 and Osx was also upregulated in BMSC cultures of transgenic mice upon osteogenic induction, whereas that of DKK1 was downregulated. Most important, BMSCs from transgenic mice were able to repair craniofacial bone defects as shown by μCT analysis, H&E staining, and osteocalcin (OCN) immunohistochemistry of newly formed bone in defects treated with BMSCs. Taken together, our results demonstrate constitutive overexpression of miR-335-5p driven by an osterix promoter in the osteoblast lineage induces osteogenic differentiation and bone formation in mice and support the potential application of miR-335-5p-modified BMSCs in craniofacial bone regeneration. © 2017 American Society for Bone and Mineral Research.

Human dental pulp cells response to mineral trioxide aggregate (MTA) and MTA Plus: cytotoxicity and gene expression analysis.

To investigate the cytotoxicity, osteogenic bioactivity and mRNA expression of osteogenic markers of bone morphogenetic protein 2 (BMP-2), osteocalcin (OC) and alkaline phosphatase (ALP) induced by the extracts of set MTA Plus (MTA P) (Avalon Biomed Inc. Bradenton, FL, USA) in comparison with MTA (Angelus, Londrina, PR, Brazil) on human dental pulp cells (hDPCs). Cell viability was assessed by mitochondrial dehydrogenase enzymatic (MTT) assay, and the mechanism of cell death was evaluated by flow cytometry. Bioactivity was evaluated by alkaline phosphatase (ALP) assay and detection of calcium deposits with alizarin red staining (ARS). The gene expression of BMP-2, OC and ALP was quantified with real-time PCR. Statistical analysis was performed with analysis of variance and Bonferroni or Tukey post-test (α=0.05). MTA and MTA P were not cytotoxic and did not induce apoptosis. MTA P had significant higher ALP activity in relation to MTA and the control (P<0.05). MTA had a significantly higher percentage of mineralized area than MTA P (P<0.05). The expression of BMP2 and OC mRNA was significantly higher in cells exposed to MTA than MTA P after 1day (P<0.05). At day 3, the mRNA expression of ALP was significantly higher in MTA P compared with MTA (P<0.05). MTA and MTA Plus were noncytotoxic, increased mineralization processes invitro and induced the expression of osteogenic markers.

BMP-9 regulates the osteoblastic differentiation and calcification of vascular smooth muscle cells through an ALK1 mediated pathway.

The process of vascular calcification shares many similarities with that of physiological skeletal mineralization, and involves the deposition of hydroxyapatite crystals in arteries. However, the cellular mechanisms responsible have yet to be fully explained. Bone morphogenetic protein (BMP-9) has been shown to exert direct effects on both bone development and vascular function. In the present study, we have investigated the role of BMP-9 in vascular smooth muscle cell (VSMC) calcification. Vessel calcification in chronic kidney disease (CKD) begins pre-dialysis, with factors specific to the dialysis milieu triggering accelerated calcification. Intriguingly, BMP-9 was markedly elevated in serum from CKD children on dialysis. Furthermore, in vitro studies revealed that BMP-9 treatment causes a significant increase in VSMC calcium content, alkaline phosphatase (ALP) activity and mRNA expression of osteogenic markers. BMP-9-induced calcium deposition was significantly reduced following treatment with the ALP inhibitor 2,5-Dimethoxy-N-(quinolin-3-yl) benzenesulfonamide confirming the mediatory role of ALP in this process. The inhibition of ALK1 signalling using a soluble chimeric protein significantly reduced calcium deposition and ALP activity, confirming that BMP-9 is a physiological ALK1 ligand. Signal transduction studies revealed that BMP-9 induced Smad2, Smad3 and Smad1/5/8 phosphorylation. As these Smad proteins directly bind to Smad4 to activate target genes, siRNA studies were subsequently undertaken to examine the functional role of Smad4 in VSMC calcification. Smad4-siRNA transfection induced a significant reduction in ALP activity and calcium deposition. These novel data demonstrate that BMP-9 induces VSMC osteogenic differentiation and calcification via ALK1, Smad and ALP dependent mechanisms. This may identify new potential therapeutic strategies for clinical intervention.

Osteogenic differentiation of bone mesenchymal stem cells regulated by osteoblasts under EMF exposure in a co-culture system

This study examined the osteogenic effect of electromagnetic fields (EMF) under the simulated in vivo conditions. Rat bone marrow mesenchymal stem cells (BMSCs) and rat osteoblasts were co-cultured and exposed to 50 Hz, 1.0 mT EMF for different terms. Unexposed single-cultured BMSCs and osteoblasts were set as controls. Cell proliferation features of single-cultured BMSCs and osteoblasts were studied by using a cell counting kit (CCK-8). For the co-culture system, cells in each group were randomly chosen for alkaline phosphatase (ALP) staining on the day 7. When EMF exposure lasted for 14 days, dishes in each group were randomly chosen for total RNA extraction and von Kossa staining. The mRNA expression of osteogenic markers was detected by using real-time PCR. Our study showed that short-term EMF exposure (2 h/day) could obviously promote proliferation of BMSCs and osteoblasts, while long-term EMF (8 h/day) could promote osteogenic differentiation significantly under co-cultured conditions. Under EMF exposure, osteogenesis-related mRNA expression changed obviously in co-cultured and single-cultured cells. It was noteworthy that most osteogenic indices in osteoblasts were increased markedly after co-culture except Bmp2, which was increased gradually when cells were exposed to EMF. Compared to other indices, the expression of Bmp2 in BMSCs was increased sharply in both single-cultured and co-cultured groups when they were exposed to EMF. The mRNA expression of Bmp2 in BMSCs was approximately four times higher in 8-h EMF group than that in the unexposed group. Our results suggest that Bmp2-mediated cellular interaction induced by EMF exposure might play an important role in the osteogenic differentiation of BMSCs.

Effect of the HDAC inhibitor vorinostat on the osteogenic differentiation of mesenchymal stem cells in vitro and bone formation in vivo

Vorinostat, a histone deacetylase (HDAC) inhibitor currently in a clinical phase III trial for multiple myeloma (MM) patients, has been reported to cause bone loss. The purpose of this study was to test whether, and to what extent, vorinostat influences the osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro and bone formation in vivo. Bone marrow-derived MSCs were prepared from both normal donors and MM patients. The MSCs were cultured in an osteogenic differentiation induction medium to induce osteogenic differentiation, which was evaluated by alkaline phosphatase (ALP) staining, Alizarin Red S staining and the mRNA expression of osteogenic markers. Naïve mice were administered vorinostat (100 mg/kg, ip) every other day for 3 weeks. After the mice were sacrificed, bone formation was assessed based on serum osteocalcin level and histomorphometric analysis. Vorinostat inhibited the viability of hMSCs in a concentration-dependent manner (the IC50 value was 15.57 μmol/L). The low concentration of vorinostat (1 μmol/L) did not significantly increase apoptosis in hMSCs, whereas pronounced apoptosis was observed following exposure to higher concentrations of vorinostat (10 and 50 μmol/L). In bone marrow-derived hMSCs from both normal donors and MM patients, vorinostat (1 μmol/L) significantly increased ALP activity, mRNA expression of osteogenic markers, and matrix mineralization. These effects were associated with upregulation of the bone-specifying transcription factor Runx2 and with the epigenetic alterations during normal hMSCs osteogenic differentiation. Importantly, the mice treated with vorinostat did not show any bone loss in response to the optimized treatment regimen. Vorinostat, known as a potent anti-myeloma drug, stimulates MSC osteogenesis in vitro. With the optimized treatment regimen, any decrease in bone formation was not observed in vivo.