Osteogenic Differentiation Of Bone Marrow Stem Cells Research Articles

Inhibition of miRNA-100 facilitates bone regeneration defects of mesenchymal stem cells in osteoporotic mice through the protein kinase B pathway

ABSTRACT Osteoporotic patients suffer from bone microstructure damage and are prone to fracture and bone defect. Due to the damage of bone healing ability, the bone repair of osteoporotic patients is usually slow. Here we aimed to explore the function and potential molecular mechanism of miR-100 in osteogenic differentiation ability of bone marrow stem cells (BMSCs). Ovariectomy was performed on mice to induce osteoporosis. BMSCs were extracted from normal and ovariectomized (OVX) mice to examine the effect of microRNA (miR)-100 on BMSC osteogenic differentiation. Hematoxylin and eosin (H&E) staining and safranin O-fast green staining assays were performed on femur tissues to reveal pathological changes. The osteogenic differentiation of BMSCs were determined by Alkaline Phosphatase and Alizarin red staining assays. The results showed that miR-100 expression was significantly upregulated in bone tissues and BMSCs from osteoporotic mice. MiR-100 knockdown partially improved osteogenic function of OVX mice-derived BMSCs. Next, mechanistic target of rapamycin kinase (MTOR) was identified as the target downstream miR-100. MiR-100 deficiency can activate the protein kinase B (AKT)/mTOR pathway. MiR-100 controlled the osteogenic function of BMSCs by the AKT/mTOR pathway. Collectively, our findings demonstrate that inhibition of miR-100 facilitates bone regeneration defects of BMSCs in osteoporotic mice through AKT pathway, indicating that miR-100 might be an effective target for the treatment of osteoporotic mandibular injury and bone defect diseases.

Effects of Transforming Growth Factor-/β 1 and Concentrated Growth Factor on Growth, Proliferation and Osteogenic Differentiation of Bone Marrow Stem Cells

Objective: We aimed to explore the effects of transforming growth factor-β1 (TGF-β1) and concentrated growth factor (CGF) on growth, proliferation and osteogenic differentiation of bone marrow stem cells (BMSCs). Methods: CGF was extracted from rat venous blood. BMSCs was isolated from rats. TGF-β1 or CGF was used to induced BMSCs to observe their effects on BMSCs. The expression levels of BMSCs surface antigens were detected by flow cytometry. ELISA was performed to test the expression of VEGF, TGF-β1, PDGF and SDF-1 in BMSCs. qRT-PCR was carried out to test the expression of COL-I, OPN, OC and ALP in BMSCs. Transwell assay was used to detect the migration rates of BMSCs. The cell morphology of BMSCs was detected by Alizarin red staining. Results: BMSCs were successfully isolated from rats. CGF could significantly increase the expression of TGF-β1, PDGF and VEGF in BMSCs. In addition, TGF-β1 and CGF notably increased the proliferation of BMSCs. And they not only improved the activity of ALD, but also increased the levels of ALP, OPN, COL-I and OCN in BMSCs to prompt the growth of BMSCs. Conclusion: CGF combined with TGF-β1 could prompt the growth, proliferation and osteogenic differentiation of BMSCs.

Glucocorticoids decreased Cx43 expression in osteonecrosis of femoral head: The effect on proliferation and osteogenic differentiation of rat BMSCs.

Glucocorticoid (GC)‐induced osteonecrosis of the femoral head (GC‐ONFH) is considered as one of the most serious side effects of long‐term or over‐dose steroid therapy. However, the underlying cause mechanisms are still not fully investigated. We firstly established a rat model of GC‐ONFH and injected lipopolysaccharide (LPS) and methylprednisolone (MPS). We found that the expressions of Cx43, Runx2, ALP and COLⅠ were more decreased than the normal group. Secondly, the isolated rat bone marrow stem cells (BMSCs) were treated with dexamethasone (Dex) in vitro, and the expressions of Cx43, Runx2, ALP and COLⅠ were decreased significantly. Moreover, the results of immunofluorescence staining, alizarin red staining, EdU assay and CCK8 showed that the osteogenic differentiation and the proliferation capacity of BMSCs were decreased after induced by Dex. A plasmid of lentivirus‐mediated Cx43 (Lv‐Cx43) gene overexpression was established to investigate the function of Cx43 in BMSCs under the Dex treatment. Findings demonstrated that the proliferation and osteogenic differentiation abilities were enhanced after Lv‐Cx43 transfected to BMSCs, and these beneficial effects of Lv‐Cx43 were significantly blocked when PD988059 (an inhibitor of ERK1/2) was used. In conclusion, the overexpression of Cx43 could promote the proliferation and osteogenic differentiation of BMSCs via activating the ERK1/2 signalling pathway, which provide a basic evidence for further study on the detailed function of Cx43 in GC‐ONFH.

Exosomes Enhance Adhesion and Osteogenic Differentiation of Initial Bone Marrow Stem Cells on Titanium Surfaces.

Successful osseointegration involves the biological behavior of bone marrow stem cells (BMSCs) on an implant surface; however, the role of BMSC-derived extracellular vesicles (EVs)/exosomes in osseointegration is little known. This study aimed to: (i) explore the interaction force between exosomes (Exo) and cells on a titanium surface; (ii) discuss whether the morphology and biological behavior of BMSCs are affected by exosomes; and (iii) preliminarily investigate the mechanism by which exosomes regulate cells on Ti surface. Exosomes secreted by rat BMSCs were collected by ultracentrifugation and analyzed using transmission electron microscopy and nanoparticle tracking analysis. Confocal fluorescence microscopy, scanning electron microscopy, Cell Counting Kit-8 (CCK-8), quantitative real-time polymerase chain reaction techniques, and alkaline phosphatase bioactivity, Alizarin Red staining, and quantification were used to investigate the exosomes that adhere to the Ti plates under different treatments as well as the morphological change, adhesion, spread, and differentiation of BMSCs. We found that exosomes were efficiently internalized and could regulate cell morphology and promoted the adhesion, spreading, and osteogenic differentiation of BMSCs. These were achieved partly by activating the RhoA/ROCK signaling pathway. Our discovery presents a new insight into the positive regulatory effect of exosomes on the biological behaviors of BMSCs on Ti surface and provides a novel route to modify the surface of a Ti implant.

Adipose-Derived Stem Cells Conditioned Media Promote In Vitro Osteogenic Differentiation of Hypothyroid Mesenchymal Stem Cells

Background: Thyroid hormones have many effects on the physiological functions of cells, including growth, differentiation, and metabolism. Objectives: Recently, studies have shown that the adipose-derived mesenchymal stem cells conditioned medium (ADMSCs-CM) has many osteogenic factors, such as IGF-1, IL-6, and FGFs. Methods: In the current study, mesenchymal stem cells (MSCs) were isolated from two sources; the adipose tissue of the testicular fat pad and the bone marrow of rat, and then characterized by flow cytometry. ADMSCs-CM was collected from the ADMSC in the healthy adult male rats. Hypothyroidism was induced by the administration of the Methimazole during 60 days and confirmed by the analysis of the serum level of T4 and TSH hormones. Cell proliferation and osteogenic differentiation potential of bone marrow stem cells (BMSCs) derived from hypothyroid rats were investigated in the presence and absence of the CM by MTT assay, alkaline phosphatase (ALP) activity, calcium content assay, and bone-related gene expression. Healthy BMSCs were assigned to the control group. Results: Although Cell proliferation was decreased in the hypothyroid BMSCs, there was no significant difference between the control and the hypothyroid-CM groups. Similarly, osteogenic potential was significantly reduced in the hypothyroid group compared to the control and hypothyroid-CM groups according to the ALP, calcium content assays, and gene expression results. There was no significant difference between the hypothyroid-CM group and control. Conclusions: Our results indicated that hypothyroidism can decrease cell proliferation and osteogenic differentiation of BMSCs. Although ADMSCs-CM improved these parameters, it may be a promising candidate for the bone regeneration of the hypothyroidism cases.

CRYAB promotes osteogenic differentiation of human bone marrow stem cells via stabilizing β-catenin and promoting the Wnt signalling.

ObjectivesThe osteogenesis differentiation of human bone marrow stem cells (BMSCs) is essential for bone formation and bone homeostasis. In this study, we aim to elucidate novel molecular targets for bone metabolism diseases.Materials and methodsThe dataset GSE80614 which includes mRNA expression profile during BMSCs osteogenic differentiation was obtained from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). The osteogenic differentiation of BMSCs was measured by ALP staining, AR staining and expression of osteogenic markers in vitro. For in vivo assay, we seeded BMSCs onto beta‐tricalcium phosphate (β‐TCP) and transplanted them into muscle pockets of nude mice. Luciferase assay, co‐immunoprecipitation assay and in vitro ubiquitination assay were carried out to investigate the molecular mechanism.ResultsWe found that α‐B‐crystallin (CRYAB) expression was elevated during the process of BMSCs osteogenic differentiation. Further studies showed that upregulation of CRYAB significantly enhanced the osteogenic differentiation, while downregulation of CRYAB suppressed it. CRYAB regulated BMSCs osteogenic differentiation mainly through the canonical Wnt/β‐catenin signalling. In addition, we found that CRYAB could physically interact with β‐catenin and protect it from ubiquitination and degradation, which stabilized β‐catenin and promoted the Wnt signalling.ConclusionsThe present study provides evidences that CRYAB is an important regulator of BMSCs osteogenic differentiation by protecting β‐catenin from ubiquitination and degradation and promoting the Wnt signalling. It may serve as a potential therapeutic target for diseases related to bone metabolism.

LMCD1 promotes osteogenic differentiation of human bone marrow stem cells by regulating BMP signaling

Human bone marrow stem cells (BMSCs) are heterogeneous progenitor cells with two defining features, self-renew and multi-lineage differentiation. As one of the differentiation directions, osteogenesis is vital for bone homeostasis. A growing body of evidences show that ubiquitin-dependent protein degradation plays an essential role in the osteogenic differentiation of BMSCs. In this study, we found that LMCD1 was upregulated during osteogenic differentiation process of BMSCs by analyzing GSE80614. In vitro and in vivo functional studies confirmed that LMCD1 was critical to the osteogenic commitment of BMSCs. Compared to those of the controls, downregulation of LMCD1 significantly restrained osteogenic differentiation and enhanced adipogenic differentiation, while upregulation of LMCD1 increased the osteogenic differentiation and suppressed adipogenic differentiation. Mechanically, we found that LMCD1 could protect RUNX2 and Smad1 protein from Smurf1-induced ubiquitination degradation thereby regulating BMP signaling. In conclusion, our findings suggest that LMCD1 is a novel regulator of osteogenic differentiation and may be a potential therapeutic target for bone metabolism related diseases.

Strontium‑containing α‑calcium sulfate hemihydrate promotes bone repair via the TGF‑β/Smad signaling pathway.

Calcium phosphate-based bone substitutes have been widely used for bone repair, augmentation and reconstruction in bone implant surgery. While some of these substitutes have shown excellent biological efficacy, there remains a need to improve the performance of the current calcium phosphate-based bone substitutes. Strontium ions (Sr) can promote new osteogenesis, inhibit osteoclast formation and increase osteoconductivity. However, the therapeutic effect and mechanism of strontium-containing α-calcium sulfate hemihydrate (Sr-CaS) remains unclear. The present study created bone injuries in rats and treated the injuries with Sr-CaS. Then Cell Counting Kit-8, soft agar colony formation, flow cytometry, Transwell and Alizarin Red staining assays were performed to assess the bone cells for their proliferation, growth, apoptosis, invasion, and osteogenic differentiation abilities. The bone reconstructive states were measured by the microCT method, hematoxylin and eosin staining and Masson staining. Bone-related factors were analyzed by the reverse transcription-quantitative PCR assay; transforming growth factor (TGF)-β, mothers against decapentaplegic homolog (Smad)2/3 and β-catenin expression was measured by western blot analysis and osteocalcin (OCN) expression was assessed by immunohistochemistry. Sr-CaS did not significantly affect the proliferation and apoptosis of bone marrow stem cells (BMSCs), but did accelerate the migration and osteogenic differentiation of BMSCs in vitro. Sr-CaS promoted bone repair and significantly increased the values for bone mineral density, bone volume fraction, and trabecular thickness, but decreased trabecular spacing in vivo in a concentration-dependent manner. In addition, Sr-CaS dramatically upregulated the expression levels of genes associated with osteogenic differentiation (Runt-related transcription factor 2, Osterix, ALP, OCN and bone sialoprotein) both in vitro and in vivo. Sr-CaS also increased Smad2/3, TGF-β and phosphorylated-β-catenin protein expression in vitro and in vivo. These results indicated that materials that contain 5 or 10% Sr can improve bone defects by regulating the TGF-β/Smad signaling pathway.

MTORC1 signaling is essential for neurofibromatosis type I gene modulated osteogenic differentiation of BMSCs.

Neurofibromatosis type I (NF1), which is caused by mutations in the NF1 gene, is a common autosomal dominant genetic disease leading to skeletal abnormalities. Both NF1 gene and mammalian target of rapamycin complex 1 (mTORC1) signaling are associated with the osteogenic differentiation of bone marrow stem cells (BMSCs). In this study, we hypothesized that mTORC1 signaling is involved in NF1-modulated osteoblast differentiation of BMSCs. Human BMSCs were cultured in an osteogenic induction medium. The expression of NF1 was either inhibited or overexpressed by transfecting NF1 with a specific small interfering RNA (siRNA) or pcDNA3.0 plasmid, respectively. In addition, an mTORC1 signaling inhibitor and agonist were used to investigate the effects of mTORC1 on NF1-modulated osteogenic differentiation of BMSCs. The results indicated that inhibiting the expression of NF1 with siRNA significantly decreased the mRNA levels of NF1, whereas overexpressing the expression of NF1 with pcDNA3.0 plasmid significantly increased the mRNA levels of NF1 at days 3, 7, 14 and 21 after culture. We observed reduced osteogenic differentiation and cell proliferation in the NF1-siRNA group and enhanced osteogenic differentiation and cell proliferation of BMSCs in the NF1-pcDNA3.0 group. The activity of mTORC1 signaling (p-mTORC1, p-S6K1, and p-4EBP1) was significantly upregulated in the NF1-siRNA group and significantly inhibited in the NF1-pcDNA3.0 group, 7 and 14 days after culture. The effects of NF1-siRNA and NF1-pcDNA3.0 on osteogenic differentiation of BMSCs and cell proliferation were reversed by mTORC1 inhibitor and agonist, respectively. In conclusion, NF1 modulates osteogenic differentiation and cell proliferation of human BMSCs and mTORC1 signaling is essential for this process.

CircRNA Expression Profiles in Human Bone Marrow Stem Cells Undergoing Osteoblast Differentiation.

Circular RNAs (circRNAs), which are produced by back-splicing events in genes, have emerged as factors in gene regulation and normal cellular homeostasis. They play an important role not only in normal development of tissues and organs, but also in disease pathogenesis and cell differentiation. However, the role of circRNAs in bone marrow stem cells (BMSCs) undergoing osteoblast differentiation remains largely unknown. We performed microarray analysis to determine the expression profiles of circRNAs during osteoblast differentiation. In total, 3938 circRNAs were upregulated and 1505 were downregulated in BMSCs at day 7 (D7) compared with day 0 (D0). About 95% of the differentially expressed circRNAs were derived from protein coding genes, and functional annotation analysis showed that the parental genes of differentially expressed circRNAs were enriched in osteogenesis-associated terms. We also analyzed the microRNA (miRNA) transcriptome since circRNAs have been suggested to interact with miRNAs. We then selected the circRNAs that were negatively correlated with miRNAs and possessed miRNA response elements to construct a circRNA-miRNA interaction network. Analysis of the hub nodes in the networks showed that the top five nodes were miRNAs. Some circRNAs were linked to miRNAs with osteogenic roles, indicating that these circRNAs potentially function in osteogenic differentiation of BMSCs. Moreover, we validated the expression of one hub miRNA, miR-199b-5p, and its linked circIGSF11. Silencing of circIGSF11 promoted osteoblast differentiation and increased the expression of miR-199b-5p. Our study suggests that circRNA-miRNA interaction actively contributes to the osteogenic differentiation of human BMSCs, suggesting potential avenues for further study.

Requirement of TGFβ Signaling for Effect of Fluoride on Osteoblastic Differentiation.

Research focused on transforming growth factor β (TGFβ) signaling in osteoblast is gradually increasing, whereas literature is rare in terms of fluorosis. This work aimed to investigate how TGFβ signaling participated in regulation of the osteoblast by different doses of fluoride treatment. Bone marrow stem cells (BMSCs) were developed into osteoblastic cells and exposed to 1, 4, and 16mg/L F- with and without 10ng/mL of TGFβ. Cell viability and differentiation state of osteoblast under different settings were measured by means of cell counting kit and analysis of alkaline phosphatase (ALP) activity as well as formation of mineral nodules. Real-time PCR was utilized to test expression of ALP and Runt-related transcription factor 2 (Runx2) at gene level. The gene expression of TGFβ signaling effectors was also investigated, such as TGFβ receptors (TβRs), smad3, and mitogen-activated protein kinases (MAPK). Results demonstrated that fluoride treatment exhibited action on osteoblast viability and osteogenic differentiation and upregulated expression of TβR2, smad3, and MAPK in this process. Administration of TGFβ strengthened ALP activity but attenuated formation of mineral nodules. Co-treatment of TGFβ and low-dose fluoride increased ALP activity compared to same dose of single fluoride treatment, whereas it inhibited mineral nodule formation. Administration of TGFβ reversed the suppression of high-dose fluoride on osteogenic differentiation of BMSCs. Taken together, studies revealed that TβR2 acted as a target for fluoride and TGFβ treatment on BMSCs, and smad3 and MAPK were involved in the mechanism of fluoride regulating osteogenic differentiation. Together, our data indicated that TGFβ receptor-mediated signaling through smad3 and MAPK was required for modulation of fluoride on osteoblast viability and differentiation, and activating TβR2-smad3 signaling pathway reversed suppression of osteoblasts differentiation by high dose of fluoride treatment.