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Background/Aims: Osteoarthritis (OA) is characterized by degradation of cartilage, sole cell type of which is chondrocytes. Bone marrow-derived mesenchymal stem cells (BMSCs) possess multipotency and can be directionally differentiated into chondrocytes under stimulation. This study was aimed to explore the possible roles of vitamin D and transforming growth factor-β1 (TGF-β1) in the chondrogenic differentiation of BMSCs. Methods: BMSCs were isolated from femurs and tibias of rats and characterized by flow cytometry. After stimulation with vitamin D, BMSC proliferation and migration were measured by Cell Counting Kit-8 (CCK-8) and Transwell assays, respectively. Chondrogenic differentiation was estimated through expression levels of specific markers by qRT-PCR and Western blot analysis. After stable transfection, the effects of aberrantly expressed TGF-β1 on vitamin D-induced alterations, including BMSC viability, migration and chondrogenic differentiation, were all evaluated utilizing CCK-8 assay, Transwell assay, qRT-PCR and Western blot analysis. Finally, the phosphorylation levels of key kinases in the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways were determined by Western blot analysis. Results: Vitamin D remarkably promoted BMSC viability, migration and chondrogenic differentiation. These alterations of BMSCs induced by vitamin D were reinforced by TGF-β1 overexpression while were reversed by TGF-β1 silencing. Additionally, the phosphorylation levels of ERK, JNK and c-Jun were enhanced by TGF-β1 overexpression but were reduced by TGF-β1 knockdown. Conclusion: Vitamin D promoted BMSC proliferation, migration and chondrogenic differentiation. TGF-β1 might be implicated in the vitamin D-induced alterations of BMSCs through regulating ERK/JNK pathway.

Background: Osteoporosis is a common endocrine metabolic bone disease, which may lead to severe consequences. However, the unknown molecular mechanism of osteoporosis, the observable side effects of present treatments and the inability to fundamentally improve bone metabolism seriously restrict the impact of prevention and treatment. The study aims to identify potential biomarkers from osteoclast progenitors, specifically peripheral blood monocytes on predicting the osteoporotic phenotype., Methods: Datasets were obtained from Gene Expression Omnibus (GEO). Based on the differentially expressed genes (DEGs) and GSEA results, GO and KEGG analyses were performed using the DAVID database and Metascape database. PPI network, TF network, drug-gene interaction network, and ceRNA network were established to determine the hub genes. Its osteogenesis, migration, and proliferation abilities in bone marrow mesenchymal stem cells (BMSCs) were validated through RT-qPCR, WB, ALP staining, VK staining, wound healing assay, transwell assay, and CCK-8 assay., Results: A total of 63 significant DEGs were screened. Functional and pathway enrichment analysis discovered that the functions of the significant DEGs (SDEGs) are mainly related to immunity and metal ions. A comprehensive evaluation of all the network analyses, PMAIP1 was defined as osteoporosis's core gene. This conclusion was further confirmed in clinical cohort data. A series of experiments demonstrated that the PMAIP1 gene can promote the osteogenesis, migration and proliferation of BMSC cells., Conclusions: All of these outcomes showed a new theoretical basis for further research in the treatment of osteoporosis, and PMAIP1 was identified as a potential biomarker for osteoporosis diagnosis and treatment.

Osteoarthritis (OA) is a chronic disease characterized by the progressive loss of cartilage and failure of the diarrheal joint. Quercetin has been reported to attenuate the development of OA. Bone marrow derived mesenchymal stem cell (BMSC)-derived exosomes are involved in OA progression. However, the role of BMSC-derived exosomes in quercetin-mediated progression of OA remains unclear. Western blotting and RT-qPCR were used to assess protein and mRNA levels, respectively. CCK8 assay was performed to assess cell viability, and cell apoptosis was assessed using flow cytometry. A dual-luciferase assay was performed to assess the relationship between miR-124-3p and TRAF6 expression. Furthermore, in vivo experiments were performed to test the function of exosomes derived from Quercetin-treated BMSCs in OA patients. IL-1β significantly inhibited the viability of chondrocytes, whereas the conditioned medium of Quercetin-treated BMSCs (BMSCs QUE -CM) reversed this phenomenon through exosomes. IL-1β notably upregulated MMP13 and ADAMT5 and reduced the expression of COL2A1 in chondrocytes, which were rescued by BMSCs QUE -CM. The effects of BMSCs QUE -CM on these three proteins were reversed in the absence of exosomes. Exosomes can be transferred from BMSCs to chondrocytes, and exosomes derived from Quercetin-treated BMSCs (BMSCs Que -Exo) can reverse the apoptotic effects of IL-1β on chondrocytes. The level of miR-124-3p in BMSCs was significantly upregulated by quercetin, and miR-124-3p was enriched in BMSCs Que -Exo. TRAF6 was identified as a direct target of miR-124-3p, and BMSCs Que -Exo abolished the IL-1β-induced activation of MAPK/p38 and NF-κB signaling. Furthermore, BMSCs Que -Exo significantly attenuated OA progression in vivo . Exosomes derived from Quercetin-treated BMSCs inhibited OA progression through the upregulation of miR-124-3p.

Bone marrow mesenchymal stem cell (BSMC)-derived extracellular vehicles (EVs) have a pivotal therapeutic potential in hepatic fibrosis (HF). Activation of hepatic stellate cells (HSCs) is the key mechanism in HF progression. Downregulation of miR-192-5p was previously observed in activated HSCs. Nonetheless, the functions of BSMC-derived exosomal miR-192-5p in activated HSCs remain unclear. In this study, transforming growth factor (TGF)-β1 was used to activate HSC-T6 cells to mimic HF in vitro. Characterization of BMSCs and BMSC-derived EVs was performed. Cell-counting kit-8 assay, flow cytometry, and western blotting revealed that TGF-β1 increased cell viability, promoted cell cycle progression, and induced upregulation of fibrosis markers in HSC-T6 cells. Overexpression of miR-192-5p or BMSC-derived exosomal miR-192-5p suppressed TGF-β1-triggered HSC-T6 cell activation. RT-qPCR revealed that protein phosphatase 2 regulatory subunit B'' alpha (PPP2R3A) was downregulated in miR-192-5p-overexpressed HSC-T6 cells. Luciferase reporter assay was used for verifying the relation between miR-192-5p and PPP2R3A, which showed that miR-192-5p targeted PPP2R3A in activated HSC-T6 cells. Collectively, BMSC-derived exosomal miR-192-5p targets PPP2R3A and inhibits activation of HSC-T6 cells.

In order to study the fatigue properties of basic Magnesium Sulfate cement reinforced concrete beams, specimens of the basic magnesium sulfate cement (BMSC) and ordinary portland cement (PO·C) were in strength grade of C40 and C50 were prepared, respectively. Response surface methodology is applied to this study. The results show that under the fatigue load, the deflection value of the specimen decreases with the increase of the number of fatigue cycles. The fatigue cycle life of the BMSC specimens with higher strength grades is more than that of the PO·C specimens, and the higher the strength grade, the more obvious. Under the fatigue load, the development of the midspan deflection of the specimen showed an S-shaped trend, and the deflection in the BMSC specimen was smaller than that of the PO·C specimen. The spacing of the cracks in BMSC columns was smaller than that of PO·C columns because of the better bond performance between the reinforcing bars and the surrounding BMS concrete. But the crack widths of BMSC columns were close to the width of PO·C columns. It shows that the BMSC specimen has a high rigidity and good resistance to deformation. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Osteoporosis (OP) has a high incidence and can be found in multiple age groups. The bone marrow mesenchymal stem cells (BMSCs) have the potential for self-renewal and multi-directional differentiation, which are often used for investigating the differentiation function of osteoporosis bone marrow mesenchymal stem cells. γ-glutamyl carboxylase (GGCX) is a carboxylase-related carboxylase and was observed to be abnormally expressed in osteoarthritis. However, the role and related mechanisms of GGCX in OP have not been fully elucidated. This work aimed to evaluate the effect of GGCX on the differentiation function of BMSCs. PATIENTS AND METHODS: Sprague-Dawley rats were randomly divided into the OP group prepared by ovariectomy and sham group. GGCX expression was tested by enzyme-linked immunosorbent assay (ELISA). BMSCs were isolated from OP rats and transfected with pcDNA-GGCX plasmids. BMSC proliferation was detected by tetrazolium salt colorimetry (MTT) assay. The osteogenic and adipogenic differentiation of BMSCs was analyzed by alizarin red staining and oil red O staining. The ALP activity was determined by alkaline phosphatase (ALP) activity colorimetric assay. Real time-PCR was used to test the expressions of osteogenesis-related genes RUNX2 and OPN mRNA. Western blot was adopted to assess the TGFß/smad signaling pathway activity. RESULTS: GGCX expression was significantly decreased in the serum of OP rats compared with the sham group (p < 0.05). The transfection of pcDNA-GGCX plasmid significantly promoted BMSC cell proliferation, increased calcified nodule formation, inhibited adipogenic differentiation, enhanced ALP activity, elevated RUNX2, and OPN mRNA expressions, and upregulated TGFß1, Smad2, and Smad7 expressions (p < 0.05). CONCLUSIONS: GGCX secretion is reduced in osteoporosis. GGCX can regulate osteoporosis via promoting the TGFß/smad signaling pathway, facilitating BMSCs osteogenic differentiation, and inhibiting BMSCs adipogenic differentiation. [ABSTRACT FROM AUTHOR]

Context: Although bone mesenchymal stem cells (BMSCs) have been used for the treatment of oral and maxillofacial defects, the survival rate and limited proliferation reduces the therapeutic efficiency of BMSC. Objective: The aim of our study is to explore the role of miR-31 in regulating survival, proliferation, and migration of BMSC in vitro. Materials and methods: LPS was used in vitro to induce BMSC damage and then miR-31 was used to incubate with BMSC. Subsequently, BMSC proliferation, survival, and migration were determined via ELISA, qPCR, western blots, and immunofluorescence. Results: The expression of miR-31 was downregulated in response to LPS stress. Interestingly, supplementation of miR-31 could reverse the survival, proliferation and migration of BMSC under LPS. Mechanically, miR-31 treatment inhibited the activation of caspase, and thus promoted BMSC survival. Besides, miR-31 upregulated the genes related to cell proliferation, an effect that was followed by an increase in the levels of migratory factors. Further, we found that miR-31 treatment activated the CXCR4/Akt pathway and blockade of CXCR4/Akt could abolish the beneficial effects of miR-31 on BMSC proliferation, survival, and migration. Conclusions: miR-31 could increase the therapeutic efficiency of BMSC via the CXCR4/Akt pathway. [ABSTRACT FROM AUTHOR]

Degeneration of retinal pigment epithelium cells (RPE) or delamination of RPE from Bruch's membrane causes visual impairment. While regeneration of RPE cells is desirable to treat age-related macular degeneration (AMD) RPE cells of mammals, which are inherently non-renewable. Here, the bone marrow derived mesenchymal stem cells (BMSCs) that have the advantage of rapid growth and ease of extraction, which are adopted in order to help regenerate RPE cell. By co-culture with RPE cell, BMSCs exhibited a tendency to differentiate into RPE cell. By irradiating with the ultraviolet (UV) light, RPE cell was inhibited from growing and the differentiation of BMSCs was induced. The B-27® supplement was used as a differentiation factor, which helps BMSCs to differentiation into RPE cells. Specifically, B-27® supplement was mixed into the growth medium, which was added only to the co-culture and BMSCs groups. In consequence, UV-irradiated RPE cells did not grow, BMSCs in the co-culture group exhibited a tendency to differentiate into RPE cells. This was demonstrated by MTT assay, SEM, confocal imaging of the live/dead cells, RT-PCR and histological staining. Cell proliferation was the highest in the co-culture group, and the differentiation of BMSCs into RPE cells was observed in RT-PCR and histological staining. Our method will allow BMSCs with B-27® supplement to offer a new direction for RPE regeneration. [ABSTRACT FROM AUTHOR]

The key signaling networks regulating bone marrow mesenchymal stem cells (BMSCs) are poorly defined. Lgr4, which belongs to the leucine‐rich repeat‐containing G protein‐coupled receptor (LGR) family, is widely expressed in multiple tissues from early embryogenesis to adulthood. We investigated whether Lgr4 functions in BMSCs and in osteogenesis, adipogenesis, and skeletal myoblasts, using mice with a β‐geo gene trap inserted into the Lgr4 gene. Abundant Lgr4 expression was detected in skeletal, adipose and muscular tissue of Lgr4+/– mice at E16.5 by β‐gal staining, and Lgr4‐deficiency promoted BMSC proliferation (16 ± 4 in wild‐type [WT] and 28 ± 2 in Lgr4−/−) using colony forming units‐fibroblast assay, while suppressing BMSC migration (from 103 ± 18 in WT to 57 ± 10 in Lgr4−/−) by transwell migration assay and apoptosis ratio (from 0.0720 ± 0.0123 to 0.0189 ± 0.0051) by annexin V staining assay. Deletion of Lgr4 decreased bone mass (BV/TV from 19.16 ± 2.14 in WT mice to 10.36 ± 1.96 in KO) and fat mass through inhibiting BMSC differentiation to osteoblasts or adipocytes. Furthermore, LGR4‐regulated osteogenic, adipogenic, and myogenic gene expression. Importantly, our data showed that loss of Lgr4‐inhibited fracture healing by suppressing osteoblast differentiation. Moreover, deletion of Lgr4 in BMSCs‐delayed fracture healing following stem cell therapy by BMSC transplantation. Together, our results demonstrated that LGR4 is essential for mesoderm‐derived tissue development and BMSC differentiation, demonstrating that LGR4 could be a promising drug target for related diseases and a critical protein for stem cell therapy. Lgr4 promoted bone mesenchymal stem cell (BMSC) migration and apoptosis and inhibited BMSC proliferation. Lgr4 deficiency led to reduced bone mass and fat mass through inhibiting BMSCs differentiation to osteoblasts or adipocytes. Furthermore, Lgr4 deficiency impaired osteoblast mineralization and healing in bone fracture model, demonstrating that LGR4 could be a promising drug target for related diseases and a critical protein for stem cell therapy. [ABSTRACT FROM AUTHOR]

Context: Bone mesenchymal stem cells (BMSC)-based regenerative therapy is critical for the craniofacial defect reconstruction. However, oxidative stress micro-environment after transplantation limits the therapeutic efficiency of BMSC. The miR-181c has been found to be associated with cell survival and proliferation. Objective: Herein, we investigated whether prior miR-181c treatment promoted BMSC proliferation and survival under oxidative stress injury. Materials and methods: Cells were treated with hydrogen peroxide (H2O2) and then cell viability was determined via MTT assay, TUNEL staining and ELISA. Western blotting and immunofluorescence assay were used to detect those alterations of mitochondrial function. Results: H2O2 treatment reduced BMSC viability and this effect could be reversed via additional supplementation of miR181-c. Mechanistically, oxidative stress increased cell apoptosis, augmented caspase-3 activity, promoted reactive oxygen species (ROS) synthesis, impaired mitochondrial potential, and induced mitochondrial dynamics imbalance. However, miR-181c pretreatment reversed these effects of oxidative stress on BMSC. Moreover, miR-181c treatment improved BMSC proliferation, migration and paracrine, which are very important for craniofacial reconstruction. In addition, we identified that AMPK-Mfn1 axis was the direct targets of miR-181c in BMSC. Mfn1 silencing impaired the protective effects miR-181c on BMSC viability and proliferation under oxidative stress environment. Conclusions: Collectively, our results indicate that miR-181c participates in oxidative stress-mediated BMSC damage by modulating the AMPK-Mfn1 signaling pathway, suggesting miR-181c-AMPK-Mfn1 axis may serves as novel therapeutic targets to facilitate craniofacial defect reconstruction. [ABSTRACT FROM AUTHOR]

In this current study, a novel multilayer porous composite scaffold was fabricated with chitosan (CS), silk fibrin (SF) and nano‐hydroxyapatite (n‐HA). Scanning electron microscope was utilized to detect the characteristics of the composed scaffold. Rat bone marrow stromal cells (rBMSC) were loaded onto the CS/SF/n‐HA scaffold and cultured in a bioreactor under an on–off dynamic compression (10% compressive strain, 0.5 Hz, [2 h action + 4 h pause]/cycle, 4 cycles/day). Metabolism of the loaded rBMSC was assessed through CCK‐8 test. Qualitative polymerase chain reaction and western blot were applied to assess the chondrogenic differentiation of the seeded cells. Compressive modulus of the cell/scaffold constructs was analyzed. Additionally, a pig model was employed to evaluate the effect of the tissue‐engineered cartilage on repairing of cartilage defect. Results showed that the four layers within the scaffold were tightly connected without gaps between porous interfaces of the layers. Scaffold porosity was 92.20% ± 1.30%. The cyclic compression upregulated chondrogenesis markers (Aggrecan, Sox‐9, and collagen II). Increased compressive modulus of the cell/scaffold complex was detected after dynamic compression. The pig bone marrow stromal cells/scaffold complex exposed to cyclic compression presented most favorable reparative effect on the mini pig femoral condyle cartilage defects. Our study suggested that the on–off dynamic compression might be a promising approach to fabricate tissue‐engineered cartilage in vitro. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1294–1302, 2019. [ABSTRACT FROM AUTHOR]

Mesenchymal stromal cells (MSCs) have been applied in prevention from allograft rejection based on their immunomodulatory effects. However, conflicting results have been presented among recent studies, for which one possibility being acknowledged is that the exact effect is determined by the microenvironment when MSCs are applied in vivo. Using a hind limb composite tissue allograft model, we investigate the influence of IFN-γ-preconditioning on the immunomodulatory effects of MSCs and the subsequent allograft survival. Firstly, different doses of IFN-γ were respectively used to incubate with bone marrow–derived MSCs (BMSCs). We found that IFN-γ altered the expression of PD-L1, a major suppressor gene in the immune system during allograft rejection, in a strictly dose-dependent manner in BMSCs. Ten nanograms per milliliter IFN-γ-incubated BMSCs significantly stimulated PD-L1 expression and suppressed T cell proliferation and differentiation, while 50 ng/mL IFN-γ-incubated BMSCs sharply reduced PD-L1 expression. Moreover, we observed that, in contrast to the naive BMSC transplantation group, BMSCs pre-conditioned with 10 ng/mL IFN-γ (BMSCs-IFN-γ) significantly delayed the allograft rejection in vivo. In vitro mixed lymphocyte reaction (MLR) indicated that BMSCs-IFN-γ inhibited T lymphocyte proliferation and activation via PD-L1. Moreover, BMSCs-IFN-γ did not influence the proliferation and activation of T lymphocytes when PD-L1 protein was neutralized by the PD-L1 antibody. These data collectively reveal a role of recipient ongoing immune microenviroment in BMSC-based immunesuppressive therapy. Graphical abstract ᅟ [ABSTRACT FROM AUTHOR]

Myocardial ischemia–reperfusion injury (MIRI) is a major obstacle in the treatment of ischemic heart disease. Recent studies have shown that exosomes—small membrane vesicles secreted by most cell types—could have a protective effect on the ischemic myocardium. In this study we explored the effect of exosomes derived from bone-marrow stromal cells (BMSC-exo) on cardiomyocyte apoptosis and MIRI. Exosomes were purified from culture media using the ExoQuick kit and observed using transmission electron microscopy. Cell viability was assessed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Cell apoptosis was analyzed by flow cytometry using the Annexin-V/PI stain. The expression levels of microRNA (miRNA), messenger RNA (mRNA) and PTEN/PI3K/AKT-pathway-related proteins were detected by qRT-PCR and western blot, respectively. Myocardial ischemia was simulated by incubating H9C2 cells in a hypoxia/reoxygenation (H/R) conditioned rat MIRI model. BMSC-exo induced the proliferation of H9C2 cells and rescued H9C2 cells from apoptosis in the H/R model, indicating that BMSC-exo has a protective effect on cardiomyocyte injury caused by H/R. Using transgenic H9C2 cells, we found that miR-486-5p in BMSC-exo suppressed the H/R-triggered apoptosis of H9C2 cells. In addition, BMSC-exo repressed the expression of PTEN in H9C2 cells via miR-486-5p, and subsequently activated the PI3K/AKT pathway in vitro. Moreover, the myocardial injury caused by ischemia/reperfusion was repaired by BMSC-exo which activates the PI3K/AKT pathway via miR-486-5p in vivo. Our results suggested that exosomes from BMSCs have a protective effect on myocardium ischemic injury. MiR-486-5p carried by BMSC-exo plays a pivotal role in the regulatory process by suppressing PTEN expression, activating the PI3K/AKT signaling pathway, and subsequently inhibiting the apoptosis of injured cardiomyocytes. • BMSC-exo suppressed cardiomyocyte apoptosis in H/R model; • BMSC-exo rescued the myocardial infarction caused by I/R in myocardium; • MiR-486-5p in BMSC-exo triggered the protective effect on ischemic myocardium; • MiR-486-5p in BMSC-exo protected ischemic myocardium via PTEN/PI3K/AKT pathway. [ABSTRACT FROM AUTHOR]

Abstract Backgrounds: Osteoactivin (OA) is a key regulator promoting bone marrow stromal cells osteogenesis progress, while Dexamethasone (Dex) could inhibit OA induced osteogenesis and lead to osteoporosis. miR-26b increased during BMSC osteogenesis but whether it participates in this progress is enigma. Osteogenesis is under regulation of canonical Wnt signaling pathway which could serve as potential target for miR-26b. It bears therapeutic potential if miR-26b could regulate osteogenesis and antagonize Dex induced Osteoporosis (OP). Methods: BMSC were isolated from bone marrow of rats and induced for osteogenesis by OA administration. We detected miR-26b mRNA level together with osteogenesis related genes or Wnt signal pathway related genes by qRT-PCR. BMSC cells with miR-26b inhibitor or mimics revealed the effect of miR-26b on osteogenesis. The osteogenesis efficiency was detected by Alizarin Red staining and ALP activity. Protein level of canonical Wnt signal pathway and other proteins were detected by Western blot. The interaction between miR-26b and GSK3β was detected by dual luciferase reporter assay. Results: We found that miR-26b was increased during OA induced BMSC osteogenesis. Inhibiting miR-26b could lead to osteogenesis inhibition while miR-26b mimics could promote this progress. The key regulator of Wnt signal pathway GSK3β is down-regulated when miR-26b was overexpressed, resulting in β-catenin activation. Since Dex could promote GSK3β expression and inhibit Wnt signal, miR-26b could also alleviate Dex induced osteogenesis inhibition. Conclusion: Our findings indicate that miR-26b promoted BMSC osteogenesis by directly targeting GSK3β and activating canonical Wnt signal pathway, suggesting miR-26b might be serve as potential therapeutic candidate of osteoporosis. [ABSTRACT FROM AUTHOR]

Abstract Aims The interplay between bone marrow stromal cells (BMSCs) and acute myeloid leukemia (AML) cells plays a critical role in AML drug resistance by secreting growth factors, cytokines, and extracellular vesicles. As kind of extracellular vesicles, exosomes consist of proteins and RNAs and regulate communication among cells. Main methods The BMSCs, HS5 cells, and AML cells were co-cultivated with transwell membranes, and treated with different doses of AML chemotherapy drug, etoposide. Key findings Findings of our research proved that co-cultivation of BMSCs with AML cells defended AML against cell death triggered via etoposide, without having an impact on cell growth. An increase in the expression of the 70 kDa heat shock proteins (HSP70) as well as lysosomal associated membrane protein 3 (CD63) was observed in the exosomes from BMSC and AML, co-cultivated in conditioned media. Exosome repression in BMSC and AML co-cultivating system rebuilt the sensitivity of the KG1A cells to apoptosis triggered via etoposide, indicating that exosome modulated drug resistance in AML. Our study proved that exosomes arising from KG1A cells could propel BMSCs to generate IL-8, which could regulate the effect of etoposide treatment. Furthermore, IL-8 inhibition by its antibody increased the sensitivity of AML cells to cell death triggered via etoposide. Significance Our results suggested that exosomes secreted by AML cells is an essential communicator for the interaction of BMSCs and AML, which can protect AML cells from chemotherapy drug induced apoptosis. Graphical abstract Unlabelled Image [ABSTRACT FROM AUTHOR]

Objective: NELL-1 is an osteogenic protein first discovered to control ossification of the cranium. NELL-1 exists in at least two isoforms. The full-length NELL-1 contains 810 amino acid (aa) (NELL-1810), the N-terminal-truncated NELL-1 isoform contains 570 aa (NELL-1570). The differences in cellular effects between NELL-1 isoforms are not well understood. Methods: Here, BMSC were derived from adult or aged mice, followed by overexpression of NELL-1810 or NELL-1570. Cell morphology, proliferation, and gene expression were examined. Results/Conclusions: Overall, the proliferative effect of NELL-1570 was age dependent, showing prominent induction in adult but not aged mice. [ABSTRACT FROM AUTHOR]

High-level quadriplegia is a devastating condition with limited treatment options. Bone marrow derived stem cells (BMSCs) are reported to have immunomodulatory and neurotrophic effects in spinal cord injury (SCI). We report a subject with complete C2 SCI who received three anatomically targeted intrathecal infusions of BMSCs under a single-patient expanded access investigational new drug (IND). She underwent intensive physical therapy and was followed for >2 years. At end-point, her American Spinal Injury Association Impairment Scale (AIS) grade improved from A to B, and she recovered focal pressure touch sensation over several body areas. We conducted serial neurophysiological testing to monitor changes in residual connectivity. Motor, sensory, and autonomic system testing included motor evoked potentials (MEPs), somatosensory evoked potentials (SSEPs), electromyography (EMG) recordings, F waves, galvanic skin responses, and tilt-table responses. The quality and magnitude of voluntary EMG activations increased over time, but remained below the threshold of clinically obvious movement. Unexpectedly, at 14 months post-injury, deep inspiratory maneuvers triggered respiratory-like EMG bursting in the biceps and several other muscles. This finding means that connections between respiratory neurons and motor neurons were newly established, or unmasked. We also report serial analysis of MRI, International Standards for Neurological Classification of SCI (ISNCSCI), pulmonary function, pain scores, cerebrospinal fluid (CSF) cytokines, and bladder assessment. As a single case, the linkage of the clinical and neurophysiological changes to either natural history or to the BMSC infusions cannot be resolved. Nevertheless, such detailed neurophysiological assessment of high cervical SCI patients is rarely performed. Our findings indicate that electrophysiology studies are sensitive to define both residual connectivity and new plasticity. [ABSTRACT FROM AUTHOR]

Deregulated proteolysis invariably underlies most human diseases including bone pathologies. Metalloproteinases constitute the largest of the five protease families, and the metzincin metalloproteinases are inhibited by the four tissue inhibitors of metalloproteinase called TIMPs. We hypothesized that Timp genes are essential for skeletal homeostasis. We bred individual Timp knockout mice to generate unique mouse models, the quadruple Timp null strain (QT) as well as mice harboring only a single Timp3 allele (QT3+/–). QT mice are grossly smaller and exhibit a dramatic reduction of trabeculae in long bones by μCT imaging with a corresponding increase in metalloproteinase activity. At the cellular level, Timp deficiency compromised differentiation markers, matrix deposition and mineralization in neonatal osteoblasts from calvariae, as well as the fibroblastic colony‐forming unit (CFU‐F) capacity of bone marrow–derived stromal cells. In contrast, we observed that osteoclasts were overactive in the Timp null state, consistent with the noted excessive bone resorption of QT bones. Immunohistochemistry (IHC) and immunofluorescence (IF) analyses of bone sections revealed higher Cathepsin K and RANKL signals upon Timp loss. Seeking the molecular mechanism, we identified abnormal TNFα bioactivity to be a central event in Timp‐deficient mice. Specifically, TNFα triggered induction of the Wnt signaling inhibitor Dkk1 in the osteoblasts at the mRNA and protein levels, with a simultaneous increase in RANKL. Neutralizing TNFα antibody was capable of rescuing the induction of Dkk1 as well as RANKL. Therefore, the generation of novel Timp‐deficient systems allowed us to uncover the essential and collective function of TIMP proteins in mammalian long‐bone homeostasis. Moreover, our study discovers a functional TIMP/metalloproteinase‐TNFα‐Dkk1/RANKL nexus for optimal control of the bone microenvironment, which dictates coexistence of the osteoblast and osteoclast lineages. © 2018 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

Background: Stargardt Disease is the most common inherited macular degeneration, typically resulting in progressive central vision loss and legal blindness at an early age. We report regarding 34 eyes with Stargardt Disease treated in the Stem Cell Ophthalmology Treatment Study (SCOTS and SCOTS2). Methods: Autologous bone marrow was processed, separating the stem cell fraction which was provided Arms using retrobulbar, subtenons, intravitreal or subretinal and intravenous. The follow-up period was one year. Results: Of the 34 treated eyes, 21 (61.8%) improved, 8 (23.5%) remained stable, and 5 (14.7%) showed continued progression of their disease. Results were statistically significant with p = 0.0004. The average central vision improvement following treatment was 17.96% (95%CI, 16.39–19.53%) and ranged up to 80.5%. Of 17 patients treated, 13 (76.5%) showed visual acuity improvement in one or both eyes, 3 patients (17.6%) showed no net loss, and 1 worsened as a consequence of disease progression; 94.1% of patients had improved vision or remained stable. There were no adverse events. Conclusions: Patients with Stargardt Disease may potentially benefit from autologous bone marrow-derived stem cells (BMSC) as provided in SCOTS. Improvement or stabilization of vision was found to occur for the vast majority of reported patients and findings were highly statistically significant.

Background/Objective: Despite recent progress in regeneration medicine, the repair of large bone defects due to trauma, inflammation and tumor surgery remains a major clinical challenge. This study was designed to produce large amounts of viable bone graft materials in a novel perfusion bioreactor to promote bone formation. Methods: Cylindrical defects were created bilaterally in the distal femurs of sheep, and titanium implants were inserted. The concentric gap around the implants was randomly filled either with allograft, granules, granules with bone marrow aspirate (BMA) or bioreactor activated granule (BAG). The viable BAG consisted of autologous bone marrow stromal cells (BMSCs) seeded upon porous scaffold granules incubated in a 3D perfusion bioreactor for 2 weeks prior to surgery. 6 weeks after, the bone formation and early implant fixation were assessed by means of micro-CT, histomorphometry, and mechanical test. Results: Microarchitectural analysis revealed that bone volume fraction and trabecular thickness in the allograft were not statistically different than those (combination of new bone and residue of granule) in the other 3 groups. The structure of the allograft group was typically plate-like, while the other 3 groups were combination of plate and rod. Histomorphometry showed that allograft induced significantly more bone and less fibrous tissue in the concentric gap than the other 3 granule groups, while the bone ingrowth to implant porous surface was not different. No significant differences among the groups were found regarding early implant mechanical fixation. Conclusion: In conclusion, despite nice bone formation and implant fixation in all groups, bioreactor activated graft material did not convincingly induce early implant fixation similar to allograft, and neither bioreactor nor by adding BMA credited additional benefit for bone formation in this model.

Bone is an active tissue where bone mineralization and resorption occur simultaneously. In the case of fracture, there are numerous factors required to facilitate bone healing including precursor cells and blood vessels. To evaluate the interaction between bone marrow-derived mesenchymal stem cells (BMSC)—the precursor cells able to differentiate into bone-forming cells and human umbilical vein endothelial cells (HUVEC)—a cell source widely used for the study of blood vessels. We performed direct coculture of BMSC and HUVEC in normoxia and chemically induced hypoxia using Cobalt(II) chloride and Dimethyloxaloylglycine and in the condition where oxygen level was maintained at 1% as well. Cell proliferation was analyzed by crystal violet staining. Osteogenesis was examined by Alizarin Red and Collagen type I staining. Expression of angiogenic factor-vascular endothelial growth factor (VEGF) and endothelial marker-von Willebrand factor (VWF) were demonstrated by immunohistochemistry and enzyme-linked immunosorbent assay. The quantitative polymerase chain reaction was also used to evaluate gene expression. The results showed that coculture in normoxia could retain both osteogenic differentiation and endothelial markers while hypoxic condition limits cell proliferation and osteogenesis but favors the angiogenic function even after 1 of day treatment.

Background: The osteogenic differentiation of vascular smooth muscle cell (VSMCs) is important for the development of vascular calcification (VC), particularly in diabetes. Exosomes derived from Mesenchymal Stromal Cells (MSCs) are effective against cardiovascular diseases, yet their role in VC remains unclear. Advanced glycation end products (AGEs) inhibit bone marrow stromal cell osteogenesis by targeting osteogenesis-associated genes. Thus, we investigated the role of exosomes derived from MSCs pretreated with AGEs-BSA in VC and its potential mechanisms.Methods: Primary VSMCs and MSCs were isolated from the aorta and bone marrow of Sprague-Dawley rats, respectively. VSMCs were cultured with AGEs-BSA to induce osteogenic differentiation. Exosomes were harvested from MSCs by ultracentrifugation. MSCs and VSMCs were cocultured in Transwells, and exosomes were added to VSMC culture medium to assess their effects on osteogenic differentiation. Double luciferase reporter assay was applied to confirm that miR-146a directly targets the 3' UTR of the thioredoxin-interacting protein (TXNIP) gene.Results: Pretreatment of VSMCs with AGEs-BSA increased the expression of thioredoxin-interacting protein (TXNIP) by inhibiting that of miR-146a, resulting in enhanced ROS production and VSMC calcification. By contrast, the expression of miR-146a in MSCs was increased by AGEs-BSA treatment. Thus, miR-146a was transferred from AGEs-BSA-pretreated or miR-146a-transfected MSCs to VSMCs via exosomes. After coculture with miR-146a-containing exosomes, the AGEs-BSA-mediated increase in VSMC calcification was diminished, accompanied by decreased TXNIP expression and ROS production. Furthermore, TXNIP overexpression counteracted the anti-calcification effects of MSC-derived miR-146a-containing exosomes. In addition, TXNIP was identified as a target gene of miR-146a, and the results of double luciferase reporter assay confirmed that TXNIP was the direct target gene of miR-146a.Conclusions: Exosomes secreted by MSCs pretreated with AGEs-BSA contained a high level of miR-146a, which was transferred to VSMCs and inhibited AGEs-BSA-induced calcification in a TXNIP-dependent manner. Thus, miR-146a-containing exosomes may be a potential therapeutic target for VC.

Backgrounds Dexamethasone (Dex) is widely used in autoimmune diseases and inflammation treatment. A sever side effect of prolonged exposure to Dex is increased risk of osteoporosis (OP) or even femoral head necrosis, which would cause much suffer to patients. To reveal the mechanism behind this phenomenon, provide therapeutic guidance and potential target, we analyzed the inhibitory mechanism of Dex on osteogenesis of rat-BMSC. Methods Rat BMSC were obtained and characterized with FACS analysis. Osteogenesis and adipogenesis abilities were detected with Oil-O-Red staining, Alizarin Red staining and ALP activity analysis. These BMSC were then treated with Dex in combination with recombinant OA or not and detected for osteogenesis related gene expression with qRT-PCR. Protein interaction and expression were detected by Co-Immunoprecipitation and western blot. Results Osteoactivin (OA) could promote integrin β 1 expression and interact with this protein physically, leading to ERK activation and promoting osteogenesis related genes’ expression including Runx2, Col1a and OCN in BMSC. Dex, however, could block expression of several upstream genes of OA and decrease OA mRNA and protein level, and eventually suppress integrin β1-ERK activation and lead to decreased osteogenesis, which could finally develop into OP. Conclusion Recombinant OA treated BMSC exerted better osteogenesis potency even with Dex administration. This is because additional OA in medium counter-acts with Dex’s influence and rescued osteoblast differentiation via up-regulating integrin β1 and activate ERK/MAPK pathway which promotes osteogenesis. Hence, OA/integrin β1 could serve as potential therapeutic target for OP. [ABSTRACT FROM AUTHOR]

Objectives: The purpose of this study was to evaluate the effects of BMP‐2 on bone‐grafting procedures for the treatment of fibrous dysplasia based on in vitro and in vivo experiments. Subjects and Methods: Proliferation of stem cells was determined by colony‐forming assay, CCK‐8 assay and BrdU staining. OCT4 and NANOG expression was analysed by flow cytometry and immunocytochemistry. Osteogenic differentiation was assessed by measuring ALP activity, Alizarin red S staining and in vivo transplantation. Gene expression of the osteogenic markers, osteocalcin and type 1 collagen, was determined by RT‐PCR. Results: FD‐BMSCs showed few calcium deposits and low ALP activity. Bone formation by transplanted FD‐BMSCs was also suppressed relative to that of normal BMSCs. However, BMP‐2 treatment enhanced osteogenic differentiation of FD‐BMSCs mixed with normal BMSCs in vitro and in vivo. Overall, BMP‐2 treatment promoted osteogenic differentiation of FD‐BMSCs mixed with normal BMSCs. Conclusions: In patients with FD, stem cells in affected bone are influenced by the mutation, resulting in weak bone formation with the proliferation of immature osteogenic cells. Current treatment of FD involves surgical removal of excess bulk lesions, which can cause facial disfigurement. Our results suggest that BMP‐2 application is a good adjunctive modality to the surgical treatment of patients with FD. [ABSTRACT FROM AUTHOR]

Emerging evidence indicates that microRNAs (miRNAs, miRs) play diverse roles in the regulation of biological processes, including osteoblastic differentiation. In this study, we found that miR-383 is a critical regulator of osteoblastic differentiation. We showed that miR-383 was downregulated during osteoblastic differentiation of rat bone marrow mesenchymal stem cells (BMSCs). Overexpression of miR-383 suppressed osteoblastic differentiation of BMSCs by downregulating alkaline phosphatase (ALP), matrix mineralization, and mRNA and protein levels of RUNX2 and OCN, whereas a knockdown of miR-383 promoted osteoblastic differentiation in vitro . The results of in vivo analysis indicated that inhibition of miR-383 expression enhanced the efficacy of new bone formation in a rat calvarial defect model. Mechanistic experiments revealed that special AT-rich-sequence–binding protein 2 ( Satb2 ) was a direct and functional target of miR-383. Knockdown of Satb2 had inhibitory effects resembling those of miR-383 on the osteoblast differentiation of rat BMSCs. In addition, the positive effect of miR-383 suppression on osteoblastic differentiation was apparently abrogated by Satb2 silencing. Collectively, these results indicate that miR-383 plays an inhibitory role in osteogenic differentiation of rat BMSCs and may act by targeting Satb2 . [ABSTRACT FROM AUTHOR]

The effect of bone marrow mesenchymal stem cells (BMSCs) in treatment for multiple organ dysfunction syndrome (MODS) remains unknown and the mechanism is still unclear. Therefore, the goal of this study is to investigate the effects of intracellular high mobility group box 1 protein (HMGB1) on BMSCs treating for MODS. The rats were given 15% blood loss plus 1 mg/kg lipopolysaccharide (LPS) via lower extremity superficial venous, then randomly allocated into four groups: sham group, MODS group, MODS plus BMSC group, MODS plus ethyl pyruvate (EP) group, MODS plus BMSCs plus EP group. Twenty-four hours later, rats in groups were sacrificed and then the blood and tissues were collected to evaluate the changes of tissue histopathology, cell apoptosis, inflammation level and organ function. The HGMB1 expression was monitored by RT-qPCR and Western blot. The expression of RAGE/TLR2/TLR4 and NF-κB at the protein levels was also assessed. BMSCs and/or EP exhibits an outstanding protective effect against LPS-induced histopathological injury by improving cell apoptosis, inflammatory response and the organ dysfunction but no effect on BMSC homing to the injury site. Moreover, BMSCs and/or EP inhibited LPS-induced upregulation of HMGB1, RAGE, TLR2 and TLR4 expression at protein levels and compromised p65 phosphorylation in the rat model of MODS. These findings suggest that HMGB1 is involved in BMSC treatment for MODS, through regulation of the TLR2, TLR4-mediated NF-κB signal pathway. It suggests that HMGB1 is an attractive potential target for the development of new therapeutic strategies for MODS. [ABSTRACT FROM AUTHOR]

Objective To investigate the functional recovery and the effect of CNTF antibody block to the bone marrow mesenchymal stem cells (BMSCs) transplantated by Abdominal aortic on the downstream signaling pathways STAT3/Caspase-9 in spinal cord ischemic reperfusion injury in rats. Methods Adult female SD rats were assigned randomly to 4 groups. The neurological functional status of the animals was assessed with BBB scores, the Motor evoked potentials (MEP) and Cortical somatosensory evoked potentials (CSEP). The IHC were used to detect the the expressional changes of CNTF, then Western blot and RT-PCR were used to detect the expressional changes of STAT3、p-STAT3、CNTF and Caspase-9 in the ischemic segments of spinal cord. Results Compared with the sham group, in the SCIRI rats, the BBB scores were markedly decreased at all time points(P<0.01), the latency and the amplitude of MEP and CSEP was longer and lower at 14d post operation(P<0.01), and this change was the most significant in the control group the second in the CNTF block group,and the last in the transplantation group, Resutts between each two groups were statistically significant (P<0.05). At 7d post operation, compared with the sham group, the immunoreactive products of CNTF were decreased in the CNTF block group (P<0.05), but were increased(P<0.05) in the control group and the transplantation group (P<0.05), and results in the transplantation group were higher than in the control group (P<0.05). At 7d post operation, compared with the sham group, the mRNA and protein level of CNTF、 STAT3、p-STAT3 were decreased obviously in CNTF block group (P<0.05), the levels were increased in the control group and the transplantation group (P<0.05), and the levels in the transplantation group were higher than that in the control group(P<0.05); but the mRNA and protein level of Caspase-9 were only decreased in the transplantation group (P<0.05), the level was increased in the CNTF block group and the control groups (P<0.05), and the level in the CNTF block group was more significantly increased than that in the control group(P<0.05). At 14d post operation, in CNTF block group, the mRNA and protein level of CNTF、STAT3、 p-STAT3 were significantly higher than that in the sham group and the control group (P<0.05), and the mRNA and protein level of caspase-9 was higher than that in the sham group (P<0.05), but lower than that in the control group (P<0.05), there were not statistically different in the level of each factor compared with transplantation group (P>0.05). Conclusions BMSCs, transplanted by the abdominal aorta, can promote the expression of CNTF in the injuried spinal cord and significantly improve the hind limb function recovery by CNTF-mediated signaling pathway downstream of STAT3 / Caspase-9 SCIRI in rats, but the role of BMSCs can be weakening by CNTF block that inhibited STAT3 / Caspase-9 signaling pathway. [ABSTRACT FROM AUTHOR]

Several studies have suggested that bone marrow stromal steam cells (BMSC) exist in a quiescent state (G0) within the in vivo niche; however, an explicit analysis of the biology of G0 state-BMSC has not been reported. We hypothesized that induction of G0 in BMSC might enhance their stem cell properties. Thus, we induced quiescence in BMSC in vitro by (a) suspension culture in a viscous medium or (b) culture on soft polyacrylamide substrate; and examined their molecular and functional phenotype. Induction of G0 was confirmed by bromo-deoxyuridine (BrdU) labelling and analysis of cell cycle gene expression. Upon reactivation and re-entry into cell cycle, G0 state-BMSC exhibited enhanced clonogenic self-renewal, preferential differentiation into osteoblastic rather than adipocytic cells and increased ectopic bone formation when implanted subcutaneously in vivo in immune-deficient mice, compared to asynchronous proliferating (pre-G0) BMSC. Global gene expression profiling revealed reprogramming of the transcriptome during G0 state including significant alterations in relevant pathways and expression of secreted factors, suggesting altered autocrine and paracrine signaling by G0 state-BMSC and a possible mechanism for enhanced bone formation. G0 state-BMSC might provide a clinically relevant model for understanding the in vivo biology of BMSC. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor (VEGF) stimulation and bone marrow mesenchymal stem cell (BMSC) transplantation have been implicated in the treatment of acute cerebral infarction for their pivotal roles in behavioral recovery, neuroprotection, neurogenesis, and angiogenesis. However, the effects of BMSC transplants are likely limited because of low transplant survival after acute cerebral infarction, and delivery of VEGF alone also has limited effects on recovery because the protein is cleared quickly. This study attempted to explore whether VEGF could be transferred into BMSC via an adenovirus and whether transplanting VEGF-transfected BMSC into the rat brain provides sufficient neuroprotection after transient middle cerebral artery occlusion. The adenovirus carried VEGF into BMSC (Ad-VEGF-BMSC), and purified adenovirus was transferred into BMSC (Ad-BMSC). Western blots were used to detect the expression of VEGF protein after transfection. Rats exposed to 90-min middle cerebral artery occlusion (MCAO) were treated with Ad-VEGF-BMSC, Ad-BMSC, BMSC and Dulbecco’s Modified Eagle’s Medium (DMEM) after ischemia reperfusion for 24 h. The Sham group only received surgery. After transplantation of Ad-VEGF-BMSC into the perifocal area of the ischemic rat brain, we found increased expression and secretion of VEGF and BDNF as well as a higher level of MAP2, increased microvascular density, improved behavioral function and enhanced BMSC survival. Our results indicated that transplantation of Ad-VEGF-BMSC improved ischemic neurological deficiency after MCAO in rats. This finding provides a potential valuable therapeutic intervention for cerebral ischemic diseases. [ABSTRACT FROM AUTHOR]

Chinese Ecliptae herba (Eclipta prostrata (L.) L.) is an ethnomedicinal herb, which is used mainly to nourish kidney and thus strengthen bones according to traditional Chinese medicine theory. Pharmacological studies have supported the ethnomedicine use, showing that Ecliptae herba extract has an anti-osteoporotic effect in vivo and promoted osteoblast proliferation and activity in vitro. However, the molecular mechanism of Ecliptae herba on osteoblast differentiation from bone marrow mesenchymal stem cells (BMSC), the progenitors of osteoblasts, is still unclear. N6-methyladenosine (m6A) mRNA epigenetic modification may play a key role in promoting osteoblastic differentiation, and thus treating osteoporosis. This study sought to assess the mechanism through which Eclipate herba and its component wedelolactone influence m6A modification during the process of osteoblastogenesis from BMSC. The alkaline phosphatase (ALP) and Alizarin red S (ARS) staining were applied to determine osteoblastogenesis from BMSC. Western blot and quantitative real-time PCR were performed. RNA sequencing analysis was used to determine the characteristics of m6A methylation. Stable knocking down of METTL3 using lentiviral-based shRNA was performed. Upon 9 d treatment of BMSC with ethyl acetate extract of Ecliptae herba (MHL), ALP activity and ossification level increased in comparison with osteogenic medium (OS)-treated control. The expression of methyltransferase METTL3 and METTL14 was significantly increased, but WTAP expression had no change in response to MHL treatment. Knocking down of METTL3 resulted in a decrease in MHL-induced ALP activity, ossification level as well as mRNA expression of Osterix and Osteocalcin , two bone formation-related markers. The level of m6A increased when BMSC was treated with MHL for 9 d. RNA sequencing analysis indicated that MHL treatment altered mRNA m6A modification of genes associated with osteoblastogenesis. By kyoto encyclopedia of genes and genomes (KEGG) pathway analysis, HIF-1α, PI3K/Akt, and Hippo signaling pathways were enriched and associated with m6A modification. The expression of m6A-modified genes including HIF-1α, VEGF-A, and RASSF1, was upregulated by MHL, but the upregulation was reversed after METTL3 knockdown. Additionally, the enhanced expression of METTL3 was also observed after treatment with wedelolactone, a component from MHL. These results suggested a previously uncharacterized mechanism of MHL and wedelolactone on osteoblastogenesis, by which METTL3-mediated m6A methylation is involved and thus contributes to the enhancement of osteoblastogenesis. [Display omitted] • m6A mechanism for Ecliptae herba-enhanced osteoblastogenesis is firstly proposed. • The mechanism relies on upregulated methyltransferase METTL3 and increased m6A level. • HIF-1α, VEGF-A and RASSF1 expression is regulated by METTL3. • Wedelolactone, a component from Ecliptae herba, contributes for METTL3-mediated m6A methylation. [ABSTRACT FROM AUTHOR]

Exosomes, small extracellular vesicles that function as a key regulator of cell-to-cell communication, are emerging as a promising candidate for bone regeneration. Here, we aimed to investigate the effect of exosomes from pre-differentiated human alveolar bone-derived bone marrow mesenchymal stromal cells (AB-BMSCs) carrying specific microRNAs on bone regeneration. Exosomes secreted from AB-BMSCs pre-differentiated for 0 and 7 days were cocultured with BMSCs in vitro to investigate their effect on the differentiation of the BMSCs. MiRNAs from AB-BMSCs at different stages of osteogenic differentiation were analyzed. BMSCs seeded on poly- L -lactic acid(PLLA) scaffolds were treated with miRNA antagonist-decorated exosomes to verify their effect on new bone regeneration. Exosomes pre-differentiated for 7 days effectively promoted the differentiation of BMSCs. Bioinformatic analysis revealed that miRNAs within the exosomes were differentially expressed, including the upregulation of osteogenic miRNAs (miR-3182, miR-1468) and downregulation of anti-osteogenic miRNAs (miR-182–5p, miR-335–3p, miR-382–5p), causing activation of the PI3K/Akt signaling pathway. The treatment of BMSC-seeded scaffolds with anti-miR-182–5p decorated exosomes demonstrated enhanced osteogenic differentiation and efficient formation of new bone. In conclusion, Osteogenic exosomes secreted from pre-differentiated AB-BMSCs were identified and the gene modification of exosomes provides great potential as a bone regeneration strategy. Data generated or analyzed in this paper partly are available in the GEO public data repository(http://www.ncbi.nlm.nih.gov/geo). [Display omitted] [ABSTRACT FROM AUTHOR]

Objective(s):OCT4 is a transcription factor required for pluripotency during early embryogenesis and the maintenance of identity of embryonic stem cells and pluripotent cells. Therefore, the effective expression regulation of this gene is highly critical. UTR regions are of great significance to gene regulation. In this study, we aimed to investigate the potential regulatory role played by 5´UTR and 3´UTR of the Oct4 gene in mouse BMSC and P19 cells. Materials and Methods: The Oct4 5´UTR and 3´UTR sequences were cloned into pGL3 luciferase plasmid which led to the generation of pGL3 5´-UTR, pGL3 5´&3´-UTRs and pGL3 3´-UTR vectors. The vectors were transfected into BMSC and P19 cells followed by luciferase assay. Results: The assay of luciferase expression exhibited a direct link between the presence of Oct4 3´- UTR and the decrease of luciferase count in both cell lines; whereas 5´UTR indicated diverse behaviors in two cells. This discrepancy could be explained in view of the difference of cellular contexts in which the Oct4 UTRs act. Conclusion: This study sheds some light on the role of UTR regions of mouse Oct4 in regulating post-transcriptional gene expression in pluripotent cells. These data represent potential to be used for the development of novel therapeutic approaches for a variety of malignancies.

Background/Aims: Osteoarthritis (OA) is characterized by degradation of cartilage, sole cell type of which is chondrocytes. Bone marrow-derived mesenchymal stem cells (BMSCs) possess multipotency and can be directionally differentiated into chondrocytes under stimulation. This study was aimed to explore the possible roles of vitamin D and transforming growth factor-β1 (TGF-β1) in the chondrogenic differentiation of BMSCs. Methods: BMSCs were isolated from femurs and tibias of rats and characterized by flow cytometry. After stimulation with vitamin D, BMSC proliferation and migration were measured by Cell Counting Kit-8 (CCK-8) and Transwell assays, respectively. Chondrogenic differentiation was estimated through expression levels of specific markers by qRT-PCR and Western blot analysis. After stable transfection, the effects of aberrantly expressed TGF-β1 on vitamin D-induced alterations, including BMSC viability, migration and chondrogenic differentiation, were all evaluated utilizing CCK-8 assay, Transwell assay, qRT-PCR and Western blot analysis. Finally, the phosphorylation levels of key kinases in the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways were determined by Western blot analysis. Results: Vitamin D remarkably promoted BMSC viability, migration and chondrogenic differentiation. These alterations of BMSCs induced by vitamin D were reinforced by TGF-β1 overexpression while were reversed by TGF-β1 silencing. Additionally, the phosphorylation levels of ERK, JNK and c-Jun were enhanced by TGF-β1 overexpression but were reduced by TGF-β1 knockdown. Conclusion: Vitamin D promoted BMSC proliferation, migration and chondrogenic differentiation. TGF-β1 might be implicated in the vitamin D-induced alterations of BMSCs through regulating ERK/JNK pathway. [ABSTRACT FROM AUTHOR]

Interleukin-15 receptor alpha (IL15RA) is an important component of interleukin-15 (IL15) pro-inflammatory signaling. In addition, IL15 and IL15RA are present in the circulation and are detected in a variety of tissues where they influence physiological functions such as muscle contractility and overall metabolism. In the skeletal system, IL15RA was previously shown to be important for osteoclastogenesis. Little is known, however, about its role in osteoblast function and bone mineralization. In this study, we evaluated bone structural and mechanical properties of an Il15ra whole-body knockout mouse (Il15ra −/− ) and used in vitro and bioinformatic analyses to understand the role IL15/IL15RA signaling on osteoblast function. We show that lack of IL15RA decreased bone mineralization in vivo and in isolated primary osteogenic cultures, suggesting a cell-autonomous effect. Il15ra −/− osteogenic cultures also had reduced Rankl / Opg mRNA ratio, indicating defective osteoblast/osteoclast coupling. We analyzed the transcriptome of primary pre-osteoblasts from normal and Il15ra −/− mice and identified 1150 genes that were differentially expressed at a FDR of 5%. Of these, 844 transcripts were upregulated and 306 were downregulated in Il15ra −/− cells. The largest functional clusters, highlighted using DAVID analysis, were related to metabolism, immune response, bone mineralization and morphogenesis. The transcriptome analysis was validated by qPCR of some of the most significant hits. Using bioinformatic approaches, we identified candidate genes, including Cd200 and Enpp1 , that could contribute to the reduced mineralization. Silencing Il15ra using shRNA in the calvarial osteoblast MC3T3-E1 cell line decreased ENPP1 activity. Taken together, these data support that IL15RA plays a cell-autonomous role in osteoblast function and bone mineralization. [ABSTRACT FROM AUTHOR]

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Objectives TGF-β1 signaling modulates epithelial mesenchymal transitions (EMT) of head and neck squamous cell carcinoma (HNSCC). Bone marrow mesenchymal stromal cells (BMSC) are able to exert a regulating influence on the expression of markers of EMT in HNSCC cells. It was thus the aim of this study to test the hypothesis that TGF-β1 modulates the interactions of tumor transition between BMSCs and HNSCC, affecting the expression of E-cadherin, Vimentin, Snail, Twist, MMP14 and beta-catenin. Furthermore, we analyzed alterations in the AKT-signaling of tumor and stroma cells. Materials and methods BMSCs were isolated from iliac bone marrow aspirates and co-cultured in trans-well permeable membrane wells with tumor cells of the established HNSCC cell line PCI-13. Following the induction with TGF-β1 under serum free conditions the expression of Vimentin and E-Cadherin was assessed via immunofluorescence. A quantitative RT-PCR analysis of tumor transition markers E-cadherin, Vimentin, Snail, Twist, MMP14 and beta-catenin was performed. Changes in AKT-Signaling were identified via protein analysis. Results In non-induced co-cultures, BMSC were able to suppress Vimentin in PCI-13 as a marker of tumor transition. In TGF-β1 induced co-cultures PCI-13 significantly increased the expression of Vimentin, Twist, Snail, MMP14, GSK3a, PRAS40, 4E-BP1, and AMPKa compared to monolayer controls. TGF-β1 co-cultured BMSC demonstrated a significant increase of Snail, PRAS40, mTOR, GSK3a/b, Bad, PDK1 and 4E-BP1. Conclusions TGF-β1 was able to attenuate the modulating influence of BMSC in co-culture and drive the co-culture towards a progressive tumor transition, affecting the expression of markers of EMT, AKT-Signaling and proliferative checkpoints. [ABSTRACT FROM AUTHOR]

Hierarchical structure mimicking the natural bone microenvironment has been considered as a promising platform to regulate cell functions. We have previously fabricated hierarchical macropore/nanowire structure and evidence has shown that it can better manipulate the cytoskeleton status and osteogenic performance of osteoblasts. However, how cues of hierarchical structure are translated and ultimately linked to BMSC lineage commitment have still remained elusive, which hinders the accurate knowledge and further development of the hierarchical structure. In this study, bone marrow-derived mesenchymal stem cells (BMSCs) fate on hierarchical structure was investigated as well as the detailed mechanisms. It was shown that well-developed cytoskeleton and focal adhesion were observed for BMSCs on hierarchical structure, which was accompanied by enhanced osteogenic and depressed adipogenic potential. Evidence of increased YAP activity and nuclear translocation were exhibited on hierarchical structure and YAP knockdown inhibited osteogenic differentiation and promoted adipogenic differentiation induced by hierarchical structure. Further remove of cytoskeleton tension inhibited YAP function, which confirmed the key role of YAP-mediated mechanotransduction in the BMSC differentiation. These results together provide information of the stem cell fate commitment on hierarchical structure and a promising approach to design advanced biomaterials by focusing on specific mechanotransduction process. [ABSTRACT FROM AUTHOR]

Creatine was reported to induce bone marrow stromal cells (BMSC) into GABAergic neuron-like cells (GNLC). In a previous study, creatine was used as a single inducer for BMSC into GNLC with low yield. In this study, BMSC-derived neurospheres (NS) have been used in generating GABAergic phenotype. The BMSC were isolated from adult rats and used in generating neurospheres and used for producing neural stem cells (NSC). A combination of all-trans-retinoic acid (RA), the ciliary neurotrophic factor (CNTF), and creatine was used in order to improve the yield of GNLC. We also used other protocols for the transdifferentiation including RA alone; RA and creatine; RA and CNTF; and RA, CNTF, and creatine. The BMSC, NSC, and GNLC were characterized by specific markers. The activity of the GNLC was evaluated using FM1-43. The isolated BMSC expressed Oct4, fibronectin, and CD44. The NS were immunoreactive to nestin and SOX2, the NSC were immunoreactive to nestin, NF68 and NF160, while the GNLC were immunoreactive to GAD1/2, VGAT, GABA, and synaptophysin. Oct4 and c-MYC, pluripotency genes, were expressed in the BMSC, while SOX2 and c-MYC were expressed in the NSC. The activity of GNLC indicates that the synaptic vesicles were released upon stimulation. The conclusion is that the combination of RA, CNTF, and creatine induced differentiation of neurosphere-derived NSC into GNLC within 1 week. This protocol gives higher yield than the other protocols used in this study. The mechanism of induction was clearly associated with several differential pluripotent genes. [ABSTRACT FROM AUTHOR]

The relationship between bone resorption and angiogenesis in peri-implantitis remains to be studied. We constructed a Beagle dog model of peri-implantitis, and extracted bone marrow mesenchymal stem cells (BMSCs) and endothelial cells (ECs) for culture. The osteogenic ability of BMSCs in the presence of ECs was investigated through an in vitro osteogenic induction model, and its mechanism was initially explored. The peri-implantitis model was verified by ligation, bone loss was observed by micro-CT, and cytokines were detected by ELISA. The isolated BMSCs and ECs were cultured to detect the expression of angiogenesis, osteogenesis-related proteins, and NF-κB signaling pathway-related proteins. 8 weeks after surgery, the peri-implant gums were swollen, and micro-CT showed bone resorption. Compared with the control group, IL-1β, TNF-α, ANGII and VEGF were markedly increased in the peri-implantitis group. In vitro studies found that the osteogenic differentiation ability of BMSCs co-cultured with IECs was decreased, and the expression of NF-κB signaling pathway-related cytokines was increased. Endothelial cells inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells through NF-κB signaling in the environment of peri-implantitis, which may become a new target for the treatment of peri-implantitis. [Display omitted] • The levels of angiogenic cytokines in peri-implant tissues were higher than those of the normal group. • Inflammatory endothelial cells inhibit the osteogenic differentiation capacity of bone marrow mesenchymal stem cells. • Inflammatory endothelial cells inhibit osteogenic differentiation by activating the NF-κB pathway. [ABSTRACT FROM AUTHOR]

Background: Chondrocytes are the primary cells responsible for maintaining cartilage integrity and function. Their role in cartilage homeostasis and response to inflammation is crucial for understanding the progression and potential therapeutic interventions for various cartilage-related disorders. Developing an accessible and cost-effective model to generate viable chondrocytes and to assess their response to different bioactive compounds can significantly advance our knowledge of cartilage biology and contribute to the discovery of novel therapeutic approaches., Objective: We developed a novel, streamlined protocol for generating chondrocytes from bone marrow-derived mesenchymal stem cells (BMSCs) in a 3D culture system that offers significant implications for the study of cartilage biology and the discovery of potential therapeutic interventions for cartilage-related and associated disorders., Methods: We developed a streamlined protocol for generating chondrocytes from BMSCs in a 3D culture system using an "in-tube" culture approach. This simple pellet-based 3D culture system allows for cell aggregation and spheroid formation, facilitating cell-cell and cell-extracellular matrix interactions that better mimic the in vivo cellular environment compared with 2D monolayer cultures. A proinflammatory chondrocyte model was created by treating the chondrocytes with lipopolysaccharide and was subsequently used to evaluate the anti-inflammatory effects of vitamin D, curcumin, and resveratrol., Results: The established protocol successfully generated a large quantity of viable chondrocytes, characterized by alcian blue and toluidine blue staining, and demonstrated versatility in assessing the anti-inflammatory effects of various bioactive compounds. The chondrocytes exhibited reduced inflammation, as evidenced by the decreased tumor necrosis factor-α levels, in response to vitamin D, curcumin, and resveratrol treatment., Conclusions: Our novel protocol offers an accessible and cost-effective approach for generating chondrocytes from BMSCs and for evaluating potential therapeutic leads in the context of inflammatory chondrocyte-related diseases. Although our approach has several advantages, further investigation is required to address its limitations, such as the potential differences between chondrocytes generated using our protocol and those derived from other established methods, and to refine the model for broader applicability and clinical translation., (©Rajashree Patnaik, Shirin Jannati, Bala Mohan Sivani, Manfredi Rizzo, Nerissa Naidoo, Yajnavalka Banerjee. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 28.07.2023.)

Bone marrow-derived mesenchymal stem cells (BMSCs) are increasingly being used in bone marrow transplantation (BMT) to enable homing of the allogeneic hematopoietic stem cells and suppress acute graft versus host disease (aGVHD). The aim of this study was to optimize the labelling of BMSCs with superparamagnetic iron oxide particles (SPIOs), and evaluate the impact of the SPIOs on the biological characteristics, gene expression profile and chemotaxis function of the BMSCs. The viability and proliferation rates of the SPIO-labeled BMSCs were analyzed by trypan blue staining and CCK-8 assay respectively, and the chemotaxis function was evaluated by the transwell assay. The expression levels of chemokine receptors were measured by RT-PCR and flow cytometry. The SPIOs had no effect on the viability of the BMSCs regardless of the labelling concentration and culture duration. The labelling rate of the cells was higher when cultured for 48 h with the SPIOs. Furthermore, cells labeled with 25 µg/ml SPIOs for 48 h had the highest proliferation rates, along with increased expression of chemokine receptor genes and proteins. However, there was no significant difference between the chemotaxis function of the labeled and unlabeled BMSCs. To summarize, labelling BMSCs with 25 µg/ml SPIOs for 48h did not affect their biological characteristics and chemotaxis function, which can be of significance for in vivo applications., Competing Interests: The authors declare there are no competing interests., (©2023 Liu et al.)