Mineralization Of Bone Marrow Mesenchymal Stem Cells Research Articles

SIRT1/Notch1 signal axis involves in the promoting effect of Segetalin B on bone formation.

Phytoestrogens are a class of potential natural medicines for treating postmenopausal osteoporosis (PMOP). Segetalin B (SB) is a cyclic peptide compound showing estrogenic activity. This study reports the effect of SB on bone formation among ovariectomized (OVX) rats. The bone marrow mesenchymal stem cells (BMSCs) from OVX rats were cultured in vitro. Alizarin Red staining was utilized to observe the effect of SB on the mineralization of BMSCs. The levels of alkaline phosphatase (ALP), osteocalcin, bone morphogenetic protein (BMP-2), and Sirtuin 1 (SIRT1) activities were detected. The OVX rats were treated with SB in vivo. Micro-CTwas utilized for imaging analysis. Urine calcium and phosphorus, and ALP activity in bone marrow were assayed. Western blot analysis and immunofluorescence were incorporated to detect protein expressions in vitro and in vivo. The results showed that SB dose-dependently promoted mineralization of OVX rat-derived BMSCs in vitro increased the level of Osteocalcin, BMP-2, ALP, and SIRT1 activity. Moreover, it upregulated expressions of Runx2, Osterix, and SIRT1, downregulated expressions of Notch intracellular domain (NICD), acetyl-NICD, and hairy and enhancer of split 1 (Hes1). In addition, SB treatment significantly decreased bone loss, inhibited calcium and phosphorus loss, elevated ALP activity, upregulated Runx2, Osterix, and SIRT1, and downregulated NICD and Hes1 in OVX rats in vivo. However, EX527, a SIRT1-selective inhibitor, could reverse the above effects of SB in vitro or in vivo. These results indicate that SB is a potential natural medicine to improve PMOP. Thus, its mechanism of promoting bone formation involves the SIRT1/Notch1 signaling axis.

LncRNA KCNQ1OT1 promotes osteogenic differentiation via miR-205-5p/RICTOR axis

Osteoporosis is a prevalent degenerative disease that is characterized by decreased bone density and strength, resulting in gradually increasing bone fragility. Osteoporosis is caused by an imbalance between osteoblastic bone formation and osteoclastic bone resorption. Recently, increasing evidence has suggested that long non-coding RNAs (lncRNAs) participate in the occurrence and development of osteoporosis. Herein, we explored the role of lncRNA KCNQ1OT1 in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). QPCR results indicated that KCNQ1OT1 and RICTOR were down-regulated, while miR-205-5p was up-regulated in the osteoporotic patients, as compared with non-osteoporotic controls. During the osteogenic differentiation of BMSCs, the expression of KCNQ1OT1 and RICTOR was upregulated, whereas miR-205-5p was downregulated. The interaction among KCNQ1OT1, miR-205-5p and RICTOR was validated by dual luciferase reporter system. KCNQ1OT1 promoted RICTOR expression via inhibiting miR-205-5p, therefore promoting osteogenesis as demonstrated by ALP assay, alizarin red staining and the increased expression of osteogenic markers (OPN, RUNX2 and OCN). Furthermore, KCNQ1OT1 overexpression or miR-205-5p inhibition could promote ALP activity and mineralization of BMSCs, while overexpressed miR-205-5p could reverse the effects of overexpressed KCNQ1OT1, and knockdown of RICTOR could reverse the effects of miR-205-5p inhibition. In conclusion, our study illustrated that KCNQ1OT1 might inhibit miR-205-5p in BMSCs, thus upregulating the expression of RICTOR and promoting osteogenic differentiation.

Brown Adipose Tissue Rescues Bone Loss Induced by Cold Exposure.

Cold temperature activates the sympathetic nervous system (SNS) to induce bone loss by altering bone remodeling. Brown adipose tissue (BAT) is influenced by the SNS in cold environments. Many studies have confirmed a positive relationship between BAT volume and bone mass, but the influence and mechanism of BAT on bone in vivo and in vitro is still unknown. Two-month-old C57/BL6j male mice were exposed to cold temperature (4°C) to induce BAT generation. BAT volume, bone remodeling and microstructure were assessed after 1 day, 14 days and 28 days of cold exposure. CTX-1, P1NP and IL-6 levels were detected in the serum by ELISA. To determine the effect of BAT on osteoclasts and osteoblasts in vitro, brown adipocyte conditional medium (BAT CM) was collected and added to the differentiation medium of bone marrow-derived macrophages (BMMs) and bone marrow mesenchymal stem cells (BMSCs). Micro-CT results showed that the bone volume fraction (BV/TV, %) significantly decreased after 14 days of exposure to cold temperature but recovered after 28 days. Double labeling and TRAP staining in vivo showed that bone remodeling was altered during cold exposure. BAT volume enlarged after 14 days of cold stimulation, and IL-6 increased. BAT CM promoted BMSC mineralization by increasing osteocalcin (Ocn), RUNX family transcription factor 2 (Runx2) and alkaline phosphatase (Alp) expression, while bone absorption was inhibited by BAT CM. In conclusion, restoration of bone volume after cold exposure may be attributed to enlarged BAT. BAT has a beneficial effect on bone mass by facilitating osteogenesis and suppressing osteoclastogenesis.

Nano-Hydroxyapatite/Polyamide Can Repair and Reconstruct Spine by Accelerating Proliferation and Mineralization of Bone Marrow Mesenchymal Stem Cells

To determine the impact of nano-hydroxyapatite/polyamide (n-HA/PA) on proliferation and mineralization of bone marrow mesenchymal stem cells (BMMSCs). An optical microscope was used to determine the number of adhesive cells, CKK-8 for determining cell proliferation, ALP activity assay along with ARS for evaluating mineralization, qRT-PCR for quantifying gene expression of osteopontin, osteonectin, collagen type I, DMP-1, lncRNA NEAT1, and miR-186, Western blotting for quantifying their protein, Starbase v.3.0 for forecasting binding sites of lncRNA NEAT1 with miR-186, and dual-luciferase reporter gene assay for verifying the binding sites. The n-HA/PA group presented enhanced the proliferation of BMMSCs and ALP activities, increased calcium deposition strength and adhesive cells, upregulated osteopontin, osteonectin, collagen type I, and DMP-1 proteins and NEAT1 expression and downregulated miR-186 than the control group not covered with n-HA/PA. Additionally, miR-186 is the downstream specific gene of lncRNA NEAT1. It can be seen from this, n-HA/PA can help repair and reconstruct the spine by accelerating the proliferation and mineralization of BMMSCs.

Enamel matrix derivative (EMD) enhances the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs)

ABSTRACT To investigate the EMD’s capacity in BMSCs osteogenic differentiation. In vivo and in vitro, BMSCs were treated with EMD, scanning electron microscopy, and Alizarin Red staining were used to detect the changes in the osteogenic ability of BMSCs, and the proliferation ability of BMSCs was evaluated by CCK8. In addition, by adding xav939, a typical inhibitor of Wnt/β-catenin signaling pathway, the regulatory function of Wnt/β-catenin signaling was clarified. The results showed that EMD promote cell proliferation and 25 μg/ml EMD had the most significant effect. Cells inducing osteogenesis for 2 and 3 even 4 weeks, the cell staining is deeper in EMD treated group than that of the control (P < 0.05) by alizarin Red staining, suggesting more mineralization of BMSCs. In vivo implanting the titanium plate wrapped with 25 μg/ml EMD treated-BMSC film into nude mice for 8 weeks, more nodules were formed on the surface of the titanium plate than that the control (P < 0.05). HE showed that there is a little blue-violet immature bone-like tissue block. Besides, the expression of RUNX Family Transcription Factor 2 (Runx2), Osterix, Osteocalcin (OCN), collagen I (COLI), alkaline phosphatase (ALP) and β-catenin were inhibited in xav939 group (P < 0.05); Inversely, all were activated in EMD group (P < 0.05). In conclusion, EMD promoted the proliferation and osteogenic differentiation of BMSCs. EMD’s function on BMSCs might be associated with the Wnt/β-catenin signaling pathway.

Macrophages enhance mesenchymal stem cell osteogenesis via down-regulation of reactive oxygen species

ObjectivesThe role played by macrophages in regulating the differentiation of mesenchymal stem cells (MSCs) during wound healing and bone regeneration is increasingly being recognized. The present study compared the pro-osteogenic effects of three co-culture methods, conditioned medium generated by macrophages (CM), indirect culture (IC) or direct culture (DC) with macrophages, on bone marrow MSCs (BMMSCs). MethodsPrimary BMMSCs were isolated, characterized and co-cultured with RAW264.7 mouse macrophages. Cell morphology and intracellular reactive oxygen species (ROS) levels were determined by scanning electron microscopy (SEM) and flow cytometry, respectively. Alkaline phosphatase (ALP) staining and assay, Alizarin red staining (ARS) and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to evaluate osteogenic differentiation. ResultsInclusion of macrophages in any of the three co-culture methods resulted in improvement in osteogenic differentiation and mineralization of BMMSCs (DC > IC > CM), as measured by ALP staining and activity, ARS and osteoblastic gene expression (Runx2, Alp, Ocn and Bmp2). The enhanced osteogenesis was reversed with hydrogen peroxide. Macrophages reduced the increased levels of intracellular ROS generated by BMMSCs during osteogenic differentiation in a manner similar to the use of an antioxidant, N-acetyl cysteine. ConclusionsMacrophages exert an osteogenesis-enhancing effect to accelerate BMMSC osteogenesis via ROS downregulation. Clinical significanceThe present findings suggest that targeting MSC-macrophage interaction is an effective strategy for regulating stem cell fate and facilitating bone regeneration.

(*)AG490 could suppress bone marrow mesenchymal stem cells migration, mineralization and bone defect healing via inhibiting Jak2-STAT3 pathway

Objective: To study the effect of Jak2-STAT3 pathway on cell proliferation, migration, mineralization and bone defect healing via simulating Jak2-STAT3 pathway inhibitor AG490 to bone marrow mesenchymal stem cells (BMSC) and bone defect mice models. Methods: The effect of AG490 on BMSC proliferation was measured by MTT (methyl thiazolyl tetrazolium) assay. Regulation of AG490 on BMSC migration was tested by scratch assay and transwell assay. The BMSC migration related gene, matrix metalloproteinase (MMP)-7, MMP-9 and CXC subfamily receptor 4 (CXCR4), regulated by AG490 was studied by real-time PCR. Western blotting was adopted to analyze the regulation of Jak2-STAT3 phosphorylation through the simulation of AG490. The alizarin red staining and alkaline phosphatase (ALP) activity assay were performed to measure the effect of AG490 on BMSC mineralization and osteogenic differentiation. Mice femur bone defect models were built to analyzed the effect of AG490 on bone remodeling. Results: AG490 significantly suppressed the migration rate of BMSC at 1 d and 2 d in the experiment group [(12.42±7.50) %, (41.8±2.6)%] compared with the control group [(55.5±9.9)%, (86.9±8.7)%] in scratch assay (P=0.000, P=0.000), the number of migrated BMSC in the experiment group (22.8± 5.9) was significantly suppressed compared with the control group (58.3±6.6) in Transwell assay (P=0.000). The expression of MMP-7, MMP-9 and CXCR4 were significantly downregulated in experiment group [(0.5± 0.1), (0.1±0.1) and (0.35±0.07)] compared with the control group [(1.1±0.1), (1.06±0.33), (1.08±0.13)] (P= 0.0003, P=0.000 and P=0.000). Also, the phosphorylation of Jak2-STAT3 was downregulated by AG490 in western blotting. After BMSCs were osteogenic induced for 14 days, the formation of mineralized nodule and the ALP activity of BMSC is significantly suppressed in experiment group (8.0±2.1) compared with the control group (35.7 ± 1.8) (P=0.0005). AG490 suppressed the bone defects healing,the expression level of phosphorylated Jak2 and phosphorylated STAT3, and the number of alkaline phosphatase positive cell at defect area in vivo is lower in experiment group than the control group. AG490 suppressed the relatively bone density at the defect area significantly (P=0.0004) at 5(th) week after the surgery. Conclusions: AG490 could suppress proliferation, migration and mineralization to of BMSCs regulate bone defect healing via inhibiting Jak2-STAT3 pathway.

The combined effects of nanotopography and Sr ion for enhanced osteogenic activity of bone marrow mesenchymal stem cells (BMSCs).

Both surface topography and chemistry have a significant influence on the biological performance of orthopedic implant coatings. In our study, a surface modification strategy embodying bioactive trace element incorporation and nanotopography construction was employed to enhance the osteogenic activity of calcium silicate (Ca-Si) coatings. We developed strontium-loaded nanolayer on plasma sprayed Ca-Si (CS) coating via hydrothermal treatment which was denoted as Sr-NT-CS. The original CS coating and the CS coating modified with similar nanotopography (NT-CS) were studied in parallel. We investigated the cellular effects of surface topography and released Sr ion on the adhesion, proliferation, differentiation, and mineralization of BMSCs and the associated molecular mechanisms. The results indicated that the nanotopography activated integrin β1, promoted the spread of BMSCs into a polygonal osteoblastic shape, and induced higher levels of collagen secretion. The Sr incorporation stimulated osteogenic differentiation and mineralization as indicated by the increases in ALP activity and mineralized nodules formation. The examination of gene expressions revealed that Sr ion exerted the effects by interacting with extracellular calcium sensitive receptor (CaSR), and combined with the nanotopographical cue for the up-regulation of osteogenic master transcription factor Runx2. The promoted Runx2 subsequently affected osteoblast (OB) marker genes (BMP-2, BSP, OPN, and OCN), thus driving BMSCs to differentiate into OBs. Moreover, the Sr incorporation inhibited osteoclastogenesis, as indicated by the down-regulation of interleukin-6 (IL-6) and the inhibition of RANKL/RANK system. Those results suggested that our developed Sr-NT-CS coating have combined the effects of nanotopography and Sr ion for enhanced osteogenic activity of BMSCs.