Differentiation Of hBMSCs Research Articles

Circular RNA CircSLC8A1 contributes to osteogenic differentiation in hBMSCs via CircSLC8A1/miR-144-3p/RUNX1 in periprosthetic osteolysis.

Circular RNAs (circRNAs) are often found in eukaryocyte and have a role in the pathogenesis of a variety of human disorders. Our related research has shown the differential expression of circRNAs in periprosthetic osteolysis (PPOL). However, the involvement of circRNAs in the exact process is yet unknown. CircSLC8A1 expression was evaluated in clinical samples and human bone marrow mesenchymal stem cells (hBMSCs) in this investigation using quantitative real-time PCR. In vitro and in vivo studies were conducted to explicate its functional role and pathway. We demonstrated CircSLC8A1 is involved in PPOL using gain- and loss-of-function methods. The association of CircSLC8A1 and miR-144-3p, along with miR-144-3p and RUNX1, was predicted using bioinformatics. RNA pull-down and luciferase assays confirmed it. The impact of CircSLC8A1 in the PPOL-mouse model was also investigated using adeno-associated virus. CircSLC8A1 was found to be downregulated in PPOL patients' periprosthetic tissues. Overexpression of CircSLC8A1 promoted osteogenic differentiation (OD) and inhibited apoptosis of hBMSCs in vitro. The osteogenic markers of RUNX1, osteopontin (OPN) and osteocalcin (OCN) were significantly upregulated in hBMSCs after miR-144-3p inhibitor was transferred. Mechanistic analysis demonstrated that CircSLC8A1 directly bound to miR-144-3p and participated in PPOL through the miR-144-3p/RUNX1 pathway in hBMSCs. Micro-CT and quantitative analysis showed that CircSLC8A1 markedly inhibited PPOL, and osteogenic markers (RUNX1, OPN and OCN) were significantly increased (P<0.05) in the mice model. Our findings prove that CircSLC8A1 exerted a regulatory role in promoting osteogenic differentiation in hBMSCs, and CircSLC8A1/miR-144-3p/RUNX1 pathway may provide a potential target for prevention of PPOL.

Bone morphogenetic protein 4 is involved in cadmium-associated bone damage.

Cadmium (Cd)is a well-characterized bone toxic agent and can induce bone damage via inhibiting osteogenic differentiation. Bone morphogenetic protein (BMP)/SMAD signaling pathway can mediate osteogenic differentiation, but the association between Cd and BMP/SMAD signaling pathway is yet to be illuminated. To understand what elements of BMPs and SMADs are affected by Cd to influence osteogenic differentiation and if BMPs can be the biomarkers of which Cd-induced osteoporosis, human bone marrow mesenchymal stem cells (hBMSCs) were treated with cadmium chloride (CdCl2) in vitro to detect the expression of BMPs and SMADs, and 134 subjects were enrolled to explore if the BMPs can be potential biomarkers of Cd-associated bone damage. Our results showed that Cd exposure significantly promoted the adipogenic differentiation of hBMSCs and inhibited its osteogenic differentiation by inhibiting the expression of BMP-2/4, SMAD4, and p-SMAD1/5/9 complex. And mediation analyses yielded that BMP-4 mediated 39.32% (95% confidence interval 7.47, 85.00) of the total association between the Cd and the risk of Cd-associated bone damage. Moreover, during differentiation, BMP-4 had the potential to enhance mineralization compared with CdCl2 only group. These results reveal that BMP-4 can be a diagnostic biomarker and therapeutic target for Cd-associated bone damage.

Barbaloin Promotes Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells: Involvement of Wnt/β-catenin Signaling Pathway.

Barbaloin, found in Aloe vera, exerts broad pharmacological activities, including antioxidant, anti-inflammatory, and anti-cancer. This study aims to investigate the effects of barbaloin on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Osteogenic induction of hBMSCs was performed in the presence or absence of barbaloin. Cell viability was determined by using the CCK-8 assay. The characteristic of hBMSCs was identified by using flow cytometry. Intracellular alkaline phosphatase (ALP) staining was performed to evaluate the ALP activity in hBMSCs. Alizarin Red S staining was performed to evaluate the matrix mineralization. The mRNA and protein levels of target genes were determined using qRT-PCR and western blotting, respectively. Treatment with barbaloin (10 and 20 μg/mL) significantly increased cell viability of hBMSCs after 72 hours. In addition, treatment with barbaloin increased the mRNA expression levels of ALP and its activities. Treatment with barbaloin also increased matrix mineralization and the mRNA and protein levels of late-differentiated osteoblast marker genes BMP2, RUNX2, and SP7 in hBMSCs. The underlying mechanisms revealed that barbaloin increased the protein expressions of Wnt/β-catenin pathway-related biomarkers. Barbaloin promotes osteogenic differentiation of hBMSCs by the regulation of the Wnt/β-catenin signaling pathway.

Functionalized Microscaffold-Hydrogel Composites Accelerating Osteochondral Repair through Endochondral Ossification.

Osteochondral regeneration remains a key challenge because of the limited self-healing ability of the bone and its complex structure and composition. Biomaterials based on endochondral ossification (ECO) are considered an attractive candidate to promote bone repair because they can effectively address the difficulties in establishing vascularization and poor bone regeneration via intramembranous ossification (IMO). However, its clinical application is limited by the complex cellular behavior of ECO and the long time required for induction of the cell cycle. Herein, functionalized microscaffold-hydrogel composites are developed to accelerate the developmental bone growth process via recapitulating ECO. The design comprises arginine-glycine-aspartic acid (RGD)-peptide-modified microscaffolds loaded with kartogenin (KGN) and wrapped with a layer of RGD- and QK-peptide-comodified alginate hydrogel. These microscaffolds enhance the proliferation and aggregation behavior of the human bone marrow mesenchymal stem cells (hBMSCs); the controlled release of kartogenin induces the differentiation of hBMSCs into chondrocytes; and the hydrogel grafted with RGD and QK peptide facilitates chondrocyte hypertrophy, which creates a vascularized niche for osteogenesis and finally accelerates osteochondral repair in vivo. The findings provide an efficient bioengineering approach by sequentially modulating cellular ECO behavior for osteochondral defect repair.

TCDD alters essential transcriptional regulators of osteogenic differentiation in multipotent mesenchymal stem cells.

Differentiation of multipotent mesenchymal stem cells (MSCs) into bone-forming osteoblasts requires strict coordination of transcriptional pathways. Aryl hydrocarbon receptor ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), have been shown to alter osteoblast differentiation in vitro and bone formation in multiple developmental in vivo models. The goal of the present study was to establish a global transcriptomic landscape during early, intermediate, and apical stages of osteogenic differentiation in vitro in response to TCDD exposure. Human bone-derived mesenchymal stem cells (hBMSCs) were cultured in growth media (GM), osteogenic differentiation media (ODM), or ODM containing 10 nM TCDD (ODM + TCDD), thus enabling a comparison of the transcriptomic profiles of undifferentiated, differentiated, and differentiated-TCDD-exposed hBMSCs, respectively. In this test system, exposure to TCDD attenuated the differentiation of hBMSCs into osteoblasts as evidenced by reduced alkaline phosphatase activity and mineralization. At various timepoints, we observed altered expression of genes that play a role in the Wnt, fibroblast growth factor, bone morphogenetic protein/transforming growth factor beta developmental pathways, as well as pathways related to extracellular matrix organization and deposition. Reconstruction of gene regulatory networks with the interactive dynamic regulatory event miner (iDREM) analysis revealed modulation of transcription factors (TFs) including POLR3G, NR4A1, RDBP, GTF2B, POU2F2, and ZEB1, which may putatively influence osteoblast differentiation and the requisite deposition and mineralization of bone extracellular matrix. We demonstrate that the combination of RNA-Seq data in conjunction with the iDREM regulatory model captures the transcriptional dynamics underlying MSC differentiation under different conditions in vitro. Model predictions are consistent with existing knowledge and provide a new tool to identify novel pathways and TFs that may facilitate a better understanding of the osteoblast differentiation process, perturbation by exogenous agents, and potential intervention strategies targeting those specific pathways.

Identification and validation of autophagy-related genes during osteogenic differentiation of bone marrow mesenchymal stem cells.

Osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is an essential stage in bone formation. Autophagy plays a pivotal role in the self-renewal potential and pluripotency of stem cells. This study aimed to explore the function of autophagy-related genes during osteogenic differentiation of BMSCs. The differentially expressed autophagy-related genes (ARGs) were obtained from the GEO and HADb databases. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using R software. The PPI and hub gene mining networks were constructed using the STRING database and Cytoscape. Finally, the RT-qPCR was conducted to validate the expression level of ARGs in BMSCs. Thirty-seven differentially expressed ARGs were finally obtained, including 12 upregulated and 25 downregulated genes. GO and KEGG enrichment analysis showed that most of these genes were enriched in apoptosis and autophagy. The PPI network revealed strong interactions between differentially expressed ARGs. The expression level of differentially expressed ARGs tested by RT-qPCR showed 6 upregulated ARGs, including FOXO1, MAP1LC3C, CTSB, FOXO3, CALCOCO2, FKBP1A, and 4 downregulated ARGs, including MAPK8IP1, NRG1, VEGFA, and ITGA6 were consistent with the expression of high-throughput sequencing data. We identified 37 ARGs during osteogenic differentiation using bioinformatics analysis. FOXO1, MAP1LC3C, CTSB, FOXO3, CALCOCO2, FKBP1A, MAPK8IP1, NRG1, VEGFA, and ITGA6 may regulate osteogenic differentiation of hBMSCs by involving autophagy pathway. This study provides new insight into the osteogenic differentiation of hBMSCs and may be available in developing therapeutic strategies for maxillofacial bone defects.

Histone H3K9 demethylase JMJD2B/KDM4B promotes osteogenic differentiation of bone marrow-derived mesenchymal stem cells by regulating H3K9me2 on RUNX2.

BackgroundA variety of proteins including epigenetic factors are involved in the differentiation of human bone marrow mesenchymal stem cells. These cells also exhibited an epigenetic plasticity that enabled them to trans-differentiate from adipocytes to osteoblasts (and vice versa) after commitment. Further in-depth study of their epigenetic alterations may make sense.MethodsChromatin Immunoprecipitation-PCR (ChIP-PCR) was used to detect the methylation enrichment status of H3K9me2 in the Runx2 promoter, alizarin red and alkaline phosphatase (ALP) staining were used to detect osteogenic differentiation and mineralization ability, western blot and quantitative RT-PCR were used to measure the differential expression of osteogenesis-related proteins and genes. Recombinant Lentivirus mediated gain-of-function and loss-of-function study. The scale of epigenetic modification was detected by laser confocal.ResultsOur results showed that compared with human bone marrow mesenchymal stem cells (hBMSCs) without osteogenic differentiation treatment, hBMSCs after osteogenic differentiation significantly promoted osteogenic differentiation and mRNA expression such as JMJD2B/KDM4B, osteogenesis-related genes like Runx2 and FAM210A in hBMSCs cells, suggesting that upregulation of JMJD2B/KDM4B is involved in the promoting effect of osteogenesis. After overexpression and silencing expression of JMJD2B, we found a completely opposite and significant difference in mRNA expression of osteogenesis-related genes and staining in hBMSCs. Overexpression of JMJD2B/KDM4B significantly promoted osteogenic differentiation, suggesting that JMJD2B/KDM4B could promote osteogenesis. In addition, ChIP-PCR showed that overexpression of JMJD2B/KDM4B significantly reversed the methylation enrichment status of H3K9me2 in Runx2 promoter. Furthermore, overexpression of JMJD2B/KDM4B significantly reverses the inhibitory effect of BIX01294 on H3K9me2, suggesting that JMJD2B/KDM4B regulates the osteogenic differentiation of hBMSCs by changing the methylation status of H3K9me2 at the Runx2 promoter.ConclusionsTaken together, these results suggest that JMJD2B/ KDM4B may induce the osteogenic differentiation of hBMSCs by regulating the methylation level of H3K9me2 at the Runx2 promoter.

Long Noncoding RNA P53 Upregulated Regulator of P53 Levels Promotes Osteogenic Differentiation in Osteoporosis Progression Through Sponging miR-135a-5p

This study aimed to explore the expression of long noncoding RNA p53 upregulated regulator of P53 levels (lncRNA PURPL) and microRNA (miR)-135a-5p in osteoporosis and their role in osteogenic differentiation. The relationship between lncRNA PURPL and miR-135a-5p was confirmed by Star-Base and luciferase reporter assay. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay was used to detect lncRNA PURPL and miR-135a-5p expression. RT-qPCR and western blot analysis were used to measure osteogenic markers expression. Alkaline phosphatase (ALP) activity was also determined. Results indicated that lncRNA PURPL binds to miR-135a-5p. lncRNA PURPL expression was decreased and miR-135a-5p expression was increased in patients with osteoporosis. In the process of osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), the expression levels of osteoblast markers including RUNX family transcription factor 2 (Runx2), ALP and Osterix, and ALP activity were significantly increased. Besides, lncRNA PURPL was improved, while miR-135a-5p was down-regulated during the osteogenic differentiation of hBMSCs. Moreover, lncRNA PURPL-siRNA significantly decreased the expression of ALP, Runx2 and Osterix, and reduced ALP activity in hBMSCs subjected to osteogenic induction, while all of these effects were reversed by miR-135a-5p inhibitor. In conclusion, lncRNA PURPL/miR-135a-5p may be a new axis for osteoporosis treatment.

Use of Gelatin as a Sacrificial Agent in Combination with Ultrasonication to Improve Cell Infiltration and Osteogenesis of Nanofibrous PCL-nHA Scaffolds for Bone Tissue Engineering.

In this study, gelatin was chosen as a novel sacrificial agent in co-electrospun with polycaprolacton-nanohydroxyapatite (PCL-nHA). After electrospinnig, gelatin was washed with water, and the prepared scaffold was ultrasonicated. Morphological and structural properties of the prepared scaffolds were studied by SEM. Fourier transform infrared (FTIR) spectroscopy and water contact angle analysis were used to evaluate the removal of gelatin. According to the SEM results, the pore size of the modified scaffolds was increased 3-folds compared to the control sample. For PCL-nHA gelatin: (80:20) after the treatment, the average cell infiltration was 42.7 μm, while there was no infiltration for the control group. The modified electrospun scaffold significantly enhanced the osteogenic differentiation of hBMSCs as verified by increased ALP activity and upregulation of runt-related transcription factor 2 (RUNX2), collagen type 1 (COL1) and osteocalcin (OCN) genes. Co-electrospun PCL-nHA with gelatin as a sacrificial agent in combination with ultrasonication may be an effective, economic and controllable method to increase the pore size in electrospun scaffolds for bone tissue engineering applications.

Contact osteogenesis by biodegradable 3D-printed poly(lactide-co-trimethylene carbonate)

BackgroundTo support bone regeneration, 3D-printed templates function as temporary guides. The preferred materials are synthetic polymers, due to their ease of processing and biological inertness. Poly(lactide-co-trimethylene carbonate) (PLATMC) has good biological compatibility and currently used in soft tissue regeneration. The aim of this study was to evaluate the osteoconductivity of 3D-printed PLATMC templates for bone tissue engineering, in comparison with the widely used 3D-printed polycaprolactone (PCL) templates.MethodsThe printability and physical properties of 3D-printed templates were assessed, including wettability, tensile properties and the degradation profile. Human bone marrow-derived mesenchymal stem cells (hBMSCs) were used to evaluate osteoconductivity and extracellular matrix secretion in vitro. In addition, 3D-printed templates were implanted in subcutaneous and calvarial bone defect models in rabbits.ResultsCompared to PCL, PLATMC exhibited greater wettability, strength, degradation, and promoted osteogenic differentiation of hBMSCs, with superior osteoconductivity. However, the higher ALP activity disclosed by PCL group at 7 and 21 days did not dictate better osteoconductivity. This was confirmed in vivo in the calvarial defect model, where PCL disclosed distant osteogenesis, while PLATMC disclosed greater areas of new bone and obvious contact osteogenesis on surface.ConclusionsThis study shows for the first time the contact osteogenesis formed on a degradable synthetic co-polymer. 3D-printed PLATMC templates disclosed unique contact osteogenesis and significant higher amount of new bone regeneration, thus could be used to advantage in bone tissue engineering.

CircZNF367 suppresses osteogenic differentiation of human bone marrow mesenchymal stromal/stem cells via reducing HuR-mediated mRNA stability of LRP5.

Osteoporosis is a highly prevalent disease characterized by bone mass loss and structural deterioration. There are evidences that altered differentiation of human bone marrow mesenchymal stromal/stem cells (hBMSCs) is a major cause for osteoporosis. Recent studies suggest that circular RNAs (circRNAs) are dysregulated in osteoporosis patients and involved in the pathogenesis of osteoporosis. In the present study, we are aimed to analyze the circRNA expression profiles in osteoporosis patients and identify potential circRNAs that involved in the differentiation of hBMSCs during osteoporosis. Transcriptome RNA-sequencing was conducted to search for differentially expressed circRNAs. Transwell assay, ARS and ALP staining, and ectopic bone formation model were performed to evaluate osteogenic differentiation of hBMSCs. RNA pull-down assay, RNA immunoprecipitation, western blot, and in vitro binding assay were conducted to evaluate the interaction of circRNAs and RNA-binding protein HuR. We found that hsa_circ_0008842 (designated as circZNF367) was upregulated in osteoporosis patients and decreased in hBMSCs during osteogenic differentiation. CircZNF367 overexpression suppressed migration, invasion and osteogenic differentiation of hBMSCs in vitro and in vivo. In comparison, knockdown of circZNF367 promoted migration, invasion and osteogenic differentiation of hBMSCs. CircZNF367 could interact with the RNA-binding protein HuR, thus reduced the mRNA stability of LRP5. Furthermore, HuR overexpression or LRP5 restoration abrogated the effects of circZNF367 overexpression on osteogenic differentiation of hBMSCs. Our results indicated that circZNF367 played a role in osteogenic differentiation of hBMSCs via reducing HuR-mediated mRNA stability of LRP5.

Enhancer RNA commits osteogenesis via microRNA-3129 expression in human bone marrow-derived mesenchymal stem cells

BackgroundHighly regulated gene expression program underlies osteogenesis of mesenchymal stem cells (MSCs), but the regulators in the program are not entirely identified. As enhancer RNAs (eRNAs) have recently emerged as a key regulator in gene expression, we assume a commitment of an eRNA in osteogenesis.MethodsWe performed in silico analysis to identify potential osteogenic microRNA (miRNA) gene predicted to be regulated by super-enhancers (SEs). SE inhibitor treatment and eRNA knocking-down were used to confirm the regulational mechanism of eRNA. miRNA function in osteogenesis was elucidated by miR mimic and inhibitor transfection experiments.ResultsmiR-3129 was found to be located adjacent in a SE (osteoblast-specific SE_46171) specifically activated in osteoblasts by in silico analysis. A RT-quantitative PCR analysis of human bone marrow-derived MSC (hBMSC) cells showed that eRNA_2S was transcribed from the SE with the expression of miR-3129. Knockdown of eRNA_2S by locked nucleic acid as well as treatment of SE inhibitors JQ1 or THZ1 resulted in low miR-3129 levels. Overexpression of miR-3129 promoted hBMSC osteogenesis, while knockdown of miR-3129 inhibited hBMSC osteogenesis. Solute carrier family 7 member 11 (SLC7A11), encoding a bone formation suppressor, was upregulated following miR-3129-5p inhibition and identified as a target gene for miR-3129 during differentiation of hBMSCs into osteoblasts.ConclusionsmiR-3129 expression is regulated by SEs via eRNA_2S and this miRNA promotes hBMSC differentiation into osteoblasts through downregulating the target gene SLC7A11. Thus, the present study uncovers a commitment of an eRNA via a miR-3129/SLC7A11 regulatory pathway during osteogenesis of hBMSCs.

MiR-215-5p regulates osteoporosis development and osteogenic differentiation by targeting XIAP

BackgroundOsteoporosis (OP) is a metabolic disease that involves microstructure destruction and fracture damage. The present study probed into the significance of miR-215-5p in OP progression.MethodsSerum samples were collected from surgical patients and healthy controls. qRT-PCR analysis was utilized to determine the miR-215-5p level in clinical samples and human bone mesenchymal stem cells (hBMSCs) induced by β-glycerol phosphate. A dual luciferase reporter assay was exploited to examine the targeted relationship between miR-215-5p and XIAP. The mineralization and calcium deposition of hBMSCs were assessed by detection of ALP activity, Alizarin red staining, and osteoblast marker expression. Protein expression was determined by western blot analysis.ResultsMiR-215-5p was significantly reduced in patients with OP and increased in hBMSCs treated with β-glycerophosphate. Enhanced miR-215-5p level triggered augment in osteoblast markers (Alkaline phosphatase/ ALP, Osteocalcin/ OCN, and Runt-Related Transcription Factor 2/ Runx2), which was accompanied by the increase of ALP activity in hBMSCs and accumulation of Calcium. Functional experiments show that XIAP was a target of miR-215-5p and negatively modulated by miR-215-5p. XIAP expression levels were increased in OP samples, and decreased XIAP in β-glycerophosphate-treated hBMSCs inhibited its’ osteogenic differentiation. Functional loss and acquisition experiments depicted that miR-215-5p promoted the differentiation of hBMSCs by inhibiting the XIAP level, playing a protective role in the pathogenesis of OP.Conclusionsβ-glycerophosphate promoted the osteogenic differentiation of hBMSCs, increased miR-215-5p level, and decreased XIAP. miR-215-5p stimulated osteogenic differentiation of hBMSCs by targeting XIAP, shedding new insights for the detection and therapy of OP.

Deubiquitinating Enzyme USP7 Is Required for Self-Renewal and Multipotency of Human Bone Marrow-Derived Mesenchymal Stromal Cells.

Ubiquitin-specific protease 7 (USP7) is highly expressed in a variety of malignant tumors. However, the role of USP7 in regulating self-renewal and differentiation of human bone marrow derived mesenchymal stromal cells (hBMSCs) remains unknown. Herein, we report that USP7 regulates self-renewal of hBMSCs and is required during the early stages of osteogenic, adipogenic, and chondrogenic differentiation of hBMSCs. USP7, a deubiquitinating enzyme (DUB), was found to be downregulated during hBMSC differentiation. Furthermore, USP7 is an upstream regulator of the self-renewal regulating proteins SOX2 and NANOG in hBMSCs. Moreover, we observed that SOX2 and NANOG are poly-ubiquitinated and their expression is downregulated in USP7-deficient hBMSCs. Overall, this study showed that USP7 is required for maintaining self-renewal and multipotency in cultured hBMSCs. Targeting USP7 might be a novel strategy to preserve the self-renewal capacity of hBMSCs intended for stem cell therapy.

The effect of AKT in extracellular matrix stiffness induced osteogenic differentiation of hBMSCs

Extracellular matrix (ECM) stiffness is an important biophysical factor in human bone marrow mesenchymal stem cells (hBMSCs) differentiation. Although there is evidence that Yes-associated protein (YAP) plays an important role in ECM elasticity induced osteogenesis, but the regulatory mechanism and signaling pathways have not been distinctly uncovered. In this study, hBMSCs were cultured on collagen-coated polydimethylsiloxane hydrogels with stiffness corresponding to Young's moduli of 0.5 kPa and 32 kPa, and gene chip analyses revealed the phosphoinositide 3-kinase (PI3K)-AKT pathway was highly correlated with ECM stiffness. Following western blots indicated that AKT phosphorylation was evidently affected in 5th–7th days after ECM stiffness stimulation, while PI3K showed little difference. The AKT activator SC79 and inhibitor MK2206 were utilized to modulate AKT phosphorylation. SC79 and MK2206 caused alteration in the mRNA expression and protein level of alkaline phosphatase (ALP), collagen type I alpha 1 (COL1A1) and runt related transcription factor 2 (RUNX2). On 32 kPa substrates, YAP enrichment in nucleus were significantly promoted by SC79 and remarkably decreased by MK2206. Besides, the ratio of YAP/p-YAP is upregulated by SC79 on both 32 kPa and 0.5 kPa substrates. In conclusion, these findings suggest that AKT is involved in the modulation of ECM stiffness induced osteogenesis, and AKT phosphorylation also influences the subcellular localization and activation of YAP.

MiR-29a-3p Inhibits Proliferation and Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells via Targeting FOXO3 and Repressing Wnt/β-Catenin Signaling in Steroid-Associated Osteonecrosis.

Background and ObjectivesThis study was to investigate the role of microRNA-29a-3p (miR-29a-3p) in human bone marrow mesenchymal stem cells (hBMSCs), and its relationship with steroid-associated osteonecrosis.Methods and ResultsThe online tool GEO2R was used to screen out the differentially expressed genes (DEGs) in GSE123568 dataset. Quantitative real time-polymerase chain reaction (qRT-PCR) was performed to detect the expression of miR-29a-3p, forkhead box O3 (FOXO3), alkaline phosphatase (ALP), bone gamma-carboxyglutamate protein (OCN) and RUNX family transcription factor 2 (Runx2) in the hBMSCs isolated from the patients with steroid-associated osteonecrosis. CCK-8 assay was executed to measure cell viability; western blot assay was utilized to detect FOXO3, ALP, Runx2, OCN and β-catenin expression. Cell apoptosis and cell cycle were detected by flow cytometry. Immunofluorescence assay was used to detect the sub-cellular localization of β-catenin. Bioinformatics analysis and luciferase reporter gene assay were performed to confirm whether miR-29a-3p can combine with FOXO3 3’UTR. MiR-29a-3p was markedly up-regulated in the hBMSCs of patients with steroid-associated osteonecrosis, while FOXO3 mRNA was significantly down-regulated. Transfection of miR-29a-3p mimics significantly inhibited the hBMSCs’ proliferation, osteogenic differentiation markers’ expressions, including ALP, Runx2, OCN, and repressed the ALP activity, as well as promoted cell apoptosis and cell-cycle arrest. FOXO3 was identified as a target gene of miR-29a-3p, and miR-29a-3p can inhibit the expression of FOXO3 and β-catenin, and inhibition of miR-29a-3p promoted translocation of β-catenin to the nucleus.ConclusionsMiR-29a-3p can modulate FOXO3 expression and Wnt/β-catenin signaling to inhibit viability and osteogenic differentiation of hBMSCs, thereby promoting the development of steroid-associated osteonecrosis.

Biofabrication of natural Au/bacterial cellulose hydrogel for bone tissue regeneration via in-situ fermentation

Bacterial cellulose (BC) possesses the desirable properties of biocompatibility, high porosity, high surface area and noticeable mechanical strength as a scaffold in bone tissue engineering. However, the lack of osteogenic activity restricts its application. In this study, gold nanoparticles (GNPs) with excellent osteogenic differentiation ability were incorporated into the network of BC hydrogel (Au/BC hydrogels) by the in-situ fermentation. The effects of GNPs on physicochemical properties of BC hydrogel and subsequently in vitro osteogenic differentiation and in vivo bone regeneration of Au/BC hydrogels were comprehensively investigated. The results showed that the increased feeding amounts of GNPs could remarkablly enhance the Au/BC hydrogels with better mechanical properties, higher porosity, larger surface area, and biocompatibility. The sustainable release of GNPs endowed the hydrogels with an outstanding biological activity in facilitating osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). Mechanism research showed that autophagy might be a potential pathway for Au/BC hydrogels-induced osteogenic differentiation of hBMSCs. In addition, Au/BC hydrogel exhibited an excellent in vivo bone repair performance in a rabbit model of femoral defect, which was evidenced by the significant newly bone formation. Overall, the multifunctional Au/BC hydrogels fabricated by in-situ fermentation could serve as a good scaffold for promoting bone tissue regeneration in clinic.

Circ_0062582 Promotes Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells in vitro by Elevating SMAD5 Expression through Sponging miR-197-3p

Circular RNAs (circRNAs) play crucial roles in many human diseases. However, the functions of circRNAs in osteoporosis (OP) are barely reported. In this study, we aimed to explore the function of circ_0062582 in osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) in vitro. circ_0062582 and SMAD5 were downregulated and miR-197-3p was upregulated in OP patients and increased in osteoblast medium (OM)-induced hBMSCs in vitro. circ_0062582 knockdown inhibited the viability and osteogenic differentiation of hBMSCs. circ_0062582 directly targeted miR-197-3p, and miR-197-3p inhibition reversed the effects of circ_0062582 on hBMSC viability and osteogenic differentiation. SMAD5 was the target gene of miR-197-3p. SMAD5 overexpression promoted the viability and osteogenic differentiation of hBMSCs and attenuated miR-197-3p-mediated suppressive roles in hBMSC viability and osteogenic differentiation. In conclusion, circ_0062582 sponged miR-197-3p to elevate SMAD5 expression, thereby inducing hBMSC proliferation and osteogenic differentiation in vitro.

LncRNA RAD51-AS1 Regulates Human Bone Marrow Mesenchymal Stem Cells via Interaction with YBX1 to Ameliorate Osteoporosis.

Long noncoding RNA (lncRNA) is a new key regulatory molecule in the occurrence of osteoporosis, but its research is still in the primary stage. In order to study the role and mechanism of lncRNA in the occurrence of osteoporosis, we reannotated the GSE35956 datasets, compared and analyzed the differential expression profiles of lncRNAs between bone marrow mesenchymal stem cells (hBMSCs) from healthy and osteoporotic patients, and then screened a lncRNA RAD51-AS1 with low expression in hBMSCs from osteoporotic patients, and its role in the occurrence of osteoporosis has not been studied. We confirmed that the expression level of lncRNA RAD51-AS1 in hBMSCs from patients with osteoporosis was significantly lower than those from healthy donors. A nuclear cytoplasmic separation experiment and RNA fluorescence in situ hybridization showed that RAD51-AS1 was mainly located in the nucleus. RAD51-AS1 knockdown significantly inhibited the proliferation and osteogenic differentiation of hBMSCs and significantly increased their apoptosis, while RAD51-AS1 overexpression significantly promoted the proliferation, osteogenic differentiation, and ectopic bone formation of hBMSCs. Mechanistically, we found that RAD51-AS1 banded to YBX1 and then activated the TGF-β signal pathway by binding to Smad7 and Smurf2 mRNA to inhibit their translation and transcription up-regulated PCNA and SIVA1 by binding to their promoter regions. In conclusion, RAD51-AS1 promoted the proliferation and osteogenic differentiation of hBMSCs by binding YBX1, inhibiting the translation of Smad7 and Smurf2, and transcriptionally up-regulated PCNA and SIVA1.

LncRNA BC083743 Silencing Exacerbated Osteoporosis by Regulating the miR-103-3p/SATB2 Axis to Inhibit Osteogenic Differentiation.

Objective The target of the present paper was to reveal the influence of LncRNA BC083743 on osteogenesis in human bone marrow mesenchymal stem cells (hBMSCs). Methods Serum specimens from osteoporotic patients and normal subjects were collected to isolate hBMSCs from femoral head tissue. The levels of BC083743 and miR-103-3p in serum and hBMSCs were measured by QRT-PCR. Alkaline phosphatase (ALP) activity test and alizarin red dyeing were used to identify ALP activity and mineralization forming ability of hBMSCs after transfection with si-BC083743 (siRNA-targeting BC083743). In addition, QRT-PCR and immunoblotting were conducted to identify the expressing levels of Runt-related transcription factor 2(Runx2), osteoprotegerin (OPG), and bone morphogenetic protein 2 (BMP2) in hBMSCs. Dual-luciferase reporter gene and RNA pull-down assays were employed to substantiate the binding of BC083743 to miR-103-3p and miR-103-3p to SATB2. Results BC083743 expression was significantly downregulated in sera from patients with osteoporosis, and osteogenic differentiation-related genes and BC083743 expression were obviously upregulated as the time to osteogenic differentiation increased. BC083743 knockdown hindered the osteogenic differentiation of hBMSCs. BC083743 was aimed at miR-103-3p and miR-103-3p inhibitors partially reversed the inhibitory effect of BC083743 downregulation on hBMSCs osteogenesis. BC083743 silencing downregulated SATB2 through uptake of miR-103-3p, thereby inhibiting hBMSCs osteogenesis to exacerbate osteoporosis. Conclusion BC083743/miR-103-3p/SATB2 axis inhibited osteogenic differentiation and exacerbated osteoporosis, which may offer brand-new molecular aims for the treatment of clinical osteoporosis.