Delivery growth factors by layer-by-layer assembly in nanofibers for enhancing bone defect repairment along with neurogenesis.

Glycosaminoglycans have been implicated in the binding and activation of a variety of growth factors, cytokines, and chemokines. In this way, glycosaminoglycans are thought to participate in events such as development and wound repair. In particular, heparin and heparan sulfate have been well studied, and specific aspects of their structure dictate their participation in a variety of activities. In contrast, although dermatan sulfate participates in many of the same biological processes as heparin and heparan sulfate, the interactions of dermatan sulfate have been less well studied. Dermatan sulfate is abundant in the wound environment and binds and activates growth factors such as fibroblast growth factor-2 (FGF-2) and FGF-7, which are present during the wound repair process. To determine the minimum size and sulfation content of active dermatan sulfate oligosaccharides, dermatan sulfate was first digested and then separated by size exclusion high pressure liquid chromatography, and the activity to facilitate FGF-2 and FGF-7 was assayed by the cellular proliferation of cell lines expressing FGFR1 or FGFR2 IIIb. The minimum size required for the activation of FGF-2 was an octasaccharide and for FGF-7 a decasaccharide. Active fractions were rich in monosulfated, primarily 4-O-sulfated, disaccharides and iduronic acid. Increasing the sulfation to primarily 2/4-O-sulfated and 2/6-O-sulfated disaccharides did not increase activity. Cell proliferation decreased or was abolished with higher sulfated dermatan sulfate preparations. This indicated a preference for specific dermatan sulfate oligosaccharides capable of promoting FGF-2- and FGF-7-dependent cell proliferation. These data identify critical oligosaccharides that promote specific members of the FGF family that are important for wound repair and angiogenesis.

TRPC1 channel modulates mechanical stretch-induced bone marrow mesenchymal stem cell proliferation through Ca2+-dependent ERK1/2 activation.

MMP-1 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells via the JNK and ERK pathway.

ERK1-mediated immunomodulation of mesenchymal stem cells ameliorates inflammatory disorders

BackgroundThis study investigated the effect of Astragalus polysaccharides (APS) on radiation-induced bystander effects (RIBE) in human bone mesenchymal stem cells (BMSCs) induced by irradiated A549 cells.Material/MethodsA549 cells were irradiated with 2 Gy X-rays to obtain conditioned medium. BMSCs were incubated with the conditioned medium or APS. The levels of reactive oxygen species (ROS) and TGF-β were detected by ELISA. Cell survival, genomic instability, and DNA damages were detected by CCK-8 assay, colony formation assay, the micronucleus test and immunofluorescence assay, respectively. The protein and phosphorylation protein expression of p38, c-Jun N-terminal kinase (JNK), extracellular regulated protein kinase (ERK1/2), P65, and cyclooxygenase-2 (COX-2) in bystander effect cells were detected by Western blot.ResultsThe expression of COX-2 and ROS increased following stimulation with conditioned medium; this effect was inhibited by pre-exposing the cells to APS. BMSCs growth and colony formation rate decreased following stimulation with conditioned medium; this effect was suppressed by pre-exposing the cells to APS. In addition, the micronucleus rate and 53BP1 foci number increased after treatment with conditioned medium; this increase in BMSCs was inhibited by APS. The levels of phosphorylated p38, JNK, ERK1/2, NF-κB P65, and COX-2 proteins were increased by conditioned medium but were decreased by pre-treatment with APS.ConclusionsRIBE in BMSCs induced by the irradiated A549 was mediated by the ROS in the conditioned medium and might be related to MAPK/NF-κB signal pathways in BMSCs. APS may block RIBE through regulating the MAPK/NF-κB pathway.

Bone marrow mesenchymal stem cells (BMSCs) have characteristics of self-renewal and multidirectional differentiation. LncRNA UCA1 regulates BMSCs differentiation. Whether LncRNA UCA1 plays a role in bone defects remains unclear. BMSCs were randomly divided into control group, radiation group (6Gy), radiation + UCA1 group followed by analysis of the expression of LncRNA UCA1, RUNX2 and OPN by real time PCR, BMSCs proliferation by MTT assay as well as ALP activity. Healthy Sprague-Dawley rats were randomly divided into control group; bone defect group; UCA1 group, in which UCA1-transfected BMSCs were infused into bone defect rats followed by analysis of bone mineral density, ALP activity as well as the formation of type II collagen by ELISA. LncRNA UCA1 expression was significantly decreased in BMSCs of irradiated group, with decreased BMSCs proliferation, reduced expression of RUNX2 and OPN as well as decreased ALP activity (P < 0.05). Transfection of UCA1 significantly up-regulated LncRNA UCA1 expression in BMSCs, promoted BMSCs proliferation, increased the expression of RUNX2 and OPN, and the activity of ALP (P < 0.05). In addition, UCA1 promoted bone mineral density, increased ALP activity and type II collagen formation in rats with bone defect. LncRNA UCA1 promotes osteogenic differentiation of BMSCs, and targeting it might be a novel approach to promote bone remodeling at the bone defect site.

Bone marrow mesenchymal stem cells (BMMSCs) have the capacity for self-renewal, and differentiation into a variety of cell types. They thus represent an attractive source of material for cell therapy. However, little is known about the mechanisms underlying the proliferation of BMMSCs. The purpose of this study was to identify the factors and signaling pathways involved in the proliferation of stem cell antigen-1(+) (Sca-1(+)) BMMSCs. Among the cytokines and growth factors examined in this study, fibroblast growth factor-2 (FGF-2) and FGF-4 significantly stimulated the proliferation of Sca-1(+) BMMSCs, as determined by bromodeoxyuridine incorporation. PI3K-Akt, ERK1/2, and JAK/STAT3 pathways were investigated after stimulation with FGF-2 or FGF-4 via Western blot analysis. No changes were observed in the total ERK1/2 and Akt; however, the pERK1/2 and pAkt levels were upregulated early within 15 min in the FGF-2- or FGF-4-treated Sca-1(+) BMMSCs. Moreover, the pERK1/2 and pAkt upregulation induced by FGF-2 and -4 were completely abolished by treatment with the MEK1/2 inhibitor, U0126 and the PI3K inhibitor, LY294002. However, no change in pJAK2 or total JAK2 levels was observed in the Sca-1(+) BMMSCs induced by FGF-2 or FGF-4. As a consequence of PI3K-Akt and ERK1/2, the upregulation of c-Jun in the Sca-1(+) BMMSCs, after stimulation with FGF-2 or FGF-4, was observed after 12 and 24 h. Moreover, the activation of c-Jun in FGF-2- and FGF-4-treated Sca-1(+) BMMSCs was significantly reduced by U0126. Taken together, these data suggest that FGF-2 and -4 promote the proliferation of Sca-1(+) BMMSCs by activation of the ERK1/2 and PI3K-Akt signaling pathways.

Bone Marrow Mesenchymal Stem Cells (BMSCs) are ideal seed cells for cell therapy, which have been through many long-term studies of in vitro and animal disease model treatment and have been widely used in preventing and treating a variety of clinical diseases caused by aging and other damage, including bone injury and regeneration, neural degenerative and injury, myocardial injury, liver cirrhosis, diabetes, lung disease and so on. BMSCs application in the treatment of orthopaedic disease is the most common and effective. By retrospective literature review, we summarize the BMSCs transplantation application in Orthopaedic disease treatment. As an ideal cell source, BMSCs was first applied in bone tissue engineering, mainly used in the treatment of long bone defect and bone nonunion; Recently, the research trends of clinical BMSCs application turned to treat degenerative diseases and genetic disorders because of its great potential, including osteoarthrits, femoral head necrosis, disc degeneration, spinal cord injury, knee varus, osteogenesis hypoplasia, hypophosphatasia and so on. The safety and efficacy of BMSCs treatment are the key issues in clinical cell therapy. There is no side effects and complications after BMSCs treatment, prove its clinical application is safe. But due to type and degree of disease and individual differences, therapeutic methods including injection method, effective concentrations, treatment window selection, cycle and therapeatic effects have a bigger difference. There is still no uniform of recognized operation standards for the treatment of the same type of disease. So BMSCs transplantation requires effective control during whole medical process and its application in orthopedic diseases is still lack of large-scale multicenter clinical study. Therefore, this review focuses on induction, summary and analysis in the research status of BMSCs in bone disease treatment, and provide new ideas and methods for BMSCs transplantation in orthopaedic disease prevention and treatment.

P17. Effect of bone marrow mesenchymal stem cell combined with concentrate growth factor (CGF) on postmenopausal bone defects

Objective To investigate the osteogenic effect of different carriers applied with rabbit bone marrow mesenchymal stem cell (BMSC) transplantation combined with HBO in the treatment of open rabbit radial bone defect associated with seawater immersion. Methods Forty-eight New Zealand white rabbits were randomly divided into 4 groups: Group A (the simple BMSC transplantation + HBO group), Group B (BMSC transplantation + Autologous bone + HBO group), Group C (the BMSC transplantation + PLGA + HBO group), and Group D (the BMSC transplantation + gelatin sponge + HBO group), each consisting of 12 animals. Bilateral bone defects with a length of 15mm were made in the middle of the radial bone, and then debridement was made 3 hours after immersion in the artificial seawater. Bone tissue transplantation was performed right after debridement, and HBO therapy was implemented 1 hour a day for a succession of 2 weeks. The animals were sacrificed 8 and 12 weeks after surgery in batches of 4 in each group, and a total of 8 radial bones were collected. The repair of bone defects in each group of animals was compared between the 4 groups, through detection by X-ray, bone callus grey value, HE staining, and biomechanical testing. Results (1) X-ray detection indicated that osteogenic efficacy of the 4 groups was group B > group C > group D > group A. (2) As shown in Figure 1, 12 weeks after injury, bone callus grey values for the 4 groups were respectively as follows: group A: 162.1±1.3; group B: 220.1±1.2; group C: 195.6±1.7 and group D: 185.3±1.6. Statistical significance could be noted, when comparisons were made between the groups(P<0.05). (3) HE staining indicated that residual material could be seen in group C and D, 4 weeks after injury. Bone repair could all be seen in the bone defect areas, with the bone repair of group B being the best, and the bone repair of group C was superior to that of group D, and the bone repair of group D was superior to that of group A. Twelve weeks after injury, the repair materials in groups C and D were all degraded. The quality of the repaired bones was best in group B, which was followed by groups C, D and A. and statistical significance could be noted, when comparisons were made between the groups(P<0.05). (4) Twelve weeks after injury, biomechanical testing indicated that significant differences could be noticed in load capacity of the radial bone in the 4 groups. The load capacity of group B was the best, which was respectively followed by groups C, D and A (P<0.05). Conclusions (1) Autologous bone, PLGA and gelatin sponge as carriers combined with BMSC and HBO could all repair bone defect, and the advantage of combined treatment was obviously superior to that of BMSC bone transplantation alone. The quality of the repaired bones in the autologous bone group was the best, and the effect of PLGA as the carrier was better than that of the gelatin sponge as the carrier. (2) In the treatment of bone defects, particularly in the treatment of large section bone defect, PLGA in replacement of autogenous bone was a practical and effective treatment method, when autologous bone was insufficient or not available. Key words: Seawater immersion; Open bone defect; Bone marrow mesenchymal stem cell; Hyperbaric oxygen; Carriers; Rabbits

Neuronal plasticity of human Wharton's jelly mesenchymal stromal cells to the dopaminergic cell type compared with human bone marrow mesenchymal stromal cells

Objective To investigate the therapeutic effect of bone marrow mesenchymal stem cells (BMSC) overexpressing basic fibroblast growth factor (bFGF) on osteoporotic fracture in rats. Methods 80 female SD rats were randomly divided into control group, model group, BMSC group and BMSC+ bFGF group. The osteoporotic fracture model was established by resection of ovariectomized in model group, BMSC group and BMSC+ bFGF group. In group BMSC and group BMSC+ bFGF, BMSC and bFGF overexpressing BMSC were transplanted at 1, 14 and 21 d after operation respectively, while rats in the control and model groups were given the same volume of saline injection. The protein level were analyzed by Western blotting in BMSC group and BMSC+ bFGF group. Determination of bone density of rats in four group were detected by bone densitometry and fracture healing was evaluated by X-ray. The changes of bone biomechanical parameters were analyzed using three point bending test. alkaline phosphatase (ALP), runt related transcription factor-2 (Runx2) and bone morphogenetic protein-2 (BMP-2) mRNA level in four groups were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Results Western blot showed that the expression of bFGF factor was up-regulated in BMSC+ bFGF group. Compared with the model group [2.02±0.37, 0.051±0.002], the X-ray scores and bone mineral density significantly increased in the BMSC [3.12±0.46, 0.800±0.004] and BMSC+ bFGF groups [4.43±0.51, 0.128±0.006], and the biological parameters (maximum load, elastic load, stiffness and maximum deflection) significantly improved (P=0.032, 0.013; P=0.000, 0.000). Compared with the BMSC group, the X-ray score and bone mineral density in the BMSC+ bFGF group significantly increased (P=0.010, 0.010) and the biological parameters (maximum load, elastic load, stiffness and maximum deflection) significantly improved (P=0.002, 0.000; P=0.000, 0.000). Compared with the model group (1.41±0.19, 1.31±0.20, 1.43±0.24), the levels of ALP, Runx2 and BMP-2 mRNA in callus tissue of BMSC group (2.16±0.33, 2.48±0.31, 3.17±0.33) and BMSC+ bFGF group (4.48±0.48, 5.13±0.41, 4.89±0.49) significantly increased (P=0.000). Compared with group BMSC, the levels of ALP, Runx2 and BMP-2 mRNA in callus tissue of BMSC+ bFGF group more significantly increased (P=0.000). Conclusion Overexpression of bFGF in BMSC can increase the expression of bone morphogenetic protein, increase bone mineral density, promote the healing of osteoporotic fracture, and enhance the biological stress of fracture. Key words: Bone marrow mesenchymal stem cells; Basic fibroblast growth factor; Osteoporotic fractures; Bone mineral density; Bone healing; Biology

Our previous studies indicated that a coculture system containing human amnion-derived mesenchymal stem cells (HAMSCs) and human bone marrow mesenchymal stem cells (HBMSCs) has the potential of application for bone regeneration. However, there is currently no enough comparative investigation between HAMSCs/HBMSCs transwell and mixed coculture systems. This study aimed to assess the phenotype and mechanisms regulated by indirect and direct coculture systems, respectively. Two in vitro models were employed with HAMSCs and HBMSCs at a ratio of 3:1, and then were analyzed by a series of processes, including flow cytometry, alkaline phosphatase (ALP) substrate assays, Alizarin red S staining, quantitative reverse transcription polymerase chain reaction (RT-qPCR), and Western blot analysis. We found that cell proliferation, ALP activity, mineralized matrix formation, and osteoblast-related mRNA expression were accelerated in transwell coculture system compared with mixed coculture system. Conditioned medium from transwell coculture system achieved an elevated level of vascular endothelial growth factor and induced more vascular structures in human umbilical vein endothelial cells than those of mixed coculture system. Moreover, we observed that transwell coculture system, promoted osteogenesis and angiogenesis by maintaining stemness through extracellular regulated protein kinases 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signaling pathway. U0126, a selective inhibitor of ERK1/2 MAPK signaling, significantly suppressed maintaining of the stemness-based effects on transwell coculture system. Taken together, our results compared the merits of two different models and clarified the role of HAMSCs/HBMSCs transwell coculture system in the development of bone tissue engineering. © 2019 IUBMB Life, 2019.

This study investigated the early mechanical adaptability and osteogenic differentiation of mouse bone marrow mesenchymal stem cells (M-BMSCs) under micro-vibration stimulation (MVS). M-BMSCs were stimulated by MVS in vitro, cell proliferation, alkaline phosphatase (ALP) activity assay, and cytoskeleton were measured, and cell apoptosis was observed by flow cytometry. Early osteoblast-associated genes, runt-related transcription factor 2 (Runx2), Collagen Ⅰ (Col-Ⅰ) and ALP, were observed by RT-PCR and the activation of extracellular regulated protein kinases 1/2 (ERK1/2) was determined by Western blotting. The results showed that MVS had no significant effect on the proliferation of M-BMSCs. The early apoptosis was induced by mechanical stimulation (for one day), but the apoptosis was decreased after cyclic stimulation for 3 days. At the same time, MVS significantly accelerated the expression of F-actin protein in cytoskeleton, the synthesis of ALP and the ERK1/2 pathway, also up-regulated the expressions of Runx2, Col-Ⅰ and ALP genes. This study indicates that MVS could regulate cellular activity, alter early adaptive structure and finally promote the early osteogenic differentiation of M-BMSCs.

BackgroundThe aging of bone marrow mesenchymal stem cells (BMSCs) impairs bone tissue regeneration, contributing to skeletal disorders. LncRNA NEAT1 is considered as a proliferative inhibitory role during cellular senescence, but the relevant mechanisms remain insufficient. This study aims to elucidate how NEAT1 regulates mitotic proteins during BMSCs aging.MethodsBMSCs were isolated from alveolar bone of human volunteers aged 26–33 (young) and 66–78 (aged). NEAT1 expression and distribution changes during aging process were observed using fluorescence in situ hybridization (FISH) in young (3 months) and aged (18 months) mice or human BMSCs. Subsequent RNA pulldown and proteomic analyses, along with single-cell analysis, immunofluorescence, RNA immunoprecipitation (RIP), and co-immunoprecipitation (Co-IP), were conducted to investigate that NEAT1 impairs the nuclear transport of mitotic FGF2 and contributes to BMSCs aging.ResultsWe reveal that NEAT1 undergoes significant upregulated and shifts from nucleus to cytoplasm in bone marrow and BMSCs during aging process. In which, the expression correlates with nuclear DNA content during karyokinesis, suggesting a link to mitogenic factor. Within NEAT1 knockdown, hallmarks of cellular aging, including senescence-associated secretory phenotype (SASP), p16, and p21, were significantly downregulated. RNA pulldown and proteomic analyses further identify NEAT1 involved in osteoblast differentiation, mitotic cell cycle, and ribosome biogenesis, highlighting its role in maintaining BMSCs differentiation and proliferation. Notably, as an essential growth factor of BMSCs, Fibroblast Growth Factor 2 (FGF2) directly abundant binds to NEAT1 and the sites enriched with nuclear localization motifs. Importantly, NEAT1 decreased the interaction between FGF2 and Karyopherin Subunit Beta 1 (KPNB1), influencing the nuclear transport of mitogenic FGF2.ConclusionsOur findings position NEAT1 as a critical regulator of mitogenic protein networks that govern BMSC aging. Targeting NEAT1 might offer novel therapeutic strategies to rejuvenate aged BMSCs.