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AMP-activated protein kinase (AMPK) signaling shows an important role in energy metabolism and has recently been involved in osteogenic and adipogenic differentiation. In this study we aimed to investigate the role of AMPK activator, A-769662, in regulating the differentiation of mesenchymal stem cells derived from bone marrow (BMSCs) into osteoblastic and adipocytic cell lineage. The effect of A-769662 on osteogenesis was assessed by quantitative alkaline phosphatase (ALP) activity, matrix mineralization stained with Alizarin red, and gene expression analysis by quantitative polymerase chain reaction (qPCR). Adipogenesis was determined by Oil Red O staining for fat droplets and qPCR analysis of adipogenic markers. A-769662 activated the phosphorylation of AMPKα1 during the osteogenesis of mBMSCs as revealed by western blot analysis. A-769662 promoted the early stage of the commitment of mouse (m) BMSCs differentiation into osteoblasts, while inhibiting their differentiation into adipocytes in a dose-dependent manner. The effects of A-769662 on stimulating osteogenesis and inhibiting adipogenesis of mBMSCs were significantly eliminated in the presence of either AMPKα1 siRNA or Compound C, an inhibitor of AMPK pathway. In conclusion, we identified A-769662 as a new compound that promotes the commitment of BMSCs into osteoblasts versus adipocytes via AMPK-dependent mechanism. Thus our data show A-769662 as a potential osteo-anabolic drug for treatment of osteoporosis., Competing Interests: The authors report no conflicts of interest in this work.

Background: After non-contact co-culture of bone marrow mesenchymal stem cells (BMSCs) with nucleus pulposus cells (NPCs), exosomes secreted by BMSCs were able to ameliorate the degree of disc degeneration. The reason for this is, at least in part, that exosomes from BMSCs achieve by affecting the level of autophagy in NPCs, while the components in exosomes are diverse and their specific mechanism of action is still unclear., Purpose: Here, we aimed to explore the therapeutic effect of co-culture of BMSCs and NPCs on NPCs and explore its specific mechanism of action., Study Design/setting: In vitro study., Methods: Rat NPCs and BMSCs were isolated and cultured in vitro. The serum deprivation experiment (using oxygen, glucose, and serum deprivation [OGD]) simulates the pathological state of low blood supply of the intervertebral disc in vivo. We used apoptotic cell staining and flow cytometry to study the effect of BMSCs on the apoptosis rate of rat NPCs, and the apoptotic proteins active-caspase-3, active-caspase-9, autophagy marker proteins LC3 and Beclin 1 were further detected using Western blot analysis. The expression levels of the pro-apoptotic protein Bax and the apoptosis-inhibiting protein Bcl2 were measured. The differentially expressed miRNAs were screened in a gene expression profiling chip. Then qRT-PCR was used to detect the effect of different treatment methods on miR-155 expression. The effect of anti-miR-155 antibodies on autophagy was studied by flow cytometry and transmission electron microscopy. A luciferase reporter assay was used to study the direct interaction between miR-155 and BACH1 mRNA, which was analyzed by TargetScan software, and the results were verified by Western blotting., Results: Compared with the OGD group, the expression level of miR-155 and the NPC autophagy level significantly increased; the HO-1 protein expression increased; and the Bach1 protein expression, degeneration index, and apoptosis index all significantly decreased in the co-culture group. After BMSCs transfected with anti-miR-155 were co-cultured with NPCs, the miR-155 expression in the cells was significantly reduced, the HO-1 protein expression and the level of cell autophagy was reduced. However, Bach1 protein expression, NPC degeneration index, and apoptosis index increased. After being inhibited by the autophagy inhibitor wortmannin, the cell degeneration index and apoptosis rate significantly improved., Conclusion: In the OGD model, BMSCs can significantly increase the viability, the level of autophagy, and reduce the level of apoptosis in rat NPCs. BMSC exosomes increase miR-155 expression in NPCs, which targets Bach1 and in turn upregulates HO-1 expression, activates autophagy in NPCs, inhibits the apoptosis level, and improves intervertebral disc degeneration., Clinical Significance: Our experiment shows that it is maybe feasible to treat disc degeneration with drugs. At the same time, compared with BMSC injection method of treatment, side effects of drug therapy are smaller, and can be controlled, it also provides a new way for intervertebral disc degeneration drug treatment., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cadmium (Cd) is a widespread environmental contaminant that causes severe bone metabolism disease, such as osteoporosis, osteoarthritis, and osteomalacia. The present review aimed to explore the molecular mechanisms of Cd-induced bone injury starting from bone cell function and teeth development. Cd inhibits the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts, and directly causes BMSC apoptosis. In the case of osteoporosis, Cd mainly affects the activation of osteoclasts and promotes bone resorption. Cd-induces osteoblast injury and oxidative stress, which causes DNA damage, mitochondrial dysfunction, and endoplasmic reticulum stress, resulting in apoptosis. In addition, the development of osteoarthritis (OA) might be related to Cd-induced chondrocyte damage. The high expression of metallothionein (MT) might reduce Cd toxicity toward osteocytes. The toxicity of Cd toward teeth mainly focuses on enamel development and dental caries. Understanding the effect of Cd on bone cell function and teeth development could contribute to revealing the mechanisms of Cd-induced bone damage. This review explores Cd-induced bone disease from cellular and molecular levels, and provides new directions for removing this heavy metal from the environment. Cd exposure causes severe bone metabolism disease. Apoptosis and differentiation play an essential role in Cd-induced osteoporosis. The development of osteoarthritis (OA) might be related to Cd-induced chondrocyte damage. The expression of MT may affect Cd-induced osteocytes damage. The toxicity of Cd toward teeth is mainly focused on enamel development and dental caries. Therefore, bone cells function and teeth development is mainly present manner of Cd toxicity. Unlabelled Image • Cd causes severe bone metabolism disease. • Cd-induces bone injury via affecting bone cell formation and function. • Apoptosis is main death manner by Cd-induced bone cell. • The toxicity of Cd toward teeth is mainly affected enamel development and dental caries. [ABSTRACT FROM AUTHOR]

Rapid and effective repair of epithelial tissue is desirable for improving the success rate of operation and reducing postoperative complications. Hydrogel is a widely studied wound repair material, especially as a wound dressing for damaged epithelial tissue. Based on the catalytic effect of thrombin on fibrinogen, in this study, a three-dimensional fibrin gel which of adequate epithelial cell compatibility was constructed by using thrombin and fibrinogen under the cross-linking action of calcium ion. Immunofluorescence staining and hematoxylin-eosin (H&E) staining showed that bone marrow mesenchymal stem cell (BMSC) was embedded in fibrin gel. Furthermore, vascular endothelial growth factor (VEGF) was used to induce BMSC to differentiate into CD31+ and vWF+ endothelial cell (EC) in fibrin gel. The results showed that the fibrin gel surface may effectively promote the adhesion and proliferation of EC and smooth muscle cell (SMC). After 15 days of culture, it was found that the BMSC embedded in the hydrogel had differentiated into EC. The results of in vivo skin wound experiment in rats further proved that the fibrin gel containing BMSC could promote wound healing and repair, and showed the potential to promote neovascularization at the injured site. The construction method of hydrogel materials proposed in this study has potential application value in the field of regenerative medicine. Unlabelled Image • Three-dimensional fibrin gel embedded with BMSC was successfully constructed. • BMSC in fibrin gel was differentiated into CD31+ and vWF+ EC via VEGF inducing. • The fibrin gel can quickly stop bleeding on wounds without secondary bleeding. • The fibrin gel containing BMSC could promote wound healing, and showed the potential to promote neovascularization. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A multitude of micro- and nano-surface structures have been developed to improve the clinical performance of endosseous titanium (Ti) implants. However, most of these surface structures only simulate the topographic elements on a micro- or nano-scale. In this study, a nano-micro hierarchical TiO 2 clustered nanotubular structure was fabricated using anodization, and then functionalized with platelet derived growth factor-BB (PDGF-BB) using PhoA (11-hydroxyundecylphosphonic acid)/CDI (carbonyldiimidazole) chemistry. The resulting 3-dimensional spatial biomimetic structure, named NTPCP, exhibited negligible cytotoxicity and satisfactory bio-activity for host cells, and significantly enhanced the attachment as well as osteogenesis-related functions (early-stage proliferation, extracellular matrix synthesis and mineralization) of human bone marrow mesenchymal stem cells (bMSCs). We observed drastically elevated expression of osteocalcin (OCN), which mirrored prominent bone formation around the NTPCP implants in a rat model. This study establishes a novel strategy to improve the osseointegration of endosseous Ti implants via surface nano-topographic modification and bio-factor covalent functionalization. [ABSTRACT FROM AUTHOR]

Synthetic polycaprolactone (PCL) was modified with various concentrations of gelatin (GL) to enhance its physical properties and biological activity for bone regeneration. A novel trisolvent mixture has been used to mix PCL and GL that were fabricated as scaffolds using 3D plotting. The scaffolds were characterized for their mechanical properties, hydrophilicity and swelling ability. In addition, the structure and morphology of the printed scaffolds were analyzed by Fourier-Transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM) and microcomputed tomography (μCT). Attachment, proliferation and osteogenic differentiation of rat bone marrow stromal cells (BMSC) cultured on the printed scaffolds were evaluated within 21 days. Increasing GL content in the scaffolds led to an enhanced hydrophilic nature, better pore size distribution and interconnected micro-pores. This resulted in better cellular attachment, proliferation and osteogenic differentiation. Although the multiple reactive sites and biochemical compatibility provided by GL improved the scaffolds' osteogenic potency, the tensile strength and elasticity of the printed scaffolds are yet challenging with increasing GL contents. [ABSTRACT FROM AUTHOR]

Hypoxic expansion has been demonstrated to enhance in vitro neuronal differentiation of bone-marrow derived mesenchymal stem cells (BMSCs). Whether adipose-derived mesenchymal stem cells (ADSCs) increase their neuronal differentiation potential following hypoxic expansion has been examined in the study. Real-time quantitative reverse transcription-polymerase chain reaction and immunofluorescence staining were employed to detect the expression of neuronal markers and compare the differentiation efficiency of hypoxic and normoxic ADSCs. A sciatic nerve injury animal model was used to analyze the gastrocnemius muscle weights as the outcomes of hypoxic and normoxic ADSC treatments, and sections of the regenerated nerve fibers taken from the conduits were analyzed by histological staining and immunohistochemical staining. Comparisons of the treatment effects of ADSCs and BMSCs following hypoxic expansion were also conducted in vitro and in vivo . Hypoxic expansion prior to the differentiation procedure promoted the expression of the neuronal markers in ADSC differentiated neuron-like cells. Moreover, the conduit connecting the sciatic nerve gap injected with hypoxic ADSCs showed the highest recovery rate of the gastrocnemius muscle weights in the animal model, suggesting a conceivable treatment for hypoxic ADSCs. The percentages of the regenerated myelinated fibers from the hypoxic ADSCs detected by toluidine blue staining and myelin basic protein (MBP) immunostaining were higher than those of the normoxic ones. On the other hand, hypoxic expansion increased the neuronal differentiation potential of ADSCs compared with that of the hypoxic BMSCs in vitro . The outcomes of animals treated with hypoxic ADSCs and hypoxic BMSCs showed similar results, confirming that hypoxic expansion enhances the neuronal differentiation potential of ADSCs in vitro and improves in vivo therapeutic potential., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wu, Liao, Hsueh, Huang, Chen, Chiang, Hsu, Tseng and Wang.)

Background: Curcumin has anti-inflammatory effects and qualifies as a potential candidate for the treatment of osteoarthritis (OA). However, curcumin has limited bioavailability. Extracellular vesicles (EVs) are released by multiple cell types and act as molecule carrier during intercellular communication. We assume that EVs can maintain bioavailability and stability of curcumin after encapsulation. Here, we evaluated modulatory effects of curcumin-primed human (h)BMSC-derived EVs (Cur-EVs) on IL-1β stimulated human osteoarthritic chondrocytes (OA-CH)., Methods: CellTiter-Blue Viability- (CTB), Caspase 3/7-, and live/dead assays were used to determine range of cytotoxic curcumin concentrations for hBMSC and OA-CH. Cur-EVs and control EVs were harvested from cell culture supernatants of hBMSC by ultracentrifugation. Western blotting (WB), transmission electron microscopy, and nanoparticle tracking analysis were performed to characterize the EVs. The intracellular incorporation of EVs derived from PHK26 labeled and curcumin-primed or control hBMSC was tested by adding the labeled EVs to OA-CH cultures. OA-CH were pre-stimulated with IL-1β, followed by Cur-EV and control EV treatment for 24 h and subsequent analysis of viability, apoptosis, and migration (scratch assay). Relative expression of selected anabolic and catabolic genes was assessed with qRT-PCR. Furthermore, WB was performed to evaluate phosphorylation of Erk1/2, PI3K/Akt, and p38MAPK in OA-CH. The effect of hsa-miR-126-3p expression on IL-1β-induced OA-CH was determined using CTB-, Caspase 3/7-, live/dead assays, and WB., Results: Cur-EVs promoted viability and reduced apoptosis of IL-1β-stimulated OA-CH and attenuated IL-1β-induced inhibition of migration. Furthermore, Cur-EVs increased gene expression of BCL2, ACAN, SOX9, and COL2A1 and decreased gene expression of IL1B, IL6, MMP13, and COL10A1 in IL-1β-stimulated OA-CH. In addition, phosphorylation of Erk1/2, PI3K/Akt, and p38 MAPK, induced by IL-1β, is prevented by Cur-EVs. Cur-EVs increased IL-1β-reduced expression of hsa-miR-126-3p and hsa-miR-126-3p mimic reversed the effects of IL-1β., Conclusion: Cur-EVs alleviated IL-1β-induced catabolic effects on OA-CH by promoting viability and migration, reducing apoptosis and phosphorylation of Erk1/2, PI3K/Akt, and p38 MAPK thereby modulating pro-inflammatory signaling pathways. Treatment of OA-CH with Cur-EVs is followed by upregulation of expression of hsa-miR-126-3p which is involved in modulation of anabolic response of OA-CH. EVs may be considered as promising drug delivery vehicles of curcumin helping to alleviate OA.

Background: Bone marrow mesenchymal stem cells are a potential resource for the clinical therapy of certain diseases. Canine, as a companion animal, living in the same space with human, is an ideal new model for human diseases research. Because of the high prevalence of diabetes, alternative transplantation islets resource (i.e. insulin producing cells) for diabetes treatment will be in urgent need, which makes our research on the transdifferentiation of Bone marrow mesenchymal stem cells into insulin producing cells become more important., Result: In this study, we completed the transdifferentiation process and achieved the transcriptome profiling of five samples with two biological duplicates, namely, "BMSCs", "islets", "stage 1", "stage 2" and "stage 3", and the latter three samples were achieved on the second, fifth and eighth day of induction. A total of 11,530 differentially expressed transcripts were revealed in the profiling data. The enrichment analysis of differentially expressed genes revealed several signaling pathways that are essential for regulating proliferation and transdifferentiation, including focal adhesion, ECM-receptor interaction, tight junction, protein digestion and absorption, and the Rap1 signaling pathway. Meanwhile, the obtained protein-protein interaction network and functional identification indicating involvement of three genes, SSTR2, RPS6KA6, and VIP could act as a foundation for further research., Conclusion: In conclusion, to the best of our knowledge, this is the first survey of the transdifferentiation of canine BMSCs into insulin-producing cells according with the timeline using next-generation sequencing technology. The three key genes we pick out may regulate decisive genes during the development of transdifferentiation of insulin producing cells.

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.

MicroRNA (miRNA) is an endogenous regulatory small molecule RNA. Growing evidence shows that miRNA plays an important regulatory role in gene expression. Although miRNA is a more intensive regulatory noncoding RNA in recent years, few studies have investigated the regulation of targeting genes involved in bone repair. Meanwhile, as a negative bone regulator, previous studies showed that casein kinase 2-interacting protein 1 (CKIP-1) is closely associated with bone formation and regeneration. However, the gene knockout method may not be suitable for clinical application. Therefore, it was hypothesized that miRNA molecules can inhibit the expression of CKIP-1 and ultimately promote the osteogenesis process. The present study revealed that let-7i-5p plays an important role in the process of fracture healing by inhibiting the expression of CKIP-1. Related research provides a novel gene target for fracture healing., Supplementary Information: The online version of this article (10.1007/s10616-020-00444-1) contains supplementary material, which is available to authorized users., (© The Author(s), under exclusive licence to Springer Nature B.V. part of Springer Nature 2021.)

Bone marrow mesenchymal stem cells (BMSCs) can be induced to process osteogenic differentiation with appropriate mechanical and/or chemical stimuli. The present study described the successful culture of murine BMSCs under mechanical strain. BMSCs were subjected to 0%, 3%, 8%, 13%, and 18% cyclic tensile strain at 0.5 Hz for 8 hr/day for 3 days. The expression of osteogenic markers and mechanosensitive ion channels was evaluated with real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blot. The expression of alkaline phosphatase (ALP) and matrix mineralization were evaluated with histochemical staining. To investigate the effects of mechanosensitive ion channel expression on cyclic tensile strain-induced osteogenic differentiation, the expression of osteogenic markers was evaluated with real-time RT-PCR in the cells without mechanosensitive ion channel expression. This study revealed a significant augment in osteogenic marker in BMSC strained at 8% compared to other treatments; therefore, an 8% strain was used for further investigations. The ALP expression and matrix mineralization were enhanced in osteogenic induced BMSCs subjected to 8% strain after 7 and 14 days, respectively. Under the same conditions, the osteogenic marker and mechanosensitive ion channel expression were significantly promoted. However, the loss function of mechanosensitive ion channels resulted in the inhibition of osteogenic marker expression. This study demonstrated that strain alone can successfully induce osteogenic differentiation in BMSCs and the expression of mechanosensitive ion channels was involved in the process. The current findings suggest that mechanical stretch could function as efficient stimuli to induce the osteogenic differentiation of BMSCs via the activation of mechanosensitive ion channels., (© 2020 Wiley Periodicals LLC.)

By establishing the Fusobacterium nucleatum (F. nucleatum) infected-bone mesenchymal stem cells (BMSCs) transplantation model in APC Min/+ mice, we investigated the role of BMSCs in the development of intestinal tumors induced by F. nucleatum. Apc Min/+ +F. nucleatum + BMSCs mice showed increased susceptibility to intestinal tumors and accelerated tumor growth. BMSCs could also enhance tumor-initiating capability, invasive traits after F. nucleatum infection in vitro, and tumorigenicity in a nude murine model. Mechanistically, BMSCs were recruited to the submucosa, migrated to the mucosal layer, and might activate the canonical Wnt/β-catenin/TGIF axis signaling. Further mechanistic results illustrated increased production of the Wnt3a protein was found in Apc Min/+ +F. nucleatum + BMSCs mice, and BMSCs were likely the major source of Wnt3a. Intriguingly, a deletion of Wnt3a via BMSC interference or antagonist analogs led to a significantly attenuated capacity of Apc Min/+ +F. nucleatum mice to generate intestinal tumors. The findings suggest that BMSCs have the potential to migrate and accelerate F. nucleatum-induced colorectal tumorigenesis by modulating Wnt3a secretion; knockdown of BMSC-derived Wnt3a or antagonist analogs could attenuate carcinogenesis. Thus, Wnt3a might be a potential pharmaceutical target for the prevention and treatment of F. nucleatum-related colorectal cancer., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Filifactor alocis, an asaccharolytic anaerobic Gram-positive rod (AAGPR), is an emerging marker of periodontitis. Severe periodontitis causes destruction of the alveolar bone that supports teeth and can even lead to tooth loss. Based on our previous report that F. alocis-derived extracellular vesicles (FA EVs) contain various effector molecules and have immunostimulatory activity, we investigated the effect of FA EVs on osteogenesis using mouse bone-derived mesenchymal stromal cells (BMSCs). FA EVs dramatically inhibited bone mineralization similar to whole bacteria and reduced the expression levels of osteogenic marker genes. The osteogenic differentiation of TLR2-deficient BMSCs was not inhibited by FA EVs, suggesting that their inhibitory effect on osteogenesis is dependent on TLR2 signaling. FA EVs effectively activated TLR2 downstream signaling of the MAPK and NF-κB pathways. In addition, FA EVs regulated RANKL and OPG gene expression, increasing the RANKL/OPG ratio in BMSCs in a TLR2-dependent manner. Our study suggests that F. alocis-derived EVs interfere with bone metabolism via TLR2 activation, providing insight into the pathogenesis of bone loss associated with periodontitis., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Transgenic therapy for central neuralgia faces the problems of low expression and weak targeting and affects superficial but not deep neurons. In this study, we generated a lentivirus vector with human preproenkephalin gene (hPPE) expression driven by the transcriptional amplification strategy system (TAS) and established a primary bone marrow-derived mesenchymal stromal cell (BMSC) line stably expressing hPPE for transplantation into a rat model of neuropathic pain rat. The paw thermal withdrawal latency assay and paw mechanical withdrawal threshold assay showed that unlike control BMSCs and BMSCs with hPPE overexpression driven by the CMV or Synapsin 1 (SYN1) promoter, TAS-hPPE BMSCs had a robust and lasting analgesic effect. The TAS-hPPE BMSC-treated group exhibited higher expression of TAS-driven hPPE and a higher ratio of BMSCs in the midbrain, spinal cord and cortex then the CMV-hPPE BMSC- and SYN1-hPPE BMSC-treated groups. Moreover, we also observed that TAS-hPPE BMSCs displayed a greater tendency to differentiate into neurons and exhibit neuronal-like distribution than CMV-hPPE or SYN1-hPPE BMSCs. In conclusion, our study shows that the TAS improves BMSC transgenic therapy for neuropathic pain treatment.

Objective: This study aimed to investigate effects of TGF-β1-containing exosomes derived from bone marrow mesenchymal stem cells (BMSC) on cell function of rotator cuff tenocytes and its implication to rotator cuff tear., Methods: The primary BMSC and rotator cuff tenocytes were extracted and cultured. Identification of BMSC were performed by observing cell morphology and measurement of surface biomarkers by flow cytometry. BMSC-derived exosomes were extracted and identified by using electron microscopy, nanoparticle-tracking analysis (NTA) and western blotting. Cell proliferation and cell cycle were measured by CCK-8 assay and flow cytometry assay, respectively. Transwell assay was used for detection of tenocytes migration. The fibrotic activity of tenocytes was determined via qPCR and western blotting assays., Results: BMSC and BMSC-derived exosomes were successfully extracted. Treatment of BMSC-derived exosomes or TGF-β1 promoted cell proliferation, migration and increased cell ratio of (S + G2/M) phases in tenocytes, as well as enhanced the expression levels of fibrotic activity associated proteins. However, inhibition of TGF-β1 by transfection of sh-TGF-β1 or treatment of TGFβR I/II inhibitor partially reversed the impact of BMSC-derived exosomes on tenocytes function., Conclusion: Taken together, TGF-β1-containing exosomes derived from BMSC promoted proliferation, migration and fibrotic activity in rotator cuff tenocytes, providing a new direction for treatment of rotator cuff tendon healing., Competing Interests: The authors have no conflicting financial interest., (© 2020 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

Multipotent bone marrow-derived mesenchymal stem/stromal cells (BMSCs) exhibit a finite life span after ex vivo expansion leading to cellular senescence. Many factors can contribute to this. Recently, our group has identified for the first time expression of the chemokine-like factor superfamily 8 (CMTM8) gene in cultured human BMSCs. In this study, we examine the role of CMTM8 in BMSC proliferation, migration, and differentiation. Functional studies using siRNA-mediated knockdown of CMTM8 in human BMSCs resulted in decreased capacity to undergo proliferation and migration and an increased capacity for osteogenic differentiation in vitro. Furthermore, reduced CMTM8 levels led to a decrease in the epidermal growth factor receptor (EGFR) signaling pathway during BMSC proliferation and migration, respectively. Supportive studies using retroviral mediated enforced expression of CMTM8 in BMSC resulted in an increased capacity for proliferation and migration but a decreased osteogenic differentiation potential. Collectively, these data suggest that CMTM8 promotes BMSC proliferation and BMSC migration through the EGFR/ERK1/2 pathway. This study provides insight into novel regulatory mechanisms of human BMSC growth and cell fate determination.

With rising demand for cartilage tissue repair and replacement, the differentiation of mesenchymal stem cells (BMSCs) into cartilage tissue forming cells provides a promising solution. Often, the BMSC-derived cartilage does not remain stable and continues maturing to bone through the process of endochondral ossification in vivo . Similar to the growth plate, invasion of blood vessels is an early hallmark of endochondral ossification and a necessary step for completion of ossification. This invasion originates from preexisting vessels that expand via angiogenesis, induced by secreted factors produced by the cartilage graft. In this study, we aimed to identify factors secreted by chondrogenically differentiated bone marrow-derived human BMSCs to modulate angiogenesis. The secretome of chondrogenic pellets at day 21 of the differentiation program was collected and tested for angiogenic capacity using in vitro endothelial migration and proliferation assays as well as the chick chorioallantoic membrane (CAM) assay. Taken together, these assays confirmed the pro-angiogenic potential of the secretome. Putative secreted angiogenic factors present in this medium were identified by comparative global transcriptome analysis between murine growth plate cartilage, human chondrogenic BMSC pellets and human neonatal articular cartilage. We then verified by PCR eight candidate angiogenesis modulating factors secreted by differentiated BMSCs. Among those, Serpin E1 and Indian Hedgehog (IHH) had a higher level of expression in BMSC-derived cartilage compared to articular chondrocyte derived cartilage. To understand the role of these factors in the pro-angiogenic secretome, we used neutralizing antibodies to functionally block them in the conditioned medium. Here, we observed a 1.4-fold increase of endothelial cell proliferation when blocking IHH and 1.5-fold by Serpin E1 blocking compared to unblocked control conditioned medium. Furthermore, endothelial migration was increased 1.9-fold by Serpin E1 blocking and 2.7-fold by IHH blocking. This suggests that the pro-angiogenic potential of chondrogenically differentiated BMSC secretome could be further augmented through inhibition of specific factors such as IHH and Serpin E1 identified as anti-angiogenic factors., (Copyright © 2020 Nossin, Farrell, Koevoet, Somoza, Caplan, Brachvogel and van Osch.)

Background: A growing body of studies have indicated that bone marrow mesenchymal stem cells (BMSCs) have powerful analgesic effects in animal models of bone cancer pain. Here, we explored the molecular mechanisms underlying how BMSCs alleviate pain sensation in a mouse model of bone cancer pain., Methods: C3H/HeN adult male mice were used to generate a bone cancer pain model. BMSCs were isolated from mouse bone marrow, modified by transfection with microRNA-9-5p (miR-9-5p), and infused into the spinal cord. Spontaneous flinches, paw withdrawal latency, limb-use score, and weight-bearing score were used to assess pain-related behaviors. ELISA, RT-PCR, western blot, and luciferase assay were used to assess gene expressions., Results: Our results show that miR-9-5p regulated the expression of both repressor element silencing transcription factor (REST) and μ-opioid receptors (MOR) by targeting REST in primary mouse BMSCs. Overexpression of miR-9-5p reversed the activation of inflammatory pathway in TNF-α- and IL-6-treated BMSCs. In addition, miR-9-5p modified BMSCs alleviated cancer pain in the sarcoma-inoculated mouse model. MiR-9-5p modified BMSCs suppressed cytokine expression in the spinal cord of sarcoma-inoculated mice by suppressing REST gene expression., Conclusions: Our results indicate that miR-9-5p modified BMSCs can relieve bone cancer pain via modulating neuroinflammation in the central nervous system, suggesting genetically modified BMSCs could be a promising cell therapy in pain management.

In indirect co-culture system, chondrocytes can induce differentiation of bone marrow mesenchymal stem cells (BMSCs) to chondrocytes without additional inducer. The participation of microRNAs (miRNAs) may take part in the chondrogenic differentiation. Present study aimed to investigate the effect and mechanism of chondrocytes-derived exosomal miRNA in BMSCs chondrogenic differentiation. Our data showed that miR-8485 was the exosomal miRNA derived from chondrocytes and transmitted to BMSCs. Functionally, miR-8485 silence in chondrocytes impaired exosome-induced chondrogenic differentiation of BMSCs. Mechanistically, exosomal miR-8485 targeted GSK3B to repress GSK-3β expression and targeted DACT1 to induce p-GSK-3β (Ser9), activating Wnt/β-catenin pathways. Our study firstly showed that chondrocytes-derived exosomal miR-8485 regulated the Wnt/β-catenin pathways to promote chondrogenic differentiation of BMSCs, providing innovative thoughts for cartilage reconstruction., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Bone mesenchymal stem cell (BMSC)-based regenerative therapy is critical for the craniofacial defect reconstruction. However, oxidative stress microenvironment after transplantation limits the therapeutic efficiency of BMSC. The miR-181c has been found to be associated with cell survival and proliferation. Herein, we investigated whether prior miR-181c treatment promoted BMSC proliferation and survival under oxidative stress injury. The results in our study demonstrated that hydrogen peroxide (H 2 O 2 ) 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 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 AMP-activated protein kinase (AMPK)-mitofusins-1 (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. 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., (© 2019 Wiley Periodicals, Inc.)

Bone loss caused by inflammatory disease, such as peri-implantitis, poses a great challenge to clinicians for restoration. Emerging evidence indicates that microRNAs (miRNAs) are indispensable regulators of bone growth, development, and formation. In the present study, we found that microRNA-128 (miR-128) was differentially up-regulated during the osteogenic differentiation of rat bone marrow stem cells (rBMSCs). Overexpression of miR-128 promoted osteogenic differentiation of rBMSCs by up-regulating alkaline phosphatase (ALP), matrix mineralization, mRNA, and protein levels of osteogenic makers (e.g. RUNX2, BMP-2, and COLIA1), whereas inhibition of miR-128 suppressed osteoblastic differentiation in vitro. Mechanistically, miR-128 directly and functionally targeted Dickkopf2 (DKK2), which is a Wnt signaling pathway antagonist, and enhanced Wnt/β-catenin signaling activity. Furthermore, the positive effect of miR-128 on osteogenic differentiation was apparently abrogated by DKK2 overexpression. Collectively, these results indicate that miR-128 promotes osteogenic differentiation of rBMSCs by targeting DKK2, which may provide a promising approach to the treatment of peri-implantitis., (© 2020 The Author(s).)

This study analyzed the influence of titanium (TiO 2 ) surface modifications with two osteogenic proteins (BMP-2, BMP-7) and an anti-osteoclastic drug (alendronic acid [AA]) on sandblasted/acid-etched (SLA) and plain TiO 2 (PT) on cell adhesion, proliferation and differentiation (alkaline phosphatase [AP] and osteocalcin [OC]) of bone-marrow derived stem cells (BMSCs) after 1, 3 and 7 days in-vitro. Initially, AA surfaces showed the highest cell number and surface coverage. At day 3 and 7, BMP and AA-modified surfaces exhibited a significantly enhanced cell growth. For proliferation, at days 3 and 7, an enhancement on BMP-2, BMP-7 and AA-surfaces was seen. At day 7, SLA also showed a higher proliferation when compared to PT. Initially, AP expression was elevated on SLA and AA surfaces. At days 3 and 7, a significant increased AP expression was seen for SLA, BMP-2, BMP-7 and AA discs. For OC, SLA and AA surfaces had the highest expression after 1 day whereas after 3 and 7 days a significant difference was recorded for SLA, BMP-2, BMP-7 and AA. In conclusion, a beneficial biological effect of a chemical immobilization method of BMP-2, BMP-7 and alendronate onto titanium surfaces on BMSCs was proven., (© 2019 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals, Inc.)

Background: To investigated the role of miR-19b-3p in regulating bone marrow mesenchymal stem cell (BMSC) proliferation and osteoblast differentiation., Methods: The expression of miR-19b-3p and lncRNA H19 were measured in postmenopausal osteoporosis patients and BMP-22 induced BMSCs using qRT-PCR. MiR-19b-3p mimic or inhibitor was transfected into BMP-2 induced BMSCs. Cell proliferation was measured by BrdU method. Protein expression of RUNX2 and COL1A1 were measured by western blot. PcDNA3.1-lncRNA H19 with or without miR-19b-3p mimic was transfected into BMP-2 induced BMSCs., Results: The expression of miR-19b-3p was significantly up-regulated in postmenopausal osteoporosis patients and BMP-2 induced BMSCs. MiR-19b-3p overexpression dramatically elevated, while miR-19b-3p inhibition decreased cell proliferation of BMSCs. Additionally, protein expression levels of RUNX2 and COL1A1, as well as ALP activity were significantly promoted by miR-19b-3p mimic transfection and inhibited by miR-19b-3p inhibitor transfection. LncRNA H19 was obviously down-regulated in postmenopausal osteoporosis patients. H19 overexpression significantly decreased cell proliferation and differentiation by down-regulating miR-19b-3p. Moreover, the expression of miR-19b-3p was inhibited, while H19 elvated in 17β-estradiol (E2) treated BMSCs in a dose-dependent manner., Conclusion: These data were the first to reveal the critical role of H19/miR-19b-3p in postmenopausal osteoporosis, and provided a new therapeutic target for OP.

EGFR-TKIs such as erlotinib and gefitinib have been introduced into the first-line treatment for patients having a mutation of deletion in exon 19 or L858R missense mutations in exon 21. Almost all patients who respond to EGFR-TKIs at first place eventually develop acquired resistance after several months of therapy. The secondary mutations and bypass signaling activation are involved in the generation of the resistance. Hypoxia in non-small cell lung cancer (NSCLC) is an important factor in treatment resistance including radiotherapy, chemotherapy and EGFR-TKI therapy. In this study, the effect of hypoxic cancer microenvironment in the bypass signaling activation was investigated. We found that bone marrow-derived mesenchymal stem cells (BMSCs) residing in the hypoxic solid cancer microenvironment highly produced molecules associated with adipocytes including adipokine leptin and IGFBPs. Leptin could induce the resistance of lung cancer cells to erlotinib through activating IGF-1R signaling. IGFBP2 counteracted the activation role of IGF-1 and induced erlotinib resistance by activating IGF-1R signaling in an IGF-1 independent manner. IGFBP2 had synergistic effect with leptin to induce erlotinib resistance. Leptin and IGFBP2 may be predictive factors for acquired resistance for EGFR-TKIs.

Demineralized bone matrix (DBM) is one of the most widely used materials for bone repair. Recently, different strategies in tissue engineering have been used to improve preparation of biomaterials from natural sources suitable for the use in bone regeneration. However, the application of DBM in tissue engineering is still a challenge, because the mechanical properties which are essential to bear tensile and load and the risk of transmission of disease by donor are still a matter of homework. A solution to this problem is to blend natural and synthetic polymers to complement defects and make them ideal biomaterials. An ideal biomaterial improves survival, adhesion, proliferation, induction, and differentiation of cells in the biomaterial after in vivo transplantation. In this review, we will look at the study of DBM made of natural and synthetic materials giving a direction for future research.

Transmembrane protein 18 (Tmem18) is an obesity-associated gene essential for adipogenesis; however, its function in the osteogenic differentiation of bone marrow–derived mesenchymal stem cells (BMSCs) is still unclear. In this study, we found that Tmem18 was significantly downregulated in rat BMSCs after osteogenic induction. TMEM18 overexpression remarkably downregulated osteo-specific genes including alkaline phosphatase (Alp), Runt˗related transcription factor 2 (Runx2), osteocalcin (Ocn), and osteopontin (Opn), and reduced the number of mineral deposits and ALP activity in vitro , whereas knockdown of Tmem18 yielded the opposite results. In vivo assays also indicated that TMEM18 knockdown BMSCs have an increased bone formation potential in a rat model of calvarial defects. Analyses of the mechanism suggested that TMEM18 overexpression decreased β-catenin expression, whereas the TMEM18 knockdown enhanced β-catenin expression and promoted its nuclear translocation. The positive effects on osteogenic differentiation of rat BMSCs owing to the TMEM18 knockdown were attenuated by β-catenin downregulation. Taken together, these results indicate that TMEM18 plays an inhibitory role in osteogenic differentiation of BMSCs via inactivation of β-catenin. • TMEM18 is downregulated during osteoblast differentiation of BMSCs. • TMEM18 inhibits osteogenic differentiation of BMSCs. • TMEM18 knockdown BMSCs have a higher bone formation potential in a rat model of calvarial defect. • TMEM18 suppresses β-catenin activity in BMSCs. • Enhanced osteogenic differentiation of BMSCs owing to TMEM18 downregulation was attenuated via suppression of β-catenin. [ABSTRACT FROM AUTHOR]

The chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) is critical for cartilage regeneration. Tissues constructed from BMSCs through cartilage tissue engineering still exhibit some histological, morphological, and biomechanical differences from normal cartilage tissues. Cyclic tensile strain (CTS) can increase chondrogenic gene expression and reduce hypertrophic gene expression in chondrocytes. miR-365 has been identified as a mechanoresponsive microRNA and is an important regulator of both chondrocyte hypertrophy and differentiation. Therefore, we hypothesized that CTS may promote the chondrogenesis of BMSCs by upregulating the expression of miR-365. BMSCs were subjected to CTS to investigate the effects and mechanism on chondrogenesis. An Agilent miRNA microarray was used to profile miRNAs in the CTS-treated BMSCs and 3D-cultured control BMSCs. miR-365 was shown to interact with HDAC4 mRNA through a luciferase reporter assay. An animal cartilage defect model was constructed and different groups of BMSCs were implanted to investigate their in vivo effect. CTS promoted BMSC chondrogenesis. miR-365 was significantly upregulated in CTS-treated cells and played an important role in CTS-mediated chondrogenesis. Luciferase assays showed that HDAC4 is a direct target of miR-365. An in vivo study showed that CTS treatment and miR-365 overexpression could promote cartilage regeneration from BMSCs. CTS can promote the expression of miR-365, a crucial mechanosensitive microRNA involved in the chondrogenesis of BMSCs, which directly inhibits the expression of HDAC4 , in turn, enhancing the chondrogenesis of BMSCs. [ABSTRACT FROM AUTHOR]