Promoted Bone Marrow Mesenchymal Stem Cells Proliferation Research Articles

Based on NF-κB and Notch1/Hes1 Signaling Pathways, the Mechanism of Artesunate on Inflammation in Osteoporosis in Ovariectomized Rats was Investigated.

Artesunate (ART) has the potential to modulate the nuclear factor kappa B (NF-κB) and Notch1/Hes1 signaling pathways, which play crucial roles in the pathogenesis of osteoporosis. This study aims to explore whether ART participates in the progression of osteoporosis by regulating these signaling pathways. In the in vitro experiments, we treated bone marrow mesenchymal stem cells (BMSCs) with different concentrations of ART (0, 3, 6, 12 µM) and evaluated osteogenic differentiation using alkaline phosphatase staining (ALP) and alizarin red S staining (ARS) staining. The expression levels of osteocalcin (OCN), RUNT-related transcription factor 2 (RUNX2), osteoprotegerin (OPG), and receptor activator of the nuclear factor kappa ligand (RANKL) were detected by real-time quantitative PCR (RT-qPCR). The effects of ART on NF-κB p65 and Notch1 protein expression were analyzed by Western blot (WB) and immunofluorescence (IF). In the in vivo experiments, a postmenopausal osteoporosis rat model was established via ovariectomy. Bone tissue pathological injury was evaluated using hematoxylin eosin (HE) staining. Serum ALP levels were measured using a kit, bone density was determined by dual-energy X-ray absorptiometry, and serum levels of bone gla protein (BGP), OPG, RANKL, tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and IL-1β were measured by enzyme-linked immunosorbent assay (ELISA). Additionally, the expression of NF-κB p65 and Notch1 in tissues was assessed by immunohistochemistry. In vitro experiments revealed that compared to the control group, ART dose-dependently promoted BMSCs proliferation and enhanced their osteogenic differentiation capability. The expression of OCN, RUNX2, and OPG significantly increased in the ART-treated group, while RANKL expression decreased significantly (p < 0.05). ART significantly inhibited the expression of NF-κB p65 and Notch1/Hes1 signaling pathway proteins (p < 0.05). Compared to ART treatment alone, combined treatment with ART and phorbol myristate acetate (PMA) or valproic acid (VPA) resulted in increased expression of NF-κB p65 and Notch1 proteins and decreased osteogenic differentiation capability (p < 0.05). In vivo experiments showed that in rats treated with ART, bone damage was significantly reduced, bone density and mineral content were restored considerably, and the expression of inflammatory factors (TNF-α, IL-6, IL-1β) decreased significantly (p < 0.05). Additionally, ART treatment significantly reduced the expression of NF-κB p65 and Notch1 proteins, increased OPG expression, and decreased BGP and RANKL levels (p < 0.05). In summary, ART facilitates the osteogenic differentiation of BMSCs by inhibiting the NF-κB and Notch1/Hes1 signaling pathways, thereby exerting significant protective effects against osteoporosis.

Bushen Huoxue recipe ameliorates ovarian function via promoting BMSCs proliferation and homing to ovaries in POI mice

BackgroundPremature ovarian insufficiency (POI) is a tricky puzzle in the field of female reproductive medicine. Bushen Huoxue recipe (BHR), a traditional Chinese medicine compound based on the combination of kidney-tonifying and blood-activating functions, has shown excellent efficacy in improving female irregular menstruation, POI, and infertility. However, the potential mechanism of BHR in POI treatment has not yet been elucidated. Bone marrow mesenchymal stem cells (BMSCs), a type of pluripotent stem cells, have received increasing attention for their significant role in improving ovarian function and restoring fertility in women with POI. PurposeThis study aimed to evaluate the therapeutic effect of BHR in POI mice and explore its potential mechanism. MethodsA POI mouse model was established with a single intraperitoneal injection of 120 mg/kg cyclophosphamide (CTX). Distilled water, BHR, or dehydroepiandrosterone was administered via gavage for 28 consecutive days. The effect of BHR on ovarian function in POI mice was evaluated by assessing the estrous cycle, ovarian morphology, follicular development, hormone levels, and angiogenesis. The proportion of BMSCs in bone marrow, peripheral blood, and ovary was analyzed via flow cytometry, and the level of molecules mediating migration and homing in ovary was measured. Cell viability assays, scratch healing assays and transwell migration assays were performed to explore the effect of BHR on BMSCs proliferation and migration in vitro, and its potential mechanism was explored. ResultsBHR significantly ameliorated estrous cycle disorders, hormone disorders, ovarian morphology, ovarian microvascular formation, and ovarian reserve in POI mice. Meanwhile, the number of BMSCs number in the bone marrow, peripheral blood, and ovary was apparently increased. Of note, BHR increased the level of hepatocyte growth factor (HGF)/cellular mesenchymal epithelial transition factor (cMET) and stromal cell-derived factor-1(SDF-1)/CXC chemokine receptor 4 (CXCR4) in the ovaries of POI mice. Moreover, BHR treatment promoted BMSCs proliferation and migration in vitro, with a significant increase in the level of proliferating cell nuclear antigen, cMET, and CXCR4. ConclusionsBHR effectively restored ovarian reserve, ovarian function, and ovarian angiogenesis in CTX-induced POI mice. In addition, BHR promoted BMSCs proliferation, migration, and homing to the ovary, which was mediated by the SDF-1/CXCR4 and HGF/cMET signaling axis. Finally, the amelioration of ovarian reserve and ovarian function in CTX-induced POI mice by BHR may be related to its promotion of endogenous BMSCs proliferation and homing.

Experimental Studies on the Effects of Glycitinregulated TGF-β Signaling Pathway on the Proliferation, Osteogenic, and Lipogenic Differentiation of BMSCs

Background Glycitin, generated from the seeds of the legume soybean, has an essential function in antioxidant, obesity inhibition, and wound healing promotion. Furthermore, recent research has shown that it has anti-inflammatory and cartilage-protective properties. Objectives This study was designed to investigate the osteogenic effects of glycitin and the transforming growth factor-β (TGF-β) signaling pathway on bone marrow mesenchymal stem cells (BMSCs), as well as their interaction. Materials and Methods We isolated rabbit BMSCs by whole bone marrow adherent method and cultured, identified, and induced differentiation. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used to determine the proliferation ability of BMSCs with different concentrations of glycitin at different times; reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression levels of osteogenesis-related genes runt-related transcription factor 2 (Runx2), collagen type I (Col-1), and osteocalcin (OC) mRNA; and a kit was used to determine the activities of alkaline phosphatase and triglycerides (TG) in BMSCs. A Western blot was used to detect the expression of TGF-β protein in each group. Results On the fifth day of BMSC primary culture, the morphology of long spindle-shaped cells might be plentiful, dominated by a daisy-like or whirling arrangement. They proliferated rapidly and fused to 70%–80% on the eighth day of culture; the third generation of BMSCs was taken for phenotyping, and the detection results of flow cytometry analysis showed that the surface antigenic markers of BMSCs, CD44, and CD90 were positive, whereas CD34 and CD45 were negative; after osteogenic induction of BMSCs, visible calcified nodules were evident. There was cartilage matrix formation after osteogenic induction; glycitin promoted BMSC proliferation in a time-dependent and dose-dependent manner, and BMSC proliferation increased significantly at 20 µM glycitin for 3–5 days; glycitin increased the expression of Runx2, Col-1, and OC mRNA in BMSCs and increased the activity of alkaline phosphatase (ALP) while decreasing the activity of TG. Meanwhile, 20 µM glycitin dramatically increased the expression of TGF-β. Conclusion These findings imply that glycitin increases BMSCs proliferation and osteogenic differentiation. By regulating the TGF-β signaling pathway, glycitin modulates the differentiation of BMSCs into osteoblasts.

NCAM mimetic peptide P2 synergizes with bone marrow mesenchymal stem cells in promoting functional recovery after stroke

The neural cell adhesion molecule (NCAM) promotes neural development and regeneration. Whether NCAM mimetic peptides could synergize with bone marrow mesenchymal stem cells (BMSCs) in stroke treatment deserves investigation. We found that the NCAM mimetic peptide P2 promoted BMSC proliferation, migration, and neurotrophic factor expression, protected neurons from oxygen-glucose deprivation through ERK and PI3K/AKT activation and anti-apoptotic mechanisms in vitro. Following middle cerebral artery occlusion (MCAO) in rats, P2 alone or in combination with BMSCs inhibited neuronal apoptosis and induced the phosphorylation of ERK and AKT. P2 combined with BMSCs enhanced neurotrophic factor expression and BMSC proliferation in the ischemic boundary zone. Moreover, combined P2 and BMSC therapy induced translocation of nuclear factor erythroid 2-related factor, upregulated heme oxygenase-1 expression, reduced infarct volume, and increased functional recovery as compared to monotreatments. Treatment with LY294002 (PI3K inhibitor) and PD98059 (ERK inhibitor) decreased the neuroprotective effects of combined P2 and BMSC therapy in MCAO rats. Collectively, P2 is neuroprotective while P2 and BMSCs work synergistically to improve functional outcomes after ischemic stroke, which may be attributed to mechanisms involving enhanced BMSC proliferation and neurotrophic factor release, anti-apoptosis, and PI3K/AKT and ERK pathways activation.

SDF-1α promotes subchondral bone sclerosis and aggravates osteoarthritis by regulating the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

BackgroundSubchondral bone sclerosis is a major feature of osteoarthritis (OA), and bone marrow mesenchymal stem cells (BMSCs) are presumed to play an important role in subchondral bone sclerosis. Accumulating evidence has shown that stromal cell-derived factor-1α (SDF-1α) plays a key role in bone metabolism-related diseases, but its role in OA pathogenesis remains largely unknown. The purpose of this study was to explore the role of SDF-1α expressed on BMSCs in subchondral bone sclerosis in an OA model.MethodsIn the present study, C57BL/6J mice were divided into the following three groups: the sham control, destabilization of the medial meniscus (DMM), and AMD3100-treated DMM (DMM + AMD3100) groups. The mice were sacrificed after 2 or 8 weeks, and samples were collected for histological and immunohistochemical analyses. OA severity was assessed by performing hematoxylin and eosin (HE) and safranin O-fast green staining. SDF-1α expression in the OA model was measured using an enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (q-PCR), and immunohistochemistry. Micro-CT was used to observe changes in subchondral bone in the OA model. CD44, CD90, RUNX2, and OCN expression in subchondral bone were measured using q-PCR and immunohistochemistry. In vitro, BMSCs were transfected with a recombinant lentivirus expressing SDF-1α, an empty vector (EV), or siRNA-SDF-1α. Western blot analysis, q-PCR, and immunofluorescence staining were used to confirm the successful transfection of BMSCs. The effect of SDF-1α on BMSC proliferation was evaluated by performing a CCK-8 assay and cell cycle analysis. The effect of SDF-1α on the osteogenic differentiation of BMSCs was assessed by performing alkaline phosphatase (ALP) and alizarin red S (ARS) staining. Cyclin D1, RUNX2 and OCN expression were measured using Western blot analysis, q-PCR, and immunofluorescence staining.ResultsSDF-1α expression in the DMM-induced OA model increased. In the DMM + AMD3100 group, subchondral bone sclerosis was alleviated, OA was effectively relieved, and CD44, CD90, RUNX2, and OCN expression in subchondral bone was decreased. In vitro, high levels of SDF-1α promoted BMSC proliferation and increased osteogenic differentiation. Cyclin D1, RUNX2, and OCN expression increased.ConclusionThe results of this study reveal a new molecular mechanism underlying the pathogenesis of OA. The targeted regulation of SDF-1α may be clinically effective in suppressing OA progression.

COX-2/PGE2/VEGF signaling promotes ERK-mediated BMSCs osteogenic differentiation under hypoxia by the paracrine action of ECs.

Understanding the crosstalk between endothelial cells (ECs) and bone-marrow mesenchymal stem cells (BMSCs) in response to hypoxic environments and deciphering of the underlying mechanisms are of great relevance for better application of BMSCs in tissue engineering. Here, we demonstrated that hypoxia promoted BMSCs proliferation, colony formation, osteogenic markers expression, mineralization, and extracellular signal-regulated protein kinase (ERK) phosphorylation, and that PD98059 (ERK inhibitor) blocked hypoxia-induced osteogenic differentiation. Hypoxia enhanced ECs migration, cyclooxygenase 2 (COX-2) and integrin αvβ3 expression, and prostaglandin E2 (PGE2), vascular endothelial growth factor (VEGF) secretion. NS398 (selective COX-2 inhibitor) and LM609 (integrin αvβ3 specific inhibitor) impaired the ECs response to hypoxia, and exogenous PGE2 partially reversed the effects of NS398. BMSCs: ECs co-culture under hypoxia upregulated BMSCs osteogenesis and ERK phosphorylation, as well as ECs migration, integrin αvβ3 expression, and PGE2 and VEGF secretion. NS398 (pretreated ECs) lessened PGE2, VEGF concentrations of the co-culture system. NS398-treated ECs and AH6809 (combined EP1/2 antagonist)/L-798106 (selective EP3 antagonist)/L-161982 (selective EP4 antagonist)/SU5416 [VEGF receptor (VEGFR) inhibitor]-treated BMSCs impaired the co-cultured ECs-induced enhancement of BMSCs osteogenic differentiation. In conclusion, hypoxia enhances BMSCs proliferation and ERK-mediated osteogenic differentiation, and augments the COX-2-dependent PGE2 and VEGF release, integrin αvβ3 expression, and migration of ECs. COX-2/PGE2/VEGF signaling is involved in intercellular BMSCs: ECs communication under hypoxia.

Proteomics reveals the potential mechanism of Tanshinone IIA in promoting the ExVivo expansion of human bone marrow mesenchymal stem cells.

Bone marrow mesenchymal stem cells (BMSCs) are a promising cell type for tissue engineering, however, the application of BMSCs is largely hampered by the limited number harvested from bone marrow cells. The methods or strategies that focused on promoting the capacity of BMSCs expansion exvivo become more and more important. Tanshinone IIA (Tan IIA), the main active components of Danshen, has been found to promote BMSCs proliferation, but the underlying mechanism is still unclear. The aim of this study is to explore the effect and underlying mechanism of Tan IIA on the expansion capacity of hBMSCs exvivo. In this present study, the effect of Tan IIA on the expansion capacity of BMSCs from human was investigated, and quantitative proteome analysis was applied furtherly to identify the differentially expressed proteins (DEPs) and the molecular signaling pathways in Tan IIA-treated hBMSCs. Finally, molecular biology skills were employed to verify the proposed mechanism of Tan IIA in promoting hBMSCs expansion. The results showed that a total of 84 DEPs were identified, of which 51 proteins were upregulated and 33 proteins were downregulated. Besides, Tan IIA could promote hBMSCs proliferation by regulating the progression of S phase via increasing the release of fibroblast growth factor 2 (FGF2), FGF-mediated PI3K/AKT signaling pathways may play an important role in Tan IIA's effect on hBMSCs expansion. This study employed molecular biology skills combined with quantitative proteome analysis, to some extent, clarified the mechanism of Tan IIA's effect on promoting hBMSCs proliferation, and will give a hint that Tan IIA may have the potential to be used for BMSCs applications in cell therapies in the future.

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

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

Therapeutic effect of platelet-rich plasma on glucocorticoid-induced rat bone marrow mesenchymal stem cells in vitro

BackgroundGlucocorticoid-induced osteonecrosis of the femoral head (GIONFH) is a progressive and disabling disease caused by long-term or high-dose glucocorticoid use. Decreased osteogenesis and proliferation of bone marrow mesenchymal stem cells (BMSCs) are the main pathogenesis of GIONFH. Platelet-rich plasma (PRP) has been shown to play a promising role in bone regeneration. However, the effects of PRP on glucocorticoid-induced BMSCs inhibition remains elusive. The objective of this study was to explore whether PRP could improve the in vitro biological activities of BMSCs inhibited by high-dose glucocorticoid in vitro.MethodsIn this study, a dexamethasone (Dex)-induced in vitro cell model was established. The effects of PRP on proliferation, migration, cell cycle and apoptosis of rat BMSCs induced with high-dose Dex compared to BMSCCTRL, using CCK-8 assay, transwell, flow cytometry and TUNEL assay, respectively. We further performed the alkaline phosphatase (ALP) and alizarin red (ALR) staining to explore the influence of PRP on osteogenic differentiation. Western Blot was used to detect the expression of Bcl-2, Caspase-3, RUNX2 apoptosis, and osteogenic-related proteins.ResultsWe observed increased apoptosis rate and Caspase-3 expression, and the decreased migration and osteogenic differentiation, and down-regulation of RUNX-2 and Bcl-2 expression in Dex-induced BMSCs. PRP could reverse these inhibitory effects of Dex, and enhance the BMSCs proliferation, migration, and osteogenic ability in vitro.ConclusionOur vitro study showed that PRP significantly protected BMSCs from Dex-induced apoptosis, and further promoted BMSCs proliferation, migration, and osteogenic differentiation. This study provides a scientific basis for the prevention and treatment of GIONFH with PRP. Meanwhile, it also lays the foundation for the application of PRP in other musculoskeletal diseases.

Stem Cell-Seeded 3D-Printed Scaffolds Combined with Self-Assembling Peptides for Bone Defect Repair.

Bone defects caused by infection, tumor, trauma, and so on remain difficult to treat clinically. Bone tissue engineering (BTE) has great application prospect in promoting bone defect repair. Polycaprolactone (PCL) is a commonly used material for creating BTE scaffolds. In addition, self-assembling peptides (SAPs) can function as the extracellular matrix and promote osteogenesis and angiogenesis. In the work, a PCL scaffold was constructed by 3D printing, then integrated with bone marrow mesenchymal stem cells (BMSCs) and SAPs. The research aimed to assess the bone repair ability of PCL/BMSC/SAP implants. BMSC proliferation in PCL/SAP scaffolds was assessed via Cell Counting Kit-8. In vitro osteogenesis of BMSCs cultured in PCL/SAP scaffolds was assessed by alkaline phosphatase staining and activity assays. Enzyme-linked immunosorbent assays were also performed to detect the levels of osteogenic factors. The effects of BMSC-conditioned medium from 3D culture systems on the migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) were assessed by scratch, transwell, and tube formation assays. After 8 weeks of in vivo transplantation, radiography and histology were used to evaluate bone regeneration, and immunohistochemistry staining was utilized to detect neovascularization. In vitro results demonstrated that PCL/SAP scaffolds promoted BMSC proliferation and osteogenesis compared to PCL scaffolds, and the PCL/BMSC/SAP conditional medium (CM) enhanced HUVEC migration and angiogenesis compared to the PCL/BMSC CM. In vivo results showed that, compared to the blank control, PCL, and PCL/BMSC groups, the PCL/BMSC/SAP group had significantly increased bone and blood vessel formation. Thus, the combination of BMSC-seeded 3D-printed PCL and SAPs can be an effective approach for treating bone defects. Impact statement Both polycaprolactone (PCL) and self-assembling peptides (SAPs) have been broadly applied in bone defect repair. However, the poor osteoinductivity of PCL and weak mechanical strength of SAPs have limited their clinical application. Here, a 3D-printed PCL scaffold was fabricated for seeding bone marrow mesenchymal stem cells (BMSCs), then combined with SAPs to construct a composite PCL/BMSC/SAP implant for treating the calvarial defect. We showed that transplantation of PCL/BMSC/SAP composite implants clearly promoted bone regeneration and neovascularization. To our knowledge, this is the first study to treat bone defects by combination of BMSC-seeded 3D-printed PCL and SAPs.

Bone marrow mesenchymal stem cells overexpressing hepatocyte growth factor ameliorate hypoxic-ischemic brain damage in neonatal rats.

ObjectivesHypoxic–ischemic brain damage (HIBD) is a major cause of brain injury in neonates. Bone marrow mesenchymal stem cells (BMSCs) show therapeutic potential for HIBD, and genetic modification may enhance their neuroprotective effects. The goal of this study was to investigate the neuroprotective effects of hepatocyte growth factor (HGF)-overexpressing BMSCs (BMSCs-HGF) against HIBD and their underlying mechanisms.Methods:BMSCs were transfected with HGF using adenoviral vectors. HIBD models were established and then BMSCs were transplanted into the brains of HIBD rats via intraventricular injection. 2,3,5-Triphenyltetrazolium chloride (TTC) staining was used to measure cerebral infarction volumes. In vitro, primary cultured cortical neurons were co-cultured with BMSCs in a Transwell plate system. Oxygen–glucose deprivation (OGD) was applied to imitate hypoxic–ischemic insult, and PD98059 was added to the culture medium to block the phosphorylation of extracellular signal-regulated kinase (ERK). Cell apoptosis was determined using TUNEL staining. The expression of HGF was measured by immunofluorescence, real-time quantitative PCR (RT-qPCR), and western blots. The expression of phosphorylated ERK (p-ERK) and B-cell lymphoma-2 (Bcl-2) was measured by western blots.ResultsHGF-gene transfection promoted BMSC proliferation. Moreover, BMSCs-HGF decreased HIBD-induced cerebral infarction volumes and enhanced the protective effects of the BMSCs against HIBD. BMSCs-HGF also increased expression of HGF, p-ERK, and Bcl-2 in brain tissues. In vitro, BMSC-HGF protected neurons against OGD-induced apoptosis. Inhibition of ERK phosphorylation abolished the neuroprotective effect of BMSCs-HGF against OGD.ConclusionsBMSCs-HGF is a potential treatment for HIBD and that the ERK/Bcl-2 pathway is involved in the underlying neuroprotective mechanism.

Polypeptides IGF-1C and P24 synergistically promote osteogenic differentiation of bone marrow mesenchymal stem cells in vitro through the p38 and JNK signaling pathways.

Insulin-like growth factor-1 (IGF-1) and bone morphogenetic protein 2 (BMP-2) both promote osteogenesis of bone marrow mesenchymal stem cells (BMSCs). IGF-1C, the C domain peptide of IGF-1, and P24, a BMP-2-derived peptide, both have similar biological activities as their parent growth factors. This study aimed to investigate the effects and mechanisms of polypeptides IGF-1C and P24 on the osteogenic differentiation of BMSCs. The optimum concentrations of IGF-IC and P24 were explored. The effects of the two polypeptides on BMSC proliferation and osteogenic differentiation were examined using a CCK-8 assay, flow cytometry, alkaline phosphatase (ALP) staining, ALP activity assay, alizarin red S staining, qPCR, and Western blotting. In addition, specific pathway inhibitors were utilized to explore whether the p38 and JNK pathways were involved in this process. The optimal concentration of both polypeptides was 50μg/ml. IGF-1C and P24 synergistically promoted BMSC proliferation, increased ALP activity and calcified nodule formation, upregulated the mRNA and protein levels of Osx, Runx2, Ocn, Opn, and Col1a1, and improved the phosphorylation levels of p38 and JNK proteins. Inhibition of the pathways significantly reduced p38 and JNK activation and blocked Runx2 expression while inhibiting ALP activity and calcified nodule formation. These findings suggest that IGF-1C and P24 synergistically promote the osteogenesis of BMSCs through activation of the p38 and JNK signaling pathways.

Bioinspired dual dynamic network hydrogels promote cartilage regeneration through regulating BMSC chondrogenic differentiation

How to improve the therapeutic efficacy of cell delivery during mechanical injection has been a great challenge for tissue engineering. Here, we present a facile strategy based on dynamic chemistry to prepare injectable hydrogels for efficient stem cell delivery using hyaluronic acid (HA) and poly(γ-glutamic acid) (γ-PGA). The combination of the guest–host (GH) complexation and dynamic hydrazone bonds enable the HA/γ-PGA hydrogels with physical and chemical dual dynamic network and endow hydrogels a stable structure, rapid self-healing ability, and injectability. The mechanical properties, self-healing ability, and adaptability can be programmed by changing the ratio of GH network to hydrazine bond cross-linked network. Benefitting from the dynamic cross-linking networks, mild preparation process, and cytocompatibility of HA/γ-PGA hydrogels, bone marrow mesenchymal stem cells (BMSCs) show high cell viability in this system following mechanical injection. Moreover, HA/γ-PGA hydrogels can promote BMSC proliferation and upregulate the expression of cartilage-critical genes. Notably, in a rabbit auricular cartilage defect model, BMSC-laden HA/γ-PGA hydrogels can effectively promote cartilage regeneration. Together, we propose a general strategy to develop injectable self-healing HA/γ-PGA hydrogels for effective stem cell delivery in cartilage tissue engineering.

Quercetin promotes bone marrow mesenchymal stem cell proliferation and osteogenic differentiation through the H19/miR-625-5p axis to activate the Wnt/\u03b2-catenin pathway

BackgroundQuercetin and H19 can promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). However, whether quercetin regulates H19 expression to promote osteogenic differentiation of BMSCs is unclear.MethodsBMSC proliferation, matrix mineralization, and alkaline phosphatase (ALP) activity were assessed using the Cell Counting Kit-8, ALP assay kit, and alizarin red staining kit, respectively. Expression of H19, miR-625-5p, BMP-2, osteocalcin, and RUNX2 were measured by qRT-PCR; β-catenin protein level was measured by western blotting.ResultsQuercetin promoted BMSC proliferation, enhanced ALP activity, and upregulated the expression of BMP-2, osteocalcin, and RUNX2 mRNAs, suggesting that it promoted osteogenic differentiation of BMSCs. Moreover, quercetin increased H19 expression, while the effect of quercetin on BMSCs was reversed by silencing H19 expression. Additionally, miR-625-5p, interacted with H19, was downregulated during quercetin-induced BMSC osteogenic differentiation, which negatively correlated with H19 expression. Silencing miR-625-5p expression promoted BMSC proliferation and osteogenic differentiation, whereas miR-625-5p overexpression weakened the effect of quercetin on BMSCs. Finally, quercetin treatment or downregulation of miR-625-5p expression increased β-catenin protein level in BMSCs. Upregulation or downregulation of miR-625-5p or H19 expression, respectively, inhibited β-catenin protein level in quercetin treated-BMSCs.ConclusionH19 promotes, while miR-625-5p inhibits BMSC osteogenic differentiation. Quercetin activates the Wnt/β-catenin pathway and promotes BMSC osteogenic differentiation via the H19/miR-625-5p axis.

Effects of Melatonin on Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in Inflammatory Environment by Regulating Mammalian Target of Rapamycin/Phosphatidylinositol 3-Kinase/Protein Kinase B Signaling

Human bone marrow mesenchymal stem cells (BMSCs) differentiation into special cell types is affected by inflammation. Melatonin has various effects such as anti-oxidation and immune regulation. However, melatonin’s effect on BMSCs osteogenic differentiation during inflammation has not been elucidated. Rat BMSCs were isolated and assigned into control group, inflammation group (1 μg/ml lipopolysaccharide, LPS) and melatonin group (100 μM melatonin was added to LPSstimulated BMSCs cells) followed by analysis of BMSCs proliferation by MTT assay, Caspase 3 and ALP activity, expression of Runx2 and OP by Real time PCR, ROS content and SOD activity, TNF-α and IL-1β secretion by ELISA and mTOR/PI3K/AKT signaling protein level by Western blot. LPS action on BMSCs significantly inhibits BMSCs proliferation, promotes Caspase 3 activity, inhibits ALP activity, decreases Runx2 and OP expression and SOD activity, increases ROS content and TNF-α and IL-1β secretion as well as reduced mTOR and p-PI3K level (P &lt;0.05). Melatonin addition significantly reversed the above changes (P &lt;0.05). Melatonin can regulate oxidative stress, inhibit inflammation, and promote BMSCs proliferation and osteogenic differentiation in inflammatory environment by activating mTOR/PI3K/AKT signaling pathway.

The Role of Tantalum Nanoparticles in Bone Regeneration Involves the BMP2/Smad4/Runx2 Signaling Pathway.

BackgroundIn recent years, nanomaterials have been increasingly developed and applied in the field of bone tissue engineering. However, there are few studies on the induction of bone regeneration by tantalum nanoparticles (Ta NPs) and no reports on the effects of Ta NPs on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and the underlying mechanisms. The main purpose of this study was to investigate the effects of Ta NPs on bone regeneration and BMSC osteogenic differentiation and the underlying mechanisms.Materials and MethodsThe effects of Ta NPs on bone regeneration were evaluated in an animal experiment, and the effects of Ta NPs on osteogenic differentiation of BMSCs and the underlying mechanisms were evaluated in cell experiments. In the animal experiment, hematoxylin-eosin (HE) staining and hard-tissue section analysis showed that Ta NPs promoted bone regeneration, and immunohistochemistry revealed elevated expression of BMP2 and Smad4 in cells cultured with Ta NPs.ResultsThe results of the cell experiments showed that Ta NPs promoted BMSC proliferation, alkaline phosphatase (ALP) activity, BMP2 secretion and extracellular matrix (ECM) mineralization, and the expression of related osteogenic genes and proteins (especially BMP2, Smad4 and Runx2) was upregulated under culture with Ta NPs. Smad4 expression, ALP activity, ECM mineralization, and osteogenesis-related gene and protein expression decreased after inhibiting Smad4.ConclusionThese data suggest that Ta NPs have an osteogenic effect and induce bone regeneration by activating the BMP2/Smad4/Runx2 signaling pathway, which in turn causes BMSCs to undergo osteogenic differentiation. This study provides insight into the molecular mechanisms underlying the effects of Ta NPs in bone regeneration.

LncRNA Urothelial Carcinoma-Associated 1 (UCA1) Regulates Rat Bone Marrow Mesenchymal Stem Cell Differentiation and Promotes Repair of Bone Defects

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 &lt; 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 &lt; 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.

Yam-Containing Serum Promotes Proliferation and Chondrogenic Differentiation of Rabbit Bone Marrow Mesenchymal Stem Cells and Synthesis of Glycosaminoglycan

The chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is affected by a variety of factors, including environmental, physical, and chemical factors and growth factors, and traditional Chinese medicine (TCM) preparations can further influence this process. In this study, the effects of different concentrations of Yam-containing serum of rabbits on BMSC proliferation and chondrogenic differentiation were investigated, as were the underlying molecular mechanisms. The growth and proliferation of BMSCs were significantly enhanced upon treatment with Yam-containing serum. Under both monolayer and micromass culture conditions, Yam-containing serum promoted the differentiation of BMSCs into chondrocytes. Toluidine blue staining results revealed that chondrocyte differentiation in the group treated by Yam-containing serum was significantly more pronounced than in the control group. Glycosaminoglycan levels, as measured by 1,2-dimethylmethylene blue (DMMB) detection, were significantly higher in cells of the Yam-containing group relative to the control group. This is the first study to our knowledge that demonstrates that Yam-containing serum can promote BMSC proliferation and chondrogenic differentiation. This study therefore lays an experimental groundwork for further application of TCM as a means of treating degenerative cartilage diseases and provides an experimental and theoretical basis for the combination of TCM and stem cells for the treatment of such diseases.

The Role of Integrin-Linked Kinase in Regulating Bone Differentiation of Metabolic Syndrome-Derived Bone Marrow Mesenchymal Stem Cells Through Autophagy

Bone marrow mesenchymal stem cells (BMSCs) can differentiate into osteogenesis. Integrin-linked kinase (ILK) regulates several biological processes. However, whether ILK affects metabolic syndrome (MS)-derived BMSCs differentiation remains unclear. SD rats were divided into control group and MS group. Diabetic rat model was prepared. BMSCs were divided into control group, MS group and ILK group, in which ILK plasmid was transfected into BMSCs from MS group followed by analysis of ILK, Bcl-2, Bax, RUNX2 and OPN expression by real time PCR, BMSCs proliferation by MTT assay, BMSCs apoptosis, expression of Beclin-3 and LC-3 by Western blot as well as secretion of IL-1β and IL-6 by ELISA. MS group showed significantly reduced BMSCs proliferation, elevated Caspase 3 activity, downregulated Bcl-2, RUNX2 and OPN expression, upregulated Bax level and increased IL-1β and IL-6 secretion as well as decreased Beclin-3 and LC-3 expression compared to control group (P &lt; 0.05). BMSCs with ILK overexpression in high glucose presented significantly promoted BMSCs proliferation, decreased Caspase 3 activity, increased Bcl-2, RUNX2 and OPN expression, decreased Bax expression and IL-1β and IL-6 secretion as well as reduced Beclin-3 and LC-3 expression compared to MS group (P &lt; 0.05). ILK expression in MS-derived BMSCs is decreased. ILK overexpression in BMSCs can promote autophagy, inhibit apoptosis and inflammation, and promote their differentiation into osteoblasts.

Osteoprotegerin Protects H2O2-Induced Bone Marrow Mesenchymal Stem Cells Damage

Oxidative stress causes damage and apoptosis of bone marrow mesenchymal stem cells (BMSCs). Osteoprotegerin (OPG) regulates bone differentiation. However, whether OPG plays a role in osteogenic/adipogenic differentiation under oxidative stress remains poorly understood. Rat BMSCs were randomly divided into control group; H2O2 group, in which H2O2 was used to prepare oxidative stress model; OPG group, in which 5 μM OPG was added to BMSCs based on oxidative stress model followed by analysis of BMSCs proliferation by MTT assay, BMSCs apoptosis, expression of RUNX2, OPN, FABP4 and PPARγ2 by real time PCR, OPG and RANKL protein expression by Western blot as well as SOD activity and ROS content by spectrophotometry. Compared with control group, H2O2 significantly decreased BMSCs proliferation, increased Caspase 3 activity, downregulated RUNX2 and OPN, upregulated FABP4 and PPARγ2, decreased SOD activity and increased ROS content (P &lt; 0.05). Under H2O2 treatment, OPG addition significantly promoted BMSCs proliferation, decreased Caspase 3 activity, increased RUNX2 and OPN expression, decreased FABP4 and PPARγ2 expression, increased SOD activity, and decreased ROS content and RANKL expression (P &lt; 0.05). OPG can promote osteogenic differentiation of BMSCs under oxidative stress, inhibit adipogenic differentiation, and protect BMSCs from damage and promote BMSCs proliferation by regulating OPG/RANKL signaling pathway.