Proliferation Of Bone Marrow Mesenchymal Stem Cells Research Articles

MiR-29b Modulates Bone Marrow Mesenchymal Stem Cells (BMSCs) Differentiation and Induces Nerve Repair in Diabetic Retina Rat Model

microRNAs are involved in diabetic retinopathy (DR). This study intends to analyze miR-29b’s role in bone marrow mesenchymal stem cells (BMSCs) differentiation in DR rat models to induce nerve repair. BMSCs from DR rat models were cultured and transfected with miR-29b mimics and inhibitors followed by measuring miR-29b level, cell proliferation and apoptosis. Retinal ganglion cells (RGC) were treated with high glucose for 24 h, and BMSCs and si-miR-29b-BMSC were cocultured for 24 h, respectively followed by assessing cell proliferation and apoptosis, inflammatory cytokines by ELISA, MDA, SOD, brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) level by ELISA. MiR-29b was up-regulated in BMSCs of DR rats. miR-29b mimics significantly up-regulated miR-29b, inhibited cell proliferation and promoted apoptosis (P < 0.05), which were reversed by miR-29b inhibitor (P < 0.05). Co-culture of BMSCs with si-miR-29b-BMSC promoted RGC proliferation, inhibited apoptosis and IL-6 secretion, decreased MDA, increased SOD, BDNF and CNTF expression (P < 0.05) with more significant changes in si-miR-29b-BMSC group (P < 0.05). In conclusion, down-regulation of miR-29b promotes BMSCs proliferation in DR rat models, inhibits BMSCs apoptosis, and promotes the recovery of retinal ganglion cell function.

Taohong Siwu decoction promotes the process of fracture healing by activating the VEGF-FAK signal pathway and systemically regulating the gut microbiota.

This study aimed to explore the effect of Taohong Siwu Decoction (THSWD) on bone marrow mesenchymal stem cells (BMSCs) at the cellular level and the possible mechanism of systemic regulation of gut microbiota on fracture recovery. Cell Counting Kit-8 (CCK-8) experiments show that THSWD effectively promotes the proliferation of BMSCs. Transwell and wound healing assays show that THSWD effectively promotes the invasion and migration of BMSCs. Alizarin red staining showed that the THSWD model enhanced the osteogenic differentiation of BMSCs. Moreover, the effect of THSWD on BMSCs is time- and concentration-dependent. RT-qPCR and western blot results showed that THSWD treatment up-regulated the expression of vascular endothelial growth factor (VEGF) and focal adhesion kinase (FAK) at mRNA and protein levels, respectively. Haematoxylin-eosin and crocin O-quick green staining showed that after 14 days of THSWD treatment, the area of callus and cartilage regeneration at the fracture site increased significantly in rats with right femoral shaft fractures. Gut microbiota was changed in fractured rats, such as the abundance of Bacteroidetes and Firmicutes was increased. THSWD showed positive regulation of both to a certain extent. THSWD up-regulates VEGF and activates the FAK signalling pathway to enhance the development and differentiation of BMSCs, and systematically regulates the gut microbiota to promote fracture healing. This study provides new insights on the cellular and systemic level to understand the mechanism of THSWD in the treatment of fractures.

Enhanced Biotribological and Anticorrosion Properties and Bioactivity of Ti6Al4V Alloys with Laser Texturing.

The poor biotribological properties and bioinertness of Ti6Al4V have restricted its application in biomedical materials. In this study, microgrooves of different widths were prepared on the surface of a Ti6Al4V alloy by laser treatment. The tribological properties under dry lubrication and simulated body fluid (SBF) lubrication conditions, the electrochemical corrosion properties in SBF solution, and the bone marrow mesenchymal stem cell (BMSC) behavior on the surfaces were systematically tested. The corresponding mechanisms were discussed. The results showed that Ti6Al4V with a microgroove width of 45 μm (Ti64-45) exhibited excellent wear resistance with decreasing wear rates of 89.79 and 85.43% under dry friction and SBF lubrication compared to the Ti64 sample, which might be due to the increase of surface microhardness. Moreover, the excellent anticorrosion performance of Ti64-45 was attributed to the grain refinement on the titanium alloy surface with a lower volume fraction ratio of β phase to α phase. In addition, the microgrooves with a width of 45 μm are more conducive to BMSC proliferation and adhesion, related to promoting cell signal transduction due to cell extrusion. These studies imply that the microgroove structures are potential for application in the medical field.

Sodium Butyrate Pre-Treatment Enhance Differentiation of Bone Marrow Mesenchymal Stem Cells (BM-MSCs) into Hepatocytes and Improve Liver Injury.

The treatment of liver failure by stem cell transplantation has attracted growing interest. Herein, we aim to explore the role of sodium butyrate (NaB) in the hepatic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) under liver-specific factors induction in vitro and vivo. We isolated BM-MSCs from the mononuclear cell fraction of rabbit bone marrow samples and identified the cells by Immunophenotypic analysis. We investigated the effects of different concentrations and induction conditions. The histone deacetylase inhibitor NaB induced hepatic differentiation of BM-MSCs under liverspecific factors induction in vitro. Morphological features, liver-specific gene and protein expression, and functional analyses in vitro and vivo were performed to evaluate the hepatic differentiation of BM-MSCs. Our results showed that pre-treated NaB inhibited the expression of the liverspecific protein in a dose-dependent manner. The induction efficiency of NaB with 24h pre-treatment was higher than that of NaB continuous intervention. 0.5 mM 24h NaB pre-treated cells can improve liver tissue damage in vivo. The liver ALB, AAT, and the serum TP were significantly increased, while the serum ALT was significantly reduced. Continuous NaB treatment can inhibit BM-MSCs proliferation in a dosedependent manner at a certain concentration range. 0.5 mM 24h pre-treatment of NaB enhanced differentiation of BM-MSCs into hepatocytes and improved liver injury in vitro and vivo.

The design for autologous bone marrow mesenchymal stem cells combined with nano-graphene material in the treatment of neuropathic pain model mice

Stem cell therapy provides a new way for central nervous system function reconstruction and nerve regeneration.In this study, the effect of mobilizing autologous bone marrow mesenchymal stem cells (BMSCs) on neural repair in an animal model of spinal cord injury (SCI).In this study, using a more convenient and efficient in vivo automatic homing method, combining nanomaterials may have integrated topographic and electrical effects on neural stem cell migration and differentiation.The effect and related mechanism of graphene nanomaterials combined with autologous BMSC transplantation in the treatment of neuropathic pain caused by spinal cord injury. Our study show Prove that the BMSCs successfully colonize and secrete neurotrophic factors. In the golden period of early repair of nerve injury, the use of a newgraphene to guide the proliferation and differentiation of autologous BMSC to achieve the continuation of nerve regeneration, may complete the function and accelerate the recovery and reduce the occurrence of neuropathic pain.

Experimental study on co-culture of DiI labeled rat bone marrow mesenchymal stem cells and polycaprolactone film in vitro to make a cell patch.

In stem cell therapy, due to the lack of an effective carrier, a large number of transplanted stem cells are lost and die. Therefore, finding a suitable carrier has become a further direction of stem cell therapy. In research on the co-culture of polycaprolactone (PCL) with 1,1'-Dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (DiI) labeled bone marrow mesenchymal stem cells (BMSCs), we observe the effect of materials on the growth and proliferation of DiI labeled stem cells, and the effect of DiI labeling on patch preparation, so as to find a kind of biomaterial suitable for the growth and proliferation of BMSCs, and find a suitable cell carrier for stem cell therapy of myocardial infarction and in vivo tracing. Clean grade Sprague Dawley rats were selected as experimental objects, BMSCs were isolated and cultured, and the surface markers were identified by flow cytometry. After the BMSCs were cultured for 3 passages, the BMSCs were stained with DiI dye, and the BMSCs DiI and PCL biomaterial film were co-cultured. After 24 hours, the cell growth was observed under fluorescence microscope, and fixed for scanning under electron microscope. The cell proliferation was detected by CCK-8 at 1, 4, 7, 10 days of culture. The measurement data conforming to normal distribution are expressed in the form of mean ± standard deviation (X¯±s). One way ANOVA was used for comparison among groups, LSD analysis was used for pairwise comparison. The difference was statistically significant (P <0.05). BMSCs were strongly positive for CD90, CD44H, but negative for CD11b/c, CD45. Under fluorescence microscope, BMSCs DiI showed red light, fusiform or polygonal. Under the scanning electron microscope, the cell patch formed by co-culture of PCL film and DiI-BMSCs had a large number of cells on the surface and normal cell state. CCK-8 assay showed that the OD value on the first day was 0.330 ± 0.025; The OD value was 0.620 ± 0.012 on the 4th day, 1.033 ± 0.144 on the 7th day and 1.223 ± 0.133 on the 10th day. There was significant difference among the time points (P <0.05). The cell patch made of PCL film and DiI labeled BMSCs can survive and proliferate on the surface, so it can be used as a scaffold material for stem cell therapy in vivo.

Effects of pre-osteogenic differentiation on the bone regeneration potentiality of marrow mesenchymal stem cells/poly(ethylene glycol)-diacrylate hydrogel using a rat cranial defect model

Transplanting cell/hydrogel constructs into a bone defect site is an effective strategy to repair the damaged tissues. However, before transplantation, there are various methods to culture cell/hydrogel constructs. Especially, the preferred pre-osteogenic differentiation period to achieve satisfied bone regeneration should be determined. To this end, Bone marrow mesenchymal stem cells (BMSCs) were firstly photo-encapsulated into poly(ethylene glycol)-diacrylate (PEGDA) hydrogel. Then the constructs were implanted in rat calvarial defects after being osteogenically induced for 0, 7, 14, and 21days. In vitro experiments demonstrated that the proliferation of BMSCs in the hydrogels deceased significantly from 0day to 7days. The activity and the gene expression of alkaline phosphatase, besides the gene expression of bone morphogenetic protein-2 peaked at day 14, whereas the gene expression of osteocalcin and the formation of calcium nodules increased with the prolongation of differentiation time. In vivo results showed that limited areas of newly formed bone were found in the day0 and day21 groups. In the day7 group, obvious new bone with bone marrow space was found, while the day14 group nearly achieved complete bone healing. Our data suggested that the period of in vitro pre-osteogenic differentiation played a crucial role for the osteogenesis of BMSCs/PEGDA hydrogels. Furthermore, we found that a pre-differentiation for 14days is preferable for bone regeneration in the rat cranial defects.

Bone Marrow Mesenchymal Stem Cells (BMSCs) Expressing Netrin-1 Alleviates Spinal Cord Injury (SCI)-Induced Inflammation

Spinal cord injury (SCI) is a common central nervous system (CNS) injury. Bone marrow mesenchymal stem cells (BMSCs) transplantation is a potential treatment for traumatic SCI. However, the role and mechanism of BMSCs with high expression of Netrin-1 on the repair and inflammation of spinal cord injury cells remains unclear. Our study intends to assess the effect of BMSCs with high Netrin-1 level on the repair of SCI cells. BMSCs or Netrin-1 transfected BMSCs were co-cultured with mechanically injured nerve cells followed by analysis of the differentiation of BMSCs by light microscope, apoptosis activity, expression of TLR-4 and NF-κB, and the TNF-α and IL-1β content in cell supernatant by ELISA. BMSCs with high Netrin-1 expression promoted the proliferation of BMSCs, inhibited apoptosis, and promoted the differentiation of nerve cells along with increased ALK activity, and the expression of GFAP and BDNF. Co-culture with BMSCs or BMSCs with high Netrin-1 expression increased mechanically damaged nerve cell proliferation, decreased apoptosis, downregulated TLR-4 and NF-κB (P &lt; 0.05) with more significant changes after co-culture with BMSCs with high Netrin-1 expression. In conclusion, Netrin-1 can promote BMSCs proliferation and differentiation, and inhibit apoptosis. By inhibiting inflammation, it can promote damaged nerve cell proliferation and repair.

EMF promote BMSCs differentiation and functional recovery in hemiparkinsonian rats

Bone marrow mesenchymal stem cells (BMSCs) have self-renewal ability while maintaining the proliferation facility. The BMSCs reproducing ability could affect by electromagnetic fields (EMFs) as a physical inducing factor. We focused on the EMF (400 µT, 75 Hz) exposed multi-potential BMSCs which differentiated and successfully implanted in the substantia nigra pars compacta (SNpc) of Parkinson's disease rat model. The purified BMSCs are exposed to sinusoidal and square waveform EMF (1 h/1 week or 7 h/1 day) then injected into the left SNpc of Parkinson’s rats. To evaluate the morphology of EMF exposed BMSCs, the cresyl violet staining labeled the Nissl bodies. After evaluation of the rat's activity by behavioral tests (open-field and rotarod tests), the brains were obtained for the preparation of SNpc blocks and carry out the cresyl violet staining. Cell morphology proved most cell differentiation to neurons in the sinusoidal EMF groups. In the sinusoidal EMF exposure groups, large and small neurons were seen with apparent synapses. Although in the square EMF exposed groups some neurons were seen, most of the differentiated cells were astrocytes, microglia, and oligodendrocyte. The results confirmed an improvement in locomotors' activity of BMSC alone and sinusoidal EMF exposed groups. We presented a low-frequency EMF (75 Hz) to promote the capability of BMSC proliferation, differentiation to neurons and glial cells, and motor coordination activity in the treatment of hemiparkinsonian rats.

Matrix-bound Cyr61/CCN1 is required to retain the properties of the bone marrow mesenchymal stem cell niche but is depleted with aging

Previously, we showed that extracellular matrices (ECMs), produced ex vivo by various types of stromal cells, direct bone marrow mesenchymal stem cells (BM-MSCs) in a tissue-specific manner and recapitulate physiologic changes characteristic of the aging microenvironment. In particular, BM-MSCs obtained from elderly donors and cultured on ECM produced by young BM stromal cells showed improved quantity, quality and osteogenic differentiation. In the present study, we searched for matrix components that are required for a functional BM-MSC niche by comparing ECMs produced by BM stromal cells from “young” (≤25 y/o) versus “elderly” (≥60 y/o) donors. With increasing donor age, ECM fibrillar organization and mechanical integrity deteriorated, along with the ability to promote BM-MSC proliferation and responsiveness to growth factors. Proteomic analyses revealed that the matricellular protein, Cyr61/CCN1, was present in young, but undetectable in elderly, BM-ECM. To assess the role of Cyr61 in the BM-MSC niche, we used genetic methods to down-regulate the incorporation of Cyr61 during production of young ECM and up-regulate its incorporation in elderly ECM. The results showed that Cyr61-depleted young ECM lost the ability to promote BM-MSC proliferation and growth factor responsiveness. However, up-regulating the incorporation of Cyr61 during synthesis of elderly ECM restored its ability to support BM-MSC responsiveness to osteogenic factors such as BMP-2 and IGF-1. We next examined aging bone and compared bone mineral density and Cyr61 content of L4-L5 vertebral bodies in “young” (9–11 m/o) and “elderly” (21–33 m/o) mice. Our analyses showed that low bone mineral density was associated with decreased amounts of Cyr61 in osseous tissue of elderly versus young mice. Our results strongly demonstrate a novel role for ECM-bound Cyr61 in the BM-MSC niche, where it is responsible for retention of BM-MSC proliferation and growth factor responsiveness, while depletion of Cyr61 from the BM niche contributes to an aging-related dysregulation of BM-MSCs. Our results also suggest new potential therapeutic targets for treating age-related bone loss by restoring specific ECM components to the stem cell niche.

Icariin protects bone marrow mesenchymal stem cells in aplastic anemia by targeting MAPK pathway.

Icariin, the main pharmacological active flavonoid extracted from Epimedi herba, can regulate cellular processes in diverse diseases. The aim of this study was to explore the effects and mechanisms of icariin on proliferation and adipogenesis of bone marrow mesenchymal stem cells (BMSCs) in aplastic anemia (AA). Bone marrow mesenchymal stem cells were isolated from posterior tibias and femurs of AA rats that were induced by benzene and cyclophosphamide and gavaged with icariin. The isolated BMSCs were characterized morphologically and immunologically by positive markers (CD29 and CD90) and negative markers (CD34 and CD45). CCK-8 assay was performed to examine the BMSCs proliferation. Cell apoptosis and cell cycle were detected by flow cytometry. Oil red O staining was carried out to evaluate the adipogenesis of BMSCs. The mRNA expression of PPARγ, C/EBP-α, and FABP4 was measured by qRT-PCR. The protein levels of p-p38/p38, p-JNK/JNK, p-ERK/ERK, PPARγ, C/EBP-α, and FABP4 were detected using Western blotting. Icariin promoted the proliferation of BMSCs from AA rats in a dose-dependent manner. The protein levels of p-p38/p38, p-JNK/JNK, and p-ERK/ERK were downregulated in BMSCs from AA rats after icariin treatment. Icariin inhibited the apoptosis and arrested cell cycle at G/S phase of BMSCs from AA rats. The adipogenesis of BMSCs from AA rats was also suppressed after icariin treatment. However, the effects of icariin on BMSCs were weakened by p38 agonist addition. Icariin promoted the proliferation and inhibited the apoptosis and adipogenesis of BMSCs in AA by suppressing MAPK pathway.

Chondrogenic differentiation of rat bone marrow mesenchymal stem cells induced by puerarin and tetrandrine

Abstract Objective: This study aims to clarify the effect of the active components puerarin and tetrandrine on the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Methods: Using network pharmacology, protein targets of puerarin and tetrandrine were predicted, and a database of cartilage formation targets was established. The protein target information related to disease was then collected, and the drug-targeting network was constructed by analyzing the protein–protein interactions. Genes related to chondrogenesis induced by puerarin and tetrandrine and chondroblast differentiation signaling pathways were searched. Finally, potential drug- and disease-related genes, as well as proteins, were screened and verified using real-time RT-PCR and western blotting. Results: Network pharmacological studies have shown that puerarin and tetrandrine are involved in BMSCs cartilage differentiation. The experimental results showed that puerarin and tetrandrine could regulate the expression of cartilage differentiation-related genes and proteins. Puerarin increased the protein expression of COL2A1, COL10A1, MMP13, and SOX-9, as well as the gene expression of Col2a1, Mmp13, Tgfb1, and Sox-9. Tetrandrine increased the protein expression of COL2A1, COL10A1, MMP13, and SOX-9, as well as the gene expression of Col10a1, Tgfb1, Sox-9, and Acan. The combination of puerarin and tetrandrine increased the protein expression of COL2A1, COL10A1, MMP13, and SOX-9 and the gene expression of Col2a1, Col10a1, Sox-9, and Acan. Conclusions: Puerarin, tetrandrine, and their combination can promote the proliferation of BMSCs and induce their differentiation into chondrocytes, and they are thus expected to be inducers of chondrogenic differentiation. These results suggest that puerarin and tetrandrine have potential therapeutic effects on osteoarthritis.

Coordination of Osteoblastogenesis and Osteoclastogenesis by the Bone Marrow Mesenchymal Stem Cell-Derived Extracellular Matrix To Promote Bone Regeneration.

Extracellular matrix (ECM)-based therapies have been developed to improve bone repair because of their abundance of bioactive components. Besides the osteogenic promotion and the immune response, the potential effect of the ECM on the coordination between osteoblastogenesis and osteoclastogenesis in vivo should also deserve great attention because both osteoblasts and osteoclasts get involved in bone regeneration and are critical for the final repair outcome. Herein, based on our previous study on decellularization, antigen removal, and growth factor retention, porous poly (lactic-co-glycolic acid) (PLGA) scaffolds decorated with the bone marrow mesenchymal stem cell (BMSC)-derived ECM were prepared, and the functions of the ECM on BMSC osteogenic differentiation and osteoclastogenesis in vitro were preferentially investigated. Afterward, bone regeneration and osteoclast formation in vivo induced by ECM-decorated PLGA scaffolds were further studied. The in vitro tests revealed that ECM-decorated PLGA scaffolds obviously facilitated BMSC proliferation and osteogenic differentiation. However, when osteoclast precursors were cultured on the BMSC-derived ECM, the number and size of osteoclasts, expression of cathepsin K and matrix metalloproteinase 9, and tartrate-resistant acid phosphatase activity were notably decreased, accompanied by the reduction in the reactive oxygen species (ROS) level. Interestingly, the addition of exogenous hydrogen peroxide elevated the osteoclast amount on the ECM and up-regulated the resorption-related enzyme levels, implying that the repressive effect of the BMSC-derived ECM on osteoclasts may be related to the intracellular ROS. After implantation into calvarial defects, the ECM-decorated PLGA scaffolds significantly increased bone volume and bone mineral density compared with bare PLGA scaffolds and did not stimulate the overmuch formation of osteoclasts in vivo. This study evidenced that the BMSC-derived ECM may coordinate osteoblastogenesis and osteoclastogenesis and promote favorable bone formation without stimulating bone resorption.

Krüppel-like factor 4 promotes the proliferation and osteogenic differentiation of BMSCs through SOX2/IGF2 pathway.

Human bone marrow mesenchymal stem cells (BMSCs) have multi-lineage differentiation potential and have been widely researched in regenerative medicine. The purpose of this research was to explore whether Krüppel-like factor 4 (KLF4) can regulate the osteogenic differentiation of BMSCs. We transfected human BMSCs with KLF4 overexpression plasmid and si-KLF4 to study the effects of KLF4. We performed cell proliferation assay, flow cytometry and Alizarin Red staining on BMSCs. Quantitative real-time PCR and western blot was performed to determined mRNA and protein expression of osteogenic differentiation markers, KLF4, SOX2 and IGF2. Bone defect animal model was created and the adenovirus containing KLF4 overexpression or knockdown plasmid was injected. Finally, HE staining was performed on tibia to assess the new bone formation. Our results showed that KLF4 promotes not only the growth of BMSCs, but also their osteogenic differentiation. Also, it mediated these effects through SOX2/IGF2 signaling pathway. In addition, KLF4 overexpression could increase the bone regeneration in in-vivo model, whereas KLF4 knockdown decreased the bone regeneration. KLF4 regulates BMSC's osteogenic differentiation via SOX2/IGF2 pathway.

Zinc and hypoxic preconditioning: a strategy to enhance the functionality and therapeutic potential of bone marrow-derived mesenchymal stem cells.

The therapeutic use of bone marrow mesenchymal stem cells (BM-MSCs) requires a large number of cells (1-100 × 106 cells/kg of body weight). Extensive in vitro growth is limited due to the aging of cultured BM-MSCs which leads to abnormal morphology and senescence. Hypoxia increases BM-MSC proliferation, but the question of whether hypoxia preconditioning is safe for clinical application of BM-MSCs remains to be answered. Zinc is essential for cell proliferation and differentiation, especially for the regulation of DNA synthesis and mitosis. It is a structural constituent of numerous proteins on a molecular level, including transcription factors and enzymes of cellular signaling machinery. All the tissues, fluids, and organs of the human body contain zinc. More than 95% of zinc is intracellular, of which 44% is involved in the transcription of DNA. We investigated the effects of ZnCl2 on proliferation, morphology, migration, population doubling time (PDT), and gene expression of BM-MSCs under hypoxic (1% O2) and normoxic (21% O2) environments. BM-MSCs were preconditioned with optimized concentrations of ZnCl2 under normoxic and hypoxic environments and further examined for morphology by the phase-contrast inverted microscope, cell proliferation by MTT assay, PDT, cell migration ability, and gene expression analysis. Zinc significantly enhanced the proliferation of BM-MSCs, and it decreases PDT under hypoxic and normoxic environments as compared to control cells. Migration of BM-MSCs toward the site of injury increased and expression of HIF1-α significantly decreased under hypoxic conditions as compared to non-treated hypoxic cells and control. At late passages (P9), the morphology of normoxic BM-MSCs was transformed into large, wide, and flat cells, and they became polygonal and lost their communication with other cells. Conversely, zinc-preconditioned BM-MSCs retained their spindle-shaped, fibroblast-like morphology at P9. The expression of proliferative genes was found significantly upregulated, while downregulation of genes OCT4 and CCNA2 was observed in zinc-treated BM-MSCs under both normoxic and hypoxic conditions. ZnCl2 treatment can be used for extensive expansion of BM-MSCs in aged populations to obtain a large number of cells required for systemic administration to produce therapeutic efficacy.

Effect of miR-144-3p-Targeted Regulation of PTEN on Proliferation, Apoptosis, and Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells under Stretch.

Objective To investigate the effects of miR-144-3p-targeted regulation of phosphatase and tensin homolog deleted on chromosome ten (PTEN) gene on proliferation, apoptosis, and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) under retraction force. Methods The BMSCs of rats were randomly divided into the tension MSC group with detrusor stimulation and the MSC group without detrusor stimulation, after which osteogenic differentiation of BMSCs was induced in both groups. Alkaline phosphatase (ALP) staining and alizarin red staining were used to detect the osteogenic differentiation ability of the two groups of cells. Real-time quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression of miR-144-3p and PTEN in the two groups of cells after osteogenic differentiation. Bioinformatics website and dual luciferase reporter were used to detect the relationship between miR-144-3p and PTEN. The tension MSC group was used as a control group, and miR-144-3p mimics (miR-144-3p mimic group), mimic controls (mimic-NC group), PTEN interferers (si-PTEN group), and interference controls (si-NC group) were transfected into BMSCs. The BMSCs were then continuously stimulated for 24 h using a Flexercell in vitro cellular mechanics loading device, applying a draft force at a frequency of 1 Hz and a deformation rate of 18%. The cell proliferation was detected by Cell Counting Kit-8 (CCK-8) colorimetric assay; the expression levels of cyclin, cyclin-dependent kinases (CDK), BCL2-associated X (BAX), B-cell lymphoma-2 (BCL-2), and other cell cycle and apoptosis related proteins were detected by western blot (WB); and the osteogenic differentiation ability of MSC cells was detected by ALP staining and alizarin red staining. Results Compared with the MSC group, the level of miR-144-3p was significantly lower and the level of PTEN was significantly higher in the tension MSC group. ALP staining showed normal activity in the MSC group and decreased ALP activity in the tension MSC group compared to the MSC group. Alizarin red staining in the MSC group showed scattered calcium nodule formation, and alizarin red staining showed red nodules with a more uniform color distribution. Compared to the MSC group, the tension MSC group showed fewer, smaller, and lighter staining mineralized nodules. Compared with the tension group and mimic-NC group (si-NC group), the proliferation rate of cells in the miR-144-3p mimic group (si-PTEN group) was significantly higher; the expression levels of PTEN and BAX were significantly lower; and the expression levels of cyclin, CDK, and BCL-2 protein were significantly higher. ALP staining results revealed that the miR-144-3p mimic group (si-PTEN group) showed significantly higher osteogenic differentiation ability and ALP activity of MSC than the tension group and mimic-NC group (si-NC group). Conclusion miR-144-3p may inhibit apoptosis and promote proliferation and osteogenic differentiation of BMSCs under tension by targeting and regulating PTEN.

Teriparatide Promotes Bone Marrow Mesenchymal Stem Cells (BMSCs) Proliferation and Differentiation via Down-Regulating miR-298

This study explored whether teriparatide promotes BMSCs proliferation and differentiation via downregulating miR-298 and provided a basis for bone repair. Based on the microarray analysis after teriparatide treatment, qRT-PCR verified the differentially expressed miRNAs and the osteogenic differentiation was assessed by transfection of miRNA overexpression plasmids and miRNA inhibitors. miRNA array analysis and qRT-PCR verification showed that miR-298 was significantly downregulated during teriparatide-induced BMSCs differentiation. miR-298 overexpression significantly inhibited ALP and OPN expression which was promoted by transfection of miR-298 inhibitor. miR-298 is a negative regulator of BMSCs differentiation induced by teriparatide. Dlx5 is the target of miR-298. Inhibition of DLX5 expression by miR-298 was involved in the osteogenic differentiation of BMSCs. In conclusion, miR-298 negatively regulates the differentiation of BMSCs induced by teriparatide by targeting DLX5, providing a possible therapeutic target for bone tissue repair and regeneration.

Effects of Low-Concentration Graphene Oxide Quantum Dots on Improving the Proliferation and Differentiation Ability of Bone Marrow Mesenchymal Stem Cells through the Wnt/β-Catenin Signaling Pathway.

Graphene oxide quantum dots (GOQDs) are considered to be a new method for regulating the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). However, there are few reports on such regulation with different concentrations of GOQDs, and the molecular mechanism has not been fully elucidated. The purposes of this study were, first, to explore the effects of GOQDs on the proliferation and differentiation of BMSCs in vitro and in vivo, and, second, to provide a theoretical basis for the repair of bone defects. Live/Dead staining, EdU staining, immunofluorescence staining, alkaline phosphatase (ALP), western blotting, and qT-PCR were used for detecting the proliferation and differentiation of BMSCs after coculture with GOQDs of different concentrations. Hematoxylin and eosin (HE) staining and Van Gieson (VG) staining were used to detect new bone regeneration in vivo. The results showed that low-concentration GOQDs (0.1 and 1 μg/mL) promoted the proliferation and differentiation of BMSCs. Compared with the 1 μg/mL GOQD group, the 0.1 μg/mL GOQD group had better ability to promote the proliferation and differentiation of BMSCs. HE and VG staining results showed the greatest proportion of new bone area on sandblasted, large-grit, and acid-etched (SLA)/GOQD scaffolds. Furthermore, the ratio of active β-catenin and the phosphorylation level of GSK-3β (p-GSK-3β) increased after BMSCs treatment with 0.1 μg/mL GOQDs. Low concentrations of GOQDs improved the osteogenic differentiation ability of BMSCs by activating the Wnt/β-catenin signaling pathway.

Effect of Olibanum Extract/Graphene Oxide on Differentiation of Bone Marrow Mesenchymal Stem Cells into Neuron-Like Cells on Freeze Dried Scaffolds.

One of the challenges in using stem cells to neural repair is to induce their differentiation into neurons and lack of glial formation. Mesenchymal stem cells have revealed great potential for neural reorganization and renewal by taking advantage of differentiation capabilities. Here we explored the potential use of olibanum extract in freeze-dried scaffolds for induction of stem cells differentiation. In this study, gelatin/ collagen/olibanum/ graphene oxide (GEL/COL/OL/GO) freeze-dried scaffolds were synthesized and then adult rat bone marrow mesenchymal stem cells (BMMSCs) were seeded on scaffolds. The viability of cells was evaluated using MTT test on days 1, 3 and 5. The morphology of the cells seeded on scaffolds was studied using SEM and specific protein expression detected by immunohistochemical analysis. Real-time PCR was applied to detect the expression of Chat, Pax6, Hb-9, Nestin, Islet-1, and neurofilament-H (NF-H). The data were analyzed using Tukey test and one-way ANOVA and the means difference was considered significant atP<0.05, P<0.01, and P<0.001. Showed that the pore size is increased in GEL/COL/OL/GO scaffolds compared with GO-free scaffolds and higher attachment and proliferation of BMMSCs on GEL/COL/OL /1.5% GO scaffolds compared to GEL/COL/OL/3% GO scaffolds. The cell viability results after 5 days of incubation showed the significant biocompatibility of GEL/COL/OL /1.5% GO freeze-dried scaffold. The results of immunohistochemicaland PCR analysis revealed positive role of GEL/COL/OL/1.5% GO scaffolds in upregulation of neuron-specific markers. These results reveal the great potential of GEL/COL/OL/GO scaffolds for nerve regeneration. Our data suggested that both OL extract and GO can regulate the MSCs differentiation into neurons.

Enamel matrix derivative expedites osteogenic differentiation of BMSCs via Wnt/β-catenin pathway in high glucose microenvironment.

The influence of enamel matrix derivative (EMD) on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) was explored in high glucose (HG) microenvironment with interaction of Wnt/β-catenin pathway. Extraction of BMSCs from Sprague-Dawley rats, culture, and identification were manifested. The cells were treated with different concentration of EMD in HG to figure out the most available concentration for proliferation and osteogenic differentiation. Then, observation of cell growth curve and cell cycle changes, and detection of Osterix, runt-related transcription factor 2 (Runx2), COL-I, early osteogenic indexes, Calcium salt deposition, and β-catenin protein in Wnt/β-catenin pathway were assured. After adding Wnt/β-catenin pathway inhibitor (XAV-939) in the cells with osteogenesis induction, detection of binding of β-catenin to Osterix was clarified. Via identification BMSCs cultured in vitro was qualified. Different concentrations of EMD could accelerate cell proliferation in HG and osteogenesis induction, and 75μg/mL EMD had the best effect. The HG augmented BMSCs proliferation and the propidium iodide index of flow cytometry cycle was elevated in HG, which were strengthened via the EMD. After BMSCs' osteogenesis induction, Osterix, Runx2, CoL-1, early osteogenic indexes, and calcium salt deposition were reduced, but elevated via EMD. β-Catenin was the lowest in the HG, but elevated after EMD. After addition of XAV-939, reduction of β-catenin and the downstream (Osterix and Runx2) were manifested. Detection of binding protein bands was in β-catenin and Osterix of the HG after EMD treatment. EMD may facilitate the osteogenic differentiation of BMSCs via activating the Wnt/β-catenin pathway in HG.