Important Role In Osteogenic Differentiation Research Articles

METTL3 promotes osteogenic differentiation of human umbilical cord mesenchymal stem cells by up-regulating m6A modification of circCTTN.

Human umbilical cord mesenchymal stem cells (hUCMSCs) are promising seed cells in bone tissue engineering. circRNA and N6-methyladenosine (m6A) RNA methylation play important roles in osteogenic differentiation. Here, we investigated the potential relevance of a critical circRNA, hsa_circ_0003376 (circCTTN), and methyltransferase-like 3 (METTL3) in osteogenic differentiation of hUCMSCs. Expression of circCTTN after hUCMSC osteogenic induction was detected by qRT-PCR. Three databases (RMBase v2.0, BERMP, and SRAMP) were used to predict m6A sites of circCTTN. RNA was enriched by methylated RNA immunoprecipitation (MeRIP), followed by quantitative real-time polymerase chain reaction to detect m6A level of circCTTN after METTL3 overexpression and osteogenic induction. RNA pull-down, Western blotting, and protein mass spectrometry were performed to investigate the potential mechanisms by which METTL3 promoted m6A modification of circCTTN. Bioinformatic analyses based on database (STRING) search and co-immunoprecipitation were used to analyze the proteins that interacted with METTL3. Overexpression of METTL3 promoted osteogenic differentiation of hUCMSCs and increased m6A level of circCTTN. Two potential m6A modification sites of circCTTN were predicted. No direct interaction between METTL3 and circCTTN was observed. Thirty-one proteins were pulled down by probes specific for circCTTN, including NOP2, and two m6A reading proteins, EIF3A and SND1. Bioinformatics analysis and co-immunoprecipitation showed that METTL3 interacted with EIF3A indirectly through NOP2. METTL3 promotes the osteogenic differentiation of hUCMSCs by increasing the m6A level of circCTTN. However, METTL3 does not bind directly to circCTTN. METTL3 interacts with circCTTN indirectly through NOP2 and EIF3A.

NEAT1/microRNA 339-5p/SPI1 Axis Feedback Loop Contributes to Osteogenic Differentiation in Acute Suppurative Osteomyelitis in Children.

Long non-coding RNA plays an important role in osteogenic differentiation. Nuclear enriched abundant transcript 1 (NEAT1) has been revealed to promote osteogenic differentiation in human bone marrow mesenchymal stem cells (hBMSCs), but the underlying regulatory mechanism remains unknown in acute suppurative osteomyelitis of children. Osteogenic medium (OM) was used to induce osteogenic differentiation. Quantitative real-time PCR and Western blotting were used to evaluate gene expression. The effects of NEAT1, microRNA 339-5p (miR-339-5p), and salmonella pathogenicity island 1 (SPI1) on osteogenic differentiation were assessed in vitro using alizarin red S staining assays and alkaline phosphatase activity. Interactions between NEAT1, miR-339-5p, and SPI1 were identified using immunoprecipitation, luciferase reporter assays, and chromatin immunoprecipitation. During osteogenic differentiation, expression of NEAT1 was up-regulated in hBMSCs, and miR-339-5p level was down during osteogenic differentiation. Knockdown of NEAT1 reduced the osteogenic differentiation of hBMSCs, and down-regulation of miR-339-5p may counteract the effect of NEAT1 silencing. SPI1 was a target of miR-339-5p by luciferase reporter assay and was also a transcription factor of NEAT1 by chromatin immunoprecipitation. A positive NEAT1-miR-339-5p-SPI1 feedback loop was found to be present during osteogenic differentiation in hBMSCs. It was the first study to reveal that the NEAT1-miR-339-5p-SPI1 feedback loop can promote osteogenic differentiation in hBMSCs and shed a new light on the role of NEAT1 during osteogenic differentiation.

Uniaxial cyclic stretch enhances osteogenic differentiation of OPLL-derived primary cells via YAP-Wnt/β-catenin axis.

The pathogenesis of posterior longitudinal ligament ossification (OPLL) remains inadequately understood. Mechanical stimulation is one of the important pathogenic factors in OPLL. As one of the mechanical stimulation transduction signals, the yes-associated protein (YAP) interacts with the Wnt/β-catenin signalling pathway, which plays an important role in osteogenic differentiation. This study aimed to demonstrate the role of YAP-Wnt/β-catenin axis in cell differentiation induced by mechanical stress. Primary cells extracted from posterior longitudinal ligament tissues from OPLL or non-OPLL patients were subjected to sinusoidal uniaxial cyclic stretch (5 %, 0.5 Hz, 3 d). The expression of runt-related transcription factor 2, collagen I, osterix, osteocalcin and alkaline phosphatase were compared between the static and the experimental groups. In addition, the cytoskeleton was detected using phalloidin staining while YAP phosphorylation states and nuclear location were identified using immunofluorescence. The results showed that mechanical stretching loading increased the expression of osteogenic genes and proteins in the OPLL group, while it had no significant effect on the control group. When OPLL cells were stretched, YAP exhibited an obvious nuclear translocation and the Wnt/β-catenin pathway was activated. Knocking down YAP or β-catenin could weaken the impact upon osteogenic differentiation induced by mechanical stimulation. YAP-mediated mechanical stimulation promoted osteogenic differentiation of OPLL cells through Wnt/β-catenin pathway and this progress was independent of the Hippo pathway.

Macrophage migration inhibitory factor (MIF) inhibitor iSO-1 promotes staphylococcal protein A-induced osteogenic differentiation by inhibiting NF-κB signaling pathway.

Osteomyelitis is among the most difficult to treat diseases in the field of orthopedics, and there is a lack of effective treatment modalities. Exploring the mechanisms of its development is beneficial for finding molecular targets for treatment. Increasing evidence suggests that macrophage migration inhibitory factor (MIF), as a proinflammatory mediator, is not only involved in various pathophysiological processes of inflammation but also plays an important role in osteogenic differentiation, while its specific regulatory mechanism in osteomyelitis remains unclear. In the present study, staphylococcal protein A (SPA)-treated rat bone marrow mesenchymal stem cells (rBMSCs) were used to construct cell models of osteomyelitis. Rat and cell models of osteomyelitis were used to validate the expression levels of MIF, and to further explore the regulatory mechanisms of the MIF inhibitor methyl ester of (S, R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid (iSO-1) and MIF knockdown on cell model of osteomyelitis toward osteogenic differentiation. We found that the expression level of MIF was upregulated in rat and cell models of osteomyelitis and subsequently demonstrated by the GSE30119 dataset that the expression level of MIF was also significantly upregulated in patients with osteomyelitis. Furthermore, SPA promotes MIF expression in rBMSCs while inhibiting the expression of osteogenic-related genes such as Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN) and collagen type-1 (COL-1) through activation of the nuclear factor kappa-B (NF-κB) pathway. In vivo, we further demonstrated that local injection of iSO-1 significantly increased the osteogenic activity in rat model of osteomyelitis. Importantly, we also demonstrated that MIF knockdown and the MIF inhibitor iSO-1 reversed the SPA-mediated inhibition of osteogenic differentiation of rBMSCs by inhibiting the activation of the NF-κB pathway, as evidenced by the upregulation of osteogenic-related gene expression and enhanced bone mineralization. ISO-1 and MIF knockdown can reverse the SPA-mediated inhibition of osteogenic differentiation in the rBMSCs model of osteomyelitis by inhibiting the NF-κB signaling pathway, providing a potential target for the treatment of osteomyelitis.

MSI1 Stabilizes MACF1 to Inhibit Apoptosis of MC3T3-E1 Cells Induced by High Glucose and Promote Osteogenic Differentiation Through Wnt/β-Catenin Signaling Pathway.

Diabetes mellitus (DM) affects bone metabolism and causes osteoporosis. Musashi 1 (MSI1), a member of the Musashi family, regulates protein expression by targeting protein mRNA and has been reported to play an important role in osteogenic differentiation. Therefore, this paper attempts to explore the role of MSI1 in diabetic osteoporosis and discussing its specific mechanism. The glucose concentration for high glucose (HG) and control MC3T3-E1 cells were 30 and 5.5mM. MC3T3-E1 cells induced by high glucose (HG) were used to simulate diabetic osteoporosis in vivo. The interaction between MSI1 and microtubule actin crosslinking factor 1 (MACF1) was confirmed by RNA Immunoprecipitation (RIP). The mRNA and protein expressions of MSI1 and MACF1 in MC3T3-E1 cells and HG-induced MC3T3-E1 cells after indicated transfection were tested by Real-time quantitative polymerase chain reaction (RT-qPCR) assay and western blot. After transfection, the proliferation, apoptosis, and osteogenic differentiation of HG-induced MC3T3-E1 cells were detected by cell counting kit (CCK)-8, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), alkaline phosphatase (ALP) activity assay, and alizarin red staining. The expression of Wnt/β-catenin signaling pathway-related proteins in HG-induced MC3T3-E1 cells after transfection was detected by western blot. This work shows that MSI1 can combine with MACF1. The expression of MSI1 and MACF1 was increased in HG-induced MC3T3-E1 cells. Upregulation of MSI1 promoted the proliferative and differentiative capabilities, but inhibited the apoptosis of HG-insulted MC3T3-E1 cells, which could be reversed by MACF1 knockdown. MSI1 stabilizes MACF1 to suppress apoptosis and promote osteogenic differentiation in HG-induced MC3T3-E1 cells by inhibiting Wnt/β-catenin signaling pathway.

Indirect Immobilised Jagged-1 Enhances Matrisome Proteins Associated with Osteogenic Differentiation of Human Dental Pulp Stem Cells: A Proteomic Study.

The indirect immobilisation of Jagged-1 (Jagged-1) promoted osteogenic differentiation of human dental pulp cells (hDPs). Furthermore, the analysis of the Reactome pathway of RNA sequencing data indicates the upregulated genes involved with the extracellular matrix (ECM). Hence, our objective was to investigate the effects of Jagged-1 on proteomic profiles of human dental pulp stem cells (hDPSC). hDPSCs were cultured on the surface coated with human IgG Fc fragment (hFc) and the surface coated with rhJagged1/Fc recombinant protein-coated surface. Cells were differentiated to the osteogenic lineage using an osteogenic differentiation medium (OM) for 14 days, and cells cultured in a growth medium were used as a control. The protein component of the cultured cells was extracted into the cytosol, membrane, nucleus, and cytoskeletal compartment. Subsequently, the proteomic analysis was performed using liquid chromatography-tandem mass spectrometry (LC-MS). Metascape gene list analysis reported that Jagged-1 stimulated the expression of the membrane trafficking protein (DOP1B), which can indirectly improve osteogenic differentiation. hDPSCs cultured on Jagged-1 surface under OM condition expressed COL27A1, MXRA5, COL7A1, and MMP16, which played an important role in osteogenic differentiation. Furthermore, common matrisome proteins of all cellular components were related to osteogenesis/osteogenic differentiation. Additionally, the gene ontology categorised by the biological process of cytosol, membrane, and cytoskeleton compartments was associated with the biomineralisation process. The gene ontology of different culture conditions in each cellular component showed several unique gene ontologies. Remarkably, the Jagged-1_OM culture condition showed the biological process related to odontogenesis in the membrane compartment. In conclusion, the Jagged-1 induces osteogenic differentiation could, mainly through the regulation of protein in the membrane compartment.

MicroRNA-loaded biomaterials for osteogenesis.

The large incidence of bone defects in clinical practice increases not only the demand for advanced bone transplantation techniques but also the development of bone substitute materials. A variety of emerging bone tissue engineering materials with osteogenic induction ability are promising strategies for the design of bone substitutes. MicroRNAs (miRNAs) are a class of non-coding RNAs that regulate intracellular protein expression by targeting the non-coding region of mRNA3′-UTR to play an important role in osteogenic differentiation. Several miRNA preparations have been used to promote the osteogenic differentiation of stem cells. Therefore, multiple functional bone tissue engineering materials using miRNA as an osteogenic factor have been developed and confirmed to have critical efficacy in promoting bone repair. In this review, osteogenic intracellular signaling pathways mediated by miRNAs are introduced in detail to provide a clear understanding for future clinical treatment. We summarized the biomaterials loaded with exogenous cells engineered by miRNAs and biomaterials directly carrying miRNAs acting on endogenous stem cells and discussed their advantages and disadvantages, providing a feasible method for promoting bone regeneration. Finally, we summarized the current research deficiencies and future research directions of the miRNA-functionalized scaffold. This review provides a summary of a variety of advanced miRNA delivery system design strategies that enhance bone regeneration.

Bioinformatics-Guided Analysis Uncovers AOX1 as an Osteogenic Differentiation-Relevant Gene of Human Mesenchymal Stem Cells.

Background: The available therapeutic options of bone defects, fracture nonunion, and osteoporosis remain limited, which are closely related to the osteogenic differentiation of bone marrow–derived mesenchymal stem cells (BMSCs). Thus, there remains an urgent demand to develop a prediction method to infer osteogenic differentiation–related genes in BMSCs. Method: We performed differential expression analysis between hBMSCs and osteogenically induced samples. Association analysis, co-expression analysis, and PPI analysis are then carried out to identify potential osteogenesis-related regulators. GO enrichment analysis and GSEA are performed to identify significantly enriched pathways associated with AOX1. qRT-PCR and Western blotting were employed to investigate the expression of genes on osteogenic differentiation, and plasmid transfection was used to overexpress the gene AOX1 in hBMSCs. Result: We identified 25 upregulated genes and 17 downregulated genes. Association analysis and PPI network analysis among these differentially expressed genes show that AOX1 is a potential regulator of osteogenic differentiation. GO enrichment analysis and GSEA show that AOX1 is significantly associated with osteoblast-related pathways. The experiments revealed that AOX1 level was higher and increased gradually in differentiated BMSCs compared with undifferentiated BMSCs, and AOX1 overexpression significantly increased the expression of osteo-specific genes, thereby clearly indicating that AOX1 plays an important role in osteogenic differentiation. Moreover, our method has ability in discriminating genes with osteogenic differentiation properties and can facilitate the process of discovery of new osteogenic differentiation–related genes. Conclusion: These findings collectively demonstrate that AOX1 is an osteogenic differentiation-relevant gene and provide a novel method established with a good performance for osteogenic differentiation-relevant genes prediction.

The Roles of Long Non-coding RNAs in Osteogenic Differentiation and Bone Diseases

Abstract Bone marrow mesenchymal stem cells play an important role in osteogenic differentiation, and they complete this important biological process through the coordination of various transcription factors and signal pathways. In recent years, studies have clearly confirmed that long non-coding RNAs (lncRNAs) are involved in osteogenic differentiation, which plays an important biological role in the occurrence and development of osteogenesis-related bone disease. This article reviews the roles and related mechanisms of lncRNAs in osteogenic differentiation, as well as their potential effects on a variety of bone diseases. This understanding may help researchers identify potential therapeutic targets and biological markers in the future.

MiRNA Expression Profiling Uncovers a Role of miR-139-5p in Regulating the Calcification of Human Aortic Valve Interstitial Cells.

Calcific aortic valve disease (CAVD) is the most common structural heart disease, and the morbidity is increased with elderly population. Several microRNAs (miRNAs) have been identified to play crucial roles in CAVD, and numerous miRNAs are still waiting to be explored. In this study, the miRNA expression signature in CAVD was analyzed unbiasedly by miRNA-sequencing, and we found that, compared with the normal control valves, 152 miRNAs were upregulated and 186 miRNAs were downregulated in calcified aortic valves. The functions of these differentially expressed miRNAs were associated with cell differentiation, apoptosis, adhesion and immune response processes. Among downregulated miRNAs, the expression level of miR-139-5p was negatively correlated with the osteogenic gene RUNX2, and miR-139-5p was also downregulated during the osteogenic differentiation of primary human aortic valve interstitial cells (VICs). Subsequent functional studies revealed that miR-139-5p overexpression inhibited the osteogenic differentiation of VICs by negatively modulating the expression of pro-osteogenic gene FZD4 and CTNNB1. In conclusion, these results suggest that miR-139-5p plays an important role in osteogenic differentiation of VICs via the Wnt/β-Catenin pathway, which may further provide a new therapeutic target for CAVD.

Osteoinduction Evaluation of Fluorinated Hydroxyapatite and Tantalum Composite Coatings on Magnesium Alloys.

Magnesium (Mg) alloys have a wide range of biomaterial applications, but their lack of biocompatibility and osteoinduction property impedes osteointegration. In order to enhance the bioactivity of Mg alloy, a composite coating of fluorinated hydroxyapatite (FHA) and tantalum (Ta) was first developed on the surface of the alloy through thermal synthesis and magnetron sputtering technologies in this study. The samples were characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), energy dispersive spectroscopy (EDS) mapping, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and water contact angle measurement (WCA), which characterized the surface alternation and confirmed the deposition of the target FHA/Ta coating. The results of cell morphology showed that the MC3T3-E1 cells on the surface of Mg/FHA/Ta samples had the largest spreading area and lamellipodia. Moreover, the FHA coating endowed the surface with superior cell viability and osteogenic properties, while Ta coating played a more important role in osteogenic differentiation. Therefore, the combination of FHA and Ta coatings could synergistically promote biological functions, thus providing a novel strategy for implant design.

Context-Specific Osteogenic Potential of Mesenchymal Stem Cells.

Despite the great progress in the field of bone tissue regeneration, the early initiating mechanisms of osteogenic differentiation are not well understood. Cells capable of osteogenic transformation vary from mesenchymal stem cells of various origins to mural cells of vessels. The mechanisms of pathological calcification are thought to be similar to those of bone formation. Notch signaling has been shown to play an important role in osteogenic differentiation, as well as in pathological calcification. Nevertheless, despite its known tissue- and context-specificity, the information about its role in the osteogenic differentiation of different cells is still limited. We compared mesenchymal stem cells from adipogenic tissue (MSCs) and interstitial cells from the aortic valve (VICs) by their ability to undergo Notch-dependent osteogenic differentiation. We showed differences between the two types of cells in their ability to activate the expression of proosteogenic genes RUNX2, BMP2, BMP4, DLX2, BGLAP, SPRY, IBSP, and SPP1 in response to Notch activation. Untargeted metabolomic profiling also confirms differences between MSCs and VICs in their osteogenic state. Analysis of the activity of RUNX2 and SPP1 promoters shows fine-tuned dose-dependency in response to Notch induction and suggests a direct link between the level of Notch activation, and the proostogenic gene expression and corresponding osteogenic induction. Our data suggest that osteogenic differentiation is a context-dependent process and the outcome of it could be cell-type dependent.

Role of 1,25-Dihydroxyvitamin D3 on Osteogenic Differentiation and Mineralization of Chicken Mesenchymal Stem Cells.

1,25-dihydroxyvitamin D3 (1,25OHD) has been suggested to play an important role in osteogenic differentiation and mineralization. However, limited data have been reported in avian species. In the present study, the direct role of 1,25OHD on osteogenic differentiation and mineralization in chicken mesenchymal stem cells (cMSCs) derived from day-old broiler bones was investigated. cMSCs were treated with control media (C), osteogenesis media (OM), OM with 1, 5, 10, and 50 nM 1,25OHD, respectively. The messenger RNA (mRNA) samples were obtained at 24 and 48 h and 3 and 7 days to examine mRNA expression of key osteogenic genes [runt related transcription factor 2 (RUNX2), bone morphogenetic protein 2 (BMP2), collagen type I alpha 2 chain (COL1A2), bone gamma-carboxyglutamate protein (BGLAP), secreted phosphoprotein 1 (SPP1), and alkaline phosphatase (ALP)]. Cells were stained at 7, 14, and 21 days using Von Kossa (mineralization), Alizarin Red (AR; mineralization), and Alkaline Phosphatase (early marker) staining methods. From the mRNA expression results, we found a time-dependent manner of 1,25OHD on osteoblast differentiation and mineralization. In general, it showed an inhibitory effect on differentiation and mineralization during the early stage (24 and 48 h), and a stimulatory effect during the late cell stage (3 and 7 days). The staining showed 1,25OHD had an inhibitory effect on ALP enzyme activities and mineralization in a dosage-dependent manner up to 14 days. However, at 21 days, there was no difference between the treatments. This study provides a novel understanding of the effects of 1,25OHD on osteogenic differentiation and mineralization of cMSCs depending on cell stage and maturity.

Characterization of Differentially Expressed miRNAs by CXCL12/SDF-1 in Human Bone Marrow Stromal Cells.

Stromal cell-derived factor 1 (SDF-1) is known to influence bone marrow stromal cell (BMSC) migration, osteogenic differentiation, and fracture healing. We hypothesize that SDF-1 mediates some of its effects on BMSCs through epigenetic regulation, specifically via microRNAs (miRNAs). MiRNAs are small non-coding RNAs that target specific mRNA and prevent their translation. We performed global miRNA analysis and determined several miRNAs were differentially expressed in response to SDF-1 treatment. Gene Expression Omnibus (GEO) dataset analysis showed that these miRNAs play an important role in osteogenic differentiation and fracture healing. KEGG and GO analysis indicated that SDF-1 dependent miRNAs changes affect multiple cellular pathways, including fatty acid biosynthesis, thyroid hormone signaling, and mucin-type O-glycan biosynthesis pathways. Furthermore, bioinformatics analysis showed several miRNAs target genes related to stem cell migration and differentiation. This study's findings indicated that SDF-1 induces some of its effects on BMSCs function through miRNA regulation.

Exosomal circLPAR1 Promoted Osteogenic Differentiation of Homotypic Dental Pulp Stem Cells by Competitively Binding to hsa-miR-31.

Human dental pulp stem cells (DPSCs) hold great promise in bone regeneration. However, the exact mechanism of osteogenic differentiation of DPSCs remains unknown, especially the role of exosomes played in. The DPSCs were cultured and received osteogenic induction; then, exosomes from osteogenic-induced DPSCs (OI-DPSC-Ex) at different time intervals were isolated and sequenced for circular RNA (circRNA) expression profiles. Gradually, increased circular lysophosphatidic acid receptor 1 (circLPAR1) expression was found in the OI-DPSC-Ex coincidentally with the degree of osteogenic differentiation. Meanwhile, results from osteogenic differentiation examinations showed that the OI-DPSC-Ex had osteogenic effect on the recipient homotypic DPSCs. To investigate the mechanism of exosomal circLPAR1 on osteogenic differentiation, we verified that circLPAR1 could competently bind to hsa-miR-31, by eliminating the inhibitory effect of hsa-miR-31 on osteogenesis, therefore promoting osteogenic differentiation of the recipient homotypic DPSCs. Our study showed that exosomal circRNA played an important role in osteogenic differentiation of DPSCs and provided a novel way of utilization of exosomes for the treatment of bone deficiencies.

Clinical plasma concentration of vinpocetine does not affect osteogenic differentiation of mesenchymal stem cells.

Vinpocetine (Vin) has long been used as a medicine to treat cerebrovascular disorders and as a dietary supplement to improve cognitive functions. Previous studies have revealed that the transcription factor nuclear factor kappa B (NF-κB) activity plays an important role in osteogenic differentiation of mesenchymal stem cells (MSC). Vin inhibits NF-κB-dependent inflammatory responses; however, the effect of Vin on the osteogenic differentiation of MSCs has not been reported. In this study, we aimed tothe investigate effect of Vin on the osteogenic differentiation of rat bone marrow-derived MSCs (BMSCs). We treated BMSCs with clinical plasma (0.17µM) or higher concentrations (5 and 20µM) of Vin with no significant effect on the cell viability. Alizarin Red S and alkaline phosphatase (ALP) stainings were used to evaluate mineralizations on days 14 and 21. Moreover, expressions of target genes were detected using qRT-PCR analysis. Osteogenic differentiation of BMSCs did not significantly change with Vin's clinical plasma concentration, but significantly decreased with higher concentrations. Calcium mineralization, ALP staining and mRNA gene expressions of Runx2 and ALP were decreased significantly with high concentrations of Vin, paticularly on day 21. Our in vitro findings suggest that clinically relevant concentration of Vin seems safe to use in elderly patients with respect to osteoporosis. On the other hand, Vin at high concentrations appears to be harmful tobone homeostasis.

Abstract 133: HUVECs Support Bone Formation Of ADSC-loaded Osteogenic Matrices In The Rat AV Loop Model

Purpose: Large volume bone defects can be challenging for the reconstructive surgeon with regard to donor side morbidity. An elegant strategy to circumvent the limiting donor side morbidity is the generation of bioartificial bone tissue. Bearing in mind that the interplay between angiogenesis and osteogenesis is a crucial prerequisite we set our focus on the microvascular endothelium. The microvascular network does not only support oxygen and nutrition supply but also plays an important role in osteogenic differentiation via heterotypic cell contacts. Using an arteriovenous fistula (AV loop), the surgically induced angiogenesis is a powerful tool to enhance intrinsic vascularization of osteogenic matrices. Due to their easy isolation, expansion and osteogenic differentiation potential adipose derived stem cells (ADSCs) are a promising cell source for bone tissue engineering applications. This study aimed at the generation of bioartificial bone tissue using osteogenic differentiated human ADSCs and human umbilical vein endothelial cells (HUVECs) embedded in a hydroxyapatite matrix vascularized by an AV loop. Methods: An osteogenic composite scaffold was generated using hydroxyapatite granula and fibrin. Four experimental groups were performed. Group A represented the control group without cells. The other groups contained 2 x 106 HUVECs (group B), ADSCs (group C) and both cell types (group D). Intrinsic vascularization was induced using an arteriovenous fistula (AV loop) between the saphenous artery and vein connected with a venous interponat. The osteogenic scaffolds and the AV loop were embedded in a porous titanium chamber, combining extrinsic and intrinsic vascularization, fixed subcutaneously in the hind leg of RNU rats. Explantation and histological analysis of the specimen was performed after 6 weeks. Results: In all experimental groups we were able to prove incipient vascularization of the matrices with newly formed fibrovasculare tissue originating from the AV loop. In all cell containing groups vascularized bone tissue was found with significantly more bone tissue in the ADSC-HUVEC coimplantation group. Interestingly, the cell implantation did not affect the matrix vascularization. Conclusion: For the first time we successfully demonstrated vascularization and human bone tissue formation in the rat AV loop model using ADSCs and/or HUVECs. Moreover, the coimplantation of ADSCs and HUVECs seems to be advantageous compared to the single cell use. Taken together the coimplantation of ADSCs and HUVECs is a promising approach for bone tissue engineering applications.

Interference of miR-212 and miR-384 promotes osteogenic differentiation via targeting RUNX2 in osteoporosis

The increasing level of osteogenic (OS) differentiation of bone marrow derived mesenchymal stem cells (MSCs) could be potentially used to relieve the signs and symptoms associated with osteoporosis (OP). Inhibition of osteoprotegerin (OPG)/Receptor Activator of Nuclear factor-Kappa B Ligand (RANKL) pathway plays an important role in OS differentiation, leading to excessive osteoclasts and reduction of osteoblasts, and finally causing OP. Recent studies revealed that microRNAs exert an essential role in regulating OS differentiation. Here, we investigated the dysregulation of miR-212 and miR-384 and the mechanism by which they are involved in OS differentiation-induced MSCs. Quantitative real-time PCR revealed that miR-212 and miR-384 were significantly upregulated in an OP animal model, but markedly downregulated in OS differentiation-induced MSCs. Interference of miR-212 and miR-384 promoted OS differentiation and alleviated OP by targeting RUNX2 in vitro and in vivo. Notably, the inhibition of miR-212 and miR-384 promoted OS differentiation via upregulating RUNX2, and activating OPG/RANKL pathway. Together, our findings demonstrated that interference of miR-212 and miR-384 alleviated OP via RUNX2/OPG/RANKL pathway, providing a novel target of treating OP.

Effect of microRNA-21 on hypoxia-inducible factor-1α in orthodontic tooth movement and human periodontal ligament cells under hypoxia.

Orthodontic tooth movement can lead to temporary hypoxia of periodontal tissues. Periodontal ligament cells (PDLCs) react to hypoxia, releasing various biological factors to promote periodontal tissue reconstruction. Hypoxia-inducible factor-1α (HIF-1α) is one of the most sensitive factors involved in the response to hypoxia. HIF-1α has been identified to be involved in osteogenic and osteoclast differentiation in vitro; however, few studies have investigated the expression of HIF-1α in the periodontal ligament (PDL) during orthodontic movement in vivo. In a previous study, microRNA-21 (miR-21) was demonstrated to be highly expressed in a rat model of orthodontic tooth movement. Additionally, miR-21 can increase the expression of HIF-1α in certain tumor cell types and is involved in tumor bioactivities. In the present study, HIF-1α exhibited expression patterns in a similar way to miR-21 in PDL samples from a rat model of orthodontic tooth movement, with expression initially increased and followed by a decrease over time. Furthermore, human PDLCs were exposed to a hypoxic environment in vitro, which induced significant upregulation of HIF-1α and miR-21 expression. Furthermore, miR-21 mimics increased HIF-1α expression and promoted osteogenic differentiation, indicated by upregulated expression of the osteogenic markers osteopontin, runt-related gene-2 and alkaline phosphatase. miR-21 inhibitors suppressed HIF-1α expression and downregulated the osteogenic markers. In conclusion, the results revealed that miR-21 has a positive effect on HIF-1α expression in PDLCs under hypoxia and has important roles in osteogenic differentiation during orthodontic tooth movement. These findings provide a theoretical basis by which to promote tissue reconstruction during orthodontic tooth movement.

MicroRNA‑223 promotes osteoblast differentiation of MC3T3‑E1 cells by targeting histone deacetylase 2.

MicroRNAs (miRNAs) have emerged as pivotal regulators in various physiological and pathological processes at the post‑transcriptional level, and may serve important roles in osteogenic differentiation. However, their roles and functions are not fully understood. In the present study, miR‑223‑5p was identified as a modulator of osteoblastic differentiation in MC3T3‑E1 cells. Reverse transcription‑quantitative polymerase chain reaction and western blotting demonstrated that miR‑223‑5p was significantly upregulated in MC3T3‑E1 cells following the induction of osteoblast differentiation. Overexpression of miR‑223‑5p promoted osteogenic differentiation both invitro and vivo. Expression of histone deacetylase2 (HDAC2), which acts as a negative regulator of osteogenesis, was regulated by miR‑223‑5p. Collectively, the results of the present study revealed a novel miR‑223‑5p/HDAC2 axis that regulates osteoblast differentiation, and may serve as a potential target for enhancing bone formation invivo.