Osteogenesis Of Bone Marrow Mesenchymal Stem Cells Research Articles

Investigation for GSK3β expression in diabetic osteoporosis and negative osteogenic effects of GSK3β on bone marrow mesenchymal stem cells under a high glucose microenvironment

Osteoporosis is a common skeletal complication of diabetes mellitus (DM). The mechanisms underlying the pathophysiology of diabetic osteoporosis are complex. Glycogen synthase kinase-3β (GSK-3β) is a widely expressed serine/threonine kinase and associated with both DM and bone metabolism, which arouse our concern. In this study, we established the diabetic mouse model by high-fat diet combined with streptozotocin injection. Decreased bone mass and reduced osteogenesis were observed in femurs of the mice. Besides, we identified that there is an activated expression of GSK3β in the bone marrow mesenchymal stem cells (BMSCs) of diabetic mice. To explore the link between GSK3β and diabetic osteoporosis, we exposed BMSCs to a high glucose microenvironment in vitro and discovered that the glucose-induced GSK3β activation has negative osteogenic effects on BMSCs by suppressing β-catenin/Tcf7/Ccn4 signaling axis. Inhibition of GSK3β by specific concentrations of LiCl could reverse the impaired osteogenesis of BMSCs and increase expression of β-catenin, Tcf7 and Ccn4. Our research indicated that abnormal activation of GSK3β plays a role in diabetic osteoporosis and might be a potential target to treat diabetic osteoporosis.

Interleukin-4 assisted calcium-strontium-zinc-phosphate coating induces controllable macrophage polarization and promotes osseointegration on titanium implant

Titanium (Ti) and its alloys are believed to be promising scaffold materials for dental and orthopedic implantation due to their ideal mechanical properties and biocompatibility. However, the host immune response always causes implant failures in the clinic. Surface modification of the Ti scaffold is an important factor in this process and has been widely studied to regulate the host immune response and to further promote bone regeneration. In this study, a calcium-strontium-zinc-phosphate (CSZP) coating was fabricated on a Ti implant surface by phosphate chemical conversion (PCC) technique, which modified the surface topography and element constituents. Here, we envisioned an accurate immunomodulation strategy via delivery of interleukin (IL)-4 to promote CSZP-mediated bone regeneration. IL-4 (0 and 40 ng/mL) was used to regulate immune response of macrophages. The mechanical properties, biocompatibility, osteogenesis, and anti-inflammatory properties were evaluated. The results showed that the CSZP coating exhibited a significant enhancement in surface roughness and hydrophilicity, but no obvious changes in proliferation or apoptosis of bone marrow mesenchymal stem cells (BMMSCs) and macrophages. In vitro, the mRNA and protein expression of osteogenic related factors in BMMSCs cultured on a CSZP coating, such as ALP and OCN, were significantly higher than those on bare Ti. In vivo, there was no enhanced bone formation but increased macrophage type 1 (M1) polarization on the CSZP coating. IL-4 could induce M2 polarization and promote osteogenesis of BMMSCs on CSZP in vivo and in vitro. In conclusion, the CSZP coating is an effective scaffold for BMMSCs osteogenesis, and IL-4 presents the additional advantage of modulating the immune response for bone regeneration on the CSZP coating in vivo.

ASPH Regulates Osteogenic Differentiation and Cellular Senescence of BMSCs.

Osteogenesis and senescence of BMSCs play great roles in age-related bone loss. However, the causes of these dysfunctions remain unclear. In this study, we identified a differentially expressed ASPH gene in middle-aged and elderly aged groups which were obtained from GSE35955. Subsequent analysis in various databases, such as TCGA, GTEx, and CCLE, revealed that ASPH had positive correlations with several osteogenic markers. The depletion of mouse Asph suppressed the capacity of osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). Notably, the expression of ASPH in vitro decreased during aging and senescence. The deficiency of Asph accelerated cellular senescence in BMSCs. Conversely, the overexpression of Asph enhanced the capacity of osteogenic differentiation and inhibited cellular senescence. Mechanistically, ASPH regulated Wnt signaling mediated by Gsk3β. Taken together, our data established that ASPH was potentially involved in the pathogenesis of age-related bone loss through regulating cellular senescence and osteogenic differentiation, which provides some new insights to treat age-related bone loss.

Effect of lentivirus-mediated silencing of P75 neurotrophin receptor gene on osteogenic differentiation of bone marrow mesenchymal stem cells in rats

To investigate the effect of small interfering RNA (siRNA) lentivirus-mediated silencing of P75 neurotrophin receptor (P75NTR) gene on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in rats. Three lentivirus-mediated P75NTR gene siRNA sequences (P75NTR-siRNA-1, 2, 3) and negative control (NC)-siRNA were designed and transfected into the 3rd generation Sprague Dawley (SD) rat BMSCs. The cells morphological changes were observed under an inverted microscope, and the expressions of P75NTR gene and protein in cells were detected by real-time fluorescence quantitative PCR and Western blot. Then the best silencing P75NTR-siRNA for subsequent osteogenic differentiation experiments was screened out. The 3rd generation SD rat BMSCs were randomly divided into experimental group, negative control group, and blank control group (normal BMSCs). The BMSCs of negative control group and experimental group were transfected with NC-siRNA and the selected P75NTR-siRNA lentiviral vector, respectively. The cells of each group were cultured by osteogenic induction. The expressions of osteogenic related proteins [osteocalcin (OCN) and Runx related transcription factor 2 (Runx2)] were detected by Western blot; the collagen type Ⅰ expression was observed by immunohistochemical staining; the osteogenesis of BMSCs was observed by alkaline phosphatase (ALP) detection and alizarin red staining. After lentivirus-mediated P75NTR transfected into BMSCs, the expressions of P75NTR mRNA and protein significantly reduced ( P<0.05), and the best silencing P75NTR-siRNA was P75NTR-siRNA-3. After P75NTR gene was silenced, MTT test showed that the cell proliferation in the experimental group was significantly faster than those in the two control groups ( P<0.05). After osteogenic induction, the relative expressions of OCN and Runx2 proteins, collagen type Ⅰ expression, and ALP activity were significantly higher in the experimental group than in the two control groups, the differences were significant ( P<0.05). With the prolongation of osteogenic induction, the mineralized nodules in the experimental group gradually increased. Silencing the P75NTR gene with siRNA lentivirus can promote the osteogenic differentiation of rat BMSCs and provide a new idea for the treatment of bone defects.

Downregulated brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein-1 inhibits osteogenesis of BMSCs through p53 in type 2 diabetes mellitus.

ABSTRACTThe bone marrow mesenchymal stem cells (BMSCs)-mediated abnormal bone metabolism can delay and impair the bone remodeling process in type 2 diabetes mellitus (T2DM). Our previous study demonstrated that the downregulation of brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1), a circadian clock protein, inhibited the Wnt/β-catenin pathway via enhanced GSK-3β in diabetic BMSCs. In this article, we confirmed that the downregulated BMAL1 in T2DM played an inhibitory role in osteogenic differentiation of BMSCs. Upregulation of BMAL1 in the diabetic BMSCs significantly recovered the expression pattern of osteogenic marker genes and alkaline phosphatase (Alp) activity. We also observed an activation of the p53 signaling pathways, exhibited by increased p53 and p21 in diabetic BMSCs. Downregulation of p53 resulting from overexpression of BMAL1 was detected, and when we applied p53 gene silencing (shRNA) and the p53 inhibitor, pifithrin-α (PFT-α), the impaired osteogenic differentiation ability of diabetic BMSCs was greatly restored. However, there was no change in the level of expression of BMAL1. Taken together, our results first revealed that BMAL1 regulated osteogenesis of BMSCs through p53 in T2DM, providing a novel direction for further exploration of the mechanism underlying osteoporosis in diabetes.

Long non-coding RNA MIR22HG promotes osteogenic differentiation of bone marrow mesenchymal stem cells via PTEN/ AKT pathway

Osteoporosis is a prevalent metabolic bone disease characterized by low bone mineral density and degenerative disorders of bone tissues. Previous studies showed the abnormal osteogenic differentiation of endogenous bone marrow mesenchymal stem cells (BMSCs) contributes to the development of osteoporosis. However, the underlying mechanisms by which BMSCs undergo osteogenic differentiation remain largely unexplored. Recently, long non-coding RNAs have been discovered to play important roles in regulating BMSC osteogenesis. In this study, we first showed MIR22HG, which has been demonstrated to be involved in the progression of several cancer types, played an important role in regulating BMSC osteogenesis. We found the expression of MIR22HG was significantly decreased in mouse BMSCs from the osteoporotic mice and it was upregulated during the osteogenic differentiation of human BMSCs. Overexpression of MIR22HG in human BMSCs enhanced osteogenic differentiation, whereas MIR22HG knockdown inhibited osteogenic differentiation both in vitro and in vivo. Mechanistically, MIR22HG promoted osteogenic differentiation by downregulating phosphatase and tensin homolog (PTEN) and therefore activating AKT signaling. Moreover, we found MIR22HG overexpression promoted osteoclastogenesis of RAW264.7 cells, which indicated that MIR22HG played a significant role in bone metabolism and could be a therapeutic target for osteoporosis and other bone-related diseases.

Effect of αCGRP on Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells Through Smad/Runx2 Signaling Pathway

The α-calcitonin gene-related peptide (α-CGRP) regulates bone metabolism and has potential applications in enhancing bone remodeling in vivo. However, α-CGRP's role in bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation remain unclear. Rat BMSCs were separated into control group, α-CGRP group and α-CGRP siRNA group, in which BMSCs were transfected with α-CGRP plasmid and α-CGRP siRNA respectively followed by analysis of α-CGRP level by real time PCR and ELISA, cell proliferation by MTT assay, Caspase 3 activity, ALP activity, formation of calcified nodules by alizarin red staining, Smad1 and Smad7 level by Western blot and Runx2 by real time PCR. αCGRP transfection into BMSCs significantly up-regulated CGRP, which could promote cell proliferation, inhibit Caspase 3 activity, promote ALP activity, increase calcified nodules formation and upregulate Smad1, Smad7 and Runx2 compared to control (P &lt; 0.05); transfection of αCGRP siRNA significantly down-regulated CGRP in BMSCs, inhibited cell proliferation, promoted Caspase 3 activity, inhibited ALP activity, inhibited calcified nodules formation and downregulate Smad1, Smad7 and Runx2 (P &lt; 0.05). αCGRP overexpression promotes the Smad/Runx2 signaling, which in turn promotes BMSCs proliferation and osteogenesis. Decreased αCGRP level inhibits Smad/Runx2 signaling, promotes BMSCs apoptosis, inhibits proliferation and osteogenic differentiation.

Increased Expression of Sox9 during Balance of BMSCs/Chondrocyte Bricks in Platelet-Rich Plasma Promotes Construction of a Stable 3-D Chondrogenesis Microenvironment for BMSCs.

Sox9 is an intrinsic transcription factor related to the determination and maintenance of chondrogenic lineage of bone marrow mesenchymal stem cells (BMSCs). In recent research, we have proved that fragmented chondrocyte aggregates (cell bricks) could promote chondrogenesis of BMSCs in vivo. However, it is still unknown whether the ratio of BMSCs/chondrocyte bricks has a significant influence on 3-D cartilage regeneration and related molecular mechanism. To address this issue, the current study subcutaneously injected three groups of cell complex with different rabbit BMSCs/chondrocyte bricks' ratios (1 : 2, 1 : 1, and 2 : 1) into nude mice. Gross morphology observation, histological and immunohistochemical assays, biochemical analysis, gene expression analysis, and western blot were used to compare the influence of different BMSCs/chondrocyte bricks' ratios on the properties of tissue-engineered cartilage and explore the related molecular mechanism. The constructs of 1 : 1 BMSCs/chondrocyte bricks, (B1CB1) group resulted in persistent chondrogenesis with appropriate morphology and adequate central nutritional perfusion without ossification. The related mechanism is that increased expression of Sox9 in the B1C1 group promoted chondrogenesis and inhibited the osteogenesis of BMSCs through upregulating Col-II as well as downregulating RUNX2 and downstream of Col-X and Col-I by upregulating Nkx3.2. This study demonstrated that BMSCs/chondrocyte bricks 1:1 should be a suitable ratio and the Sox9-Nkx3.2-RUNX2 pathway was a related mechanism which played an important role in the niche for stable chondrogenesis of BMSCs constructed by chondrocyte bricks and PRP.

Long Noncoding RNA Metastasis-Associated Lung Carcinoma Transcript 1 (MALAT1) Inhibits Bone Marrow Mesenchymal Stem Cell Formation Through Regulating Smad/Runx2 Signaling Pathway

Osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is controlled by elaborate genetic programs. lncRNA MALAT1 plays an important role in many diseases. However, the role of lncRNA MALAT1 in BMSCs remains unclear. Isolated Rat BMSCs were cultured and randomly divided into control group, MALAT1 group and MALAT1 siRNA group, in which lncRNA MALAT1 plasmid and lncRNA MALAT1 siRNA were transfected into BMSCs followed by analysis of lncRNA MALAT1 expression by real time PCR, cell proliferation by MTT assay, Caspase 3 activity, ALP activity was analyzed, calcified nodules by alizarin red staining, expression of Smad1 and Smad7 by Western blot as well as Runx2 expression by real time PCR. In MALAT1 group, MALAT1 expression was significantly increased along with significantly inhibited cell proliferation, increased Caspase 3 activity, decreased ALP activity and calcified nodules, reduced expression of Smad1, Smad7 and Runx2 compared with control group (P &lt; 0 05). MALAT1 expression in MALAT1 siRNA group was decreased with significantly promoted cell proliferation, decreased Caspase 3 activity, increased ALP activity and calcified nodules, as well as significantly elevated expression of Smad1, Smad7 and Runx2 compared with control group (P &lt; 0 05). Up-regulation of lncRNA MALAT1 expression inhibits the Smad/Runx2 signaling pathway, thereby inhibiting BMSCs proliferation and osteogenesis. Down-regulation of lncRNA MALAT1 expression promotes Smad/Runx2 signaling pathway activation, inhibits BMSCs apoptosis, promotes proliferation and osteogenic differentiation.

Low expression of miR-1 promotes osteogenic repair of bone marrow mesenchymal stem cells by targeting TLR1.

Bone marrow mesenchymal stem cells (BMSCs) promote bone tissue repair. MiR-1 regulates myogenic and osteogenic differentiation of human adipose tissue stem cells. However, miR-1's effect on BMSCs osteogenesis is unclear. Rat BMSCs were isolated and divided into control group, miR-1 group, and si-miR-1 group respectively transfected with miR-1 plasmid and miR-1 siRNA followed by analysis of cell proliferation by MTT assay and Caspase 3 activity. The expression of osteogenic genes Runx2 and OPN was measured by Real Time-PCR. Healthy male Sprague-Dawley rats were separated into fracture group, NC group, and si-miR-1 group followed by analysis of bone mineral density, miR-1 level by Real Time-PCR, type I collagen, and BMP-2 by enzyme-linked immunosorbent assay (ELISA), and TLR1 expression by Western blot. Transfection of miR-1 siRNA into BMSCs significantly downregulated miR-1 expression, promoted BMSCs cell proliferation, inhibited Caspase 3 activity, as well as promoted osteogenic genes Runx2 and OPN expression and decreased TLR1 expression (p<0.05). The upregulation of miR-1 expression significantly reversed the above changes. TLR1 is a target of miR-1. Downregulation of miR-1 expression in BMSCs of fractured rats significantly increased bone density and ALP activity, promoted type I collagen and BMP-2 expression, and decreased TLR1 expression (p<0.05). The downregulation of miR-1 promotes BMSCs osteogenic differentiation via targeting TLR1, which promotes osteogenic differentiation and bone healing.

Vasoactive Intestinal Peptide Stimulates Bone Marrow-Mesenchymal Stem Cells Osteogenesis Differentiation by Activating Wnt/β-Catenin Signaling Pathway and Promotes Rat Skull Defect Repair.

Bone defect regeneration is a complex process that involves the coordination of a variety of different type of cells. As bone tissues are innervated and rich in nerve fibers, the neuropeptides released from various never fibers could regulate bone development, metabolism, and remodeling. Among all the neuropeptides, vasoactive intestinal peptide (VIP) could modulate the functions of both osteoblasts and osteoclasts, and may play a vital role in bone marrow mesenchymal stem cell (BMSC) osteogenesis during bone repair. In this study, we investigated the role of VIP in bone formation and the mechanisms of VIP in mediating BMSC osteogenic differentiation, and its possibility in clinical application of bone defect reconstruction. Our in vitro study results indicated that VIP promoted BMSC osteogenic differentiation by activating Wnt/β-catenin signaling pathway in BMSCs. VIP could also stimulate tube formation of EA.hy926 endothelial cell and increase vascular endothelial growth factor (VEGF) expression in BMSCs. Furthermore, in the rat skull defect model, VIP-conjugated functionalized hydrogel significantly enhanced cranial bone defect repair compared with the control group, with increased bone formation and angiogenesis. Taken together, as a member of neuropeptides, VIP could promote the BMSCs osteogenesis and angiogenesis differentiation in vitro and stimulate bone repair in vivo by activating Wnt/β-catenin signaling pathway. The knowledge obtained from this study emphasized the close association between innervation and bone repair process, and VIP may be a potential therapeutic agent for augmenting bone repair.

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.

RAI3 knockdown enhances osteogenic differentiation of bone marrow mesenchymal stem cells via STAT3 signaling pathway

Bone marrow mesenchymal stem cells (BMSCs), which have multipotential differentiation and self-renewal ability, have been becoming an attractive source of seed cells for bone tissue engineering. Nonetheless, the precise underlying mechanisms of osteogenesis of BMSCs have not been fully understood. Retinoic acid-induced gene 3 (RAI3) has been found to play important roles in mesenchymal stem cells (MSCs) adipogenesis in our previous study. However, its function in the osteogenic differentiation of BMSCs remains unknown. In this study, we found that RAI3 was significantly reduced in osteogenically differentiated BMSCs; RAI3 knockdown promoted osteogenesis of BMSCs both in vitro and in vivo. Moreover, we found RAI3 knockdown significantly upregulated the expression level of phosphorylated signal transducer and activator of transcription 3 (p-STAT3), and AG-490 which can inhibit the STAT3 signaling reversed the enhancing effect of RAI3 knockdown on the osteogenic differentiation of BMSCs. These results suggest that RAI3 plays important roles in BMSCs osteogenesis with an involvement of the STAT3 signaling, which might open a new avenue to explore BMSCs osteogenesis for the application of BMSCs in bone regeneration.

Zoledronic acid accelerates osteogenesis of bone marrow mesenchymal stem cells by attenuating oxidative stress via the SIRT3/SOD2 pathway and thus alleviates osteoporosis.

The aim of this study was to clarify the potential effect of zoledronic acid on alleviating oxidative stress and promoting bone marrow mesenchymal stem cells (BMSCs) osteogenesis through the SIRT3/SOD2 pathway, thus alleviating the progression of osteoporosis. Relative expression levels of osteogenesis-related genes (ALP, RUNX2, and Bglap) were determined. Meanwhile, ALP activity and capacity of mineralization in BMSCs treated with different doses of zoledronic acid were measured. Subsequently, viability and ROS level in H2O2-induced BMSCs influenced by zoledronic acid treatment were assessed. The regulatory effect of zoledronic acid on the SIRT3/SOD2 pathway was detected by Western blot. Furthermore, the involvement of the SIRT3/SOD2 pathway in zoledronic acid-mediated BMSCs osteogenesis was evaluated. Zoledronic acid treatment significantly up-regulated the levels of ALP, RUNX2, and Bglap. Meanwhile, it improved ALP activity and capacity of mineralization in BMSCs dose-dependently. H2O2 induction markedly suppressed viability and enhanced ROS level in BMSCs, which were reversed by zoledronic acid treatment. Besides, zoledronic acid protected H2O2-induced SIRT3 down-regulation and AC-SOD2/SOD2 up-regulation in BMSCs. In addition, silence of SIRT3 reversed the protective effects of zoledronic acid on osteogenesis of BMSCs. Zoledronic acid alleviates the progression of osteoporosis. Meanwhile, it accelerates BMSCs osteogenesis by inhibiting oxidative stress via the SIRT3/SOD2 pathway.

Overexpression of Bone Morphogenetic Protein-1 Promotes Osteogenesis of Bone Marrow Mesenchymal Stem Cells In Vitro.

BackgroundOsteogenesis of bone marrow mesenchymal stem cells (BMSCs) is an important research topic in the application of bone tissue engineering. Bone morphogenetic protein-1 (BMP-1) is important in bone formation and stability, but its effects on the osteogenesis of BMSCs are unclear. This study aimed to investigate the association of BMP-1 with the osteogenic capacity of BMSCs.Material/MethodsPrimary rabbit BMSCs were cultured and divided into a BMP-1-overexpressing group, a Green Fluorescent Protein-expressing (GFP) group, and a Control group. The transfection efficiency of BMP-1 was tested by Western blotting. Cell viabilities, alkaline phosphatase (ALP) activities, Ca2+ concentrations, and gross examinations of BMSC sheets were examined at different times. The osteogenic marker collagen I was assessed by immunohistochemical analysis.ResultsThe cell viability, ALP activity, and Ca2+ content of the BMP1-overexpressed group were significantly enhanced compared with the GFP group and Control group. Immunohistochemistry staining results showed that BMP-1 promoted the expression of type I collagen in BMSCs sheets.ConclusionsOur results suggest that the overexpression of BMP-1 can promote the osteogenesis of BMSCs and provides an improved method of cell-based tissue engineering.

LncRNA MALAT1 Inhibits Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells by Down-Regulating WNT5A

ncRNA involves in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). WNT5A participates in the growth and development of osteogenic differentiation. This study aims to investigate whether lncRNA MALAT1 regulates BMSCs osteogenesis through WNT5A. qRT-PCR was done to detect the lncRNA MALAT1 level and osteogenic markers in osteoporosis patients and control groups. The above markers and WNT5A protein levels were detected by Western blot. The degree of osteogenic differentiation was detected by ALP activity assay and ALP staining. The differentiation ability of BMSCs after lncRNA MALAT1 overexpression was detected by ARS staining. The binding site of lncRNA MALAT1 to WNT5A was determined by dual luciferase reporter assay. lncRNA MALAT1 expression was higher in patients with osteoporosis, and decreased significantly with increased osteogenic induction. Overexpression of lncRNA MALAT1 in BMSCS reduced WNT5A level, while interference with lncRNA MALAT1 increased WNT5A levels. In cells with overexpression of lncRNA MALAT1, transfection of si-WNT5A can significantly downregulate the RUNX2, OSX, ALP, OCN, OPN, and COL1A1, thereby inhibiting osteogenic differentiation, interfering with the regulation of WNT signaling pathway and regulating BMSCs osteogenic differentiation. lncRNA MALAT1 and WNT5A can regulate BMSCs osteogenesis, thus accelerating the progression of osteoporosis.

Exosomes derived from maxillary BMSCs enhanced the osteogenesis in iliac BMSCs.

Secondary alveolar bone grafting is an essential part in the treatment of alveolar cleft deformity. Autologous iliac bone is the most favorable grafting source. However, the factors regulating postoperative bone formation are unclear. Investigations are needed to found whether the alveolar bone niche and bone marrow mesenchymal stem cells (BMSCs) derived from the jaw bone (BMSCs-J) affected the osteogenesis of BMSCs from the ilium (BMSCs-I). The effect of BMSCs-J on BMSCs-I was investigated using a co-culture model. The exosomes were purified by sequential centrifugation. The osteoblastic differentiation of BMSCs was analyzed in vitro and in vivo. Co-culture with BMSCs-J increased the alkaline phosphatase (ALP) activity, Alizarin Red S (ARS) staining, and osteogenic gene expression in BMSCs-I. Transmission electron microscopy and nanoparticle tracking analysis verified the presence of exosomes in the culture supernatants of BMSCs. Exosomes secreted by BMSCs-J enhanced the ALP activity, ARS staining, osteogenic gene expression of BMSCs-I in vitro, and new bone formation in vivo. Blocking the secretion of exosomes using siRNA for Rab27a inhibited the effect of BMSCs-J. Exosomes played a role in the interaction between BMSCs-J and BMSCs-I, thereby leading to the enhanced osteogenic capacity of BMSCs-I and bone formation.

Melatonin Rescued Reactive Oxygen Species-Impaired Osteogenesis of Human Bone Marrow Mesenchymal Stem Cells in the Presence of Tumor Necrosis Factor-Alpha.

Accumulation of reactive oxygen species (ROS), which can be induced by inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), can significantly inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). This process can contribute to the imbalance of bone remodeling, which ultimately leads to osteoporosis. Therefore, reducing the ROS generation during osteogenesis of BMSCs may be an effective way to reverse the impairment of osteogenesis. Melatonin (MLT) has been reported to act as an antioxidant during cell proliferation and differentiation, but its antioxidant effect and mechanism of action during osteogenesis of MSCs in the inflammatory microenvironment, especially in the presence of TNF-α, remain unknown and need further study. In our study, we demonstrate that melatonin can counteract the generation of ROS and the inhibitory osteogenesis of BMSCs induced by TNF-α, by upregulating the expression of antioxidases and downregulating the expression of oxidases. Meanwhile, MLT can inhibit the phosphorylation of p65 protein and block the degradation of IκBα protein, thus decreasing the activity of the NF-κB pathway. This study confirmed that melatonin can inhibit the generation of ROS during osteogenic differentiation of BMSCs and reverse the inhibition of osteogenic differentiation of BMSCs in vitro, suggesting that melatonin can antagonize TNF-α-induced ROS generation and promote the great effect of osteogenic differentiation of BMSCs. Accordingly, these findings provide more evidence that melatonin can be used as a candidate drug for the treatment of osteoporosis.

Large tumor suppressor gene 2-mediated Hippo signaling pathway regulates the biological behavior of mesenchymal stem cells in vitro

To investigate the regulatory effect of Hippo signaling pathway mediated by large tumor suppressor gene 2 (LATS2) on biological behavior of mice bone marrow mesenchymal stem cells (BMSCs) in vitro. BMSCs of C57BL/6 mice were cultured in vitro and passed to the 6th to 7th generations for experiment. BMSCs with activated and inactivated LATS2 were constructed with lentiviral vectors transfections. The BMSCs were allocated to blank control group (MSC group), empty vector control group (MSC-eGFP group), LATS2-overexpressing group (MSC-LATS2 group), empty vector without LATS2 shRNA control group (MSC-shcontrol group) and LATS2-underexpressing group (MSC-shLATS2 group). The transduction efficiencies mediated by the lentiviral vectors were evaluated by flow cytometry. The mRNA and protein expressions of LATS2, phosphorylation of Yes associated protein (p-YAP) and 14-3-3 were quantified by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot respectively. Osteogenic and adipogenic differentiation of BMSCs were evaluated through alizarin red and oil red O staining. Proliferation of BMSCs was evaluated using the CCK-8 assay. The effect of Hippo pathway on horizontal and vertical migration ability of BMSCs was measured by the scratch test and Transwell chamber test. The transduction efficiencies mediated by the lentiviral vectors were 93.1%-97.1%. Compared with MSC-eGFP group, the expressions of LATS2, p-YAP and 14-3-3 in MSC-LATS2 group were significantly elevated [LATS2 mRNA (2-ΔΔCT): 2.55±0.13 vs. 1.08±0.05, LATS2/GAPDH: 2.63±0.11 vs. 1.06±0.08, p-YAP/total YAP: 1.67±0.11 vs. 1.00±0.04, 14-3-3/β-actin: 2.22±0.20 vs. 0.98±0.06, all P < 0.05], however, compared with MSC-shcontrol group, the expressions in MSC-shLATS2 group were significantly reduced [LATS2 mRNA (2-ΔΔCT): 0.10±0.01 vs. 1.01±0.05, LATS2/GAPDH: 0.09±0.01 vs. 1.05±0.06, p-YAP/total YAP: 0.10±0.02 vs. 1.10±0.09, 14-3-3/β-actin: 0.05±0.01 vs. 0.90±0.08, all P < 0.05]. It suggested that high and low expression of LATS2 could activate or inhibit Hippo pathway. The osteogenic and adipogenic differentiation and proliferation rate of BMSCs in MSC-LATS2 group were significantly lower than those in MSC-eGFP group, however, those in MSC-shLATS2 group were significantly higher than MSC-shontrol group (all P < 0.05). It suggested that high and low expression of LATS2 could inhibit or promote osteogenesis, adipogenesis and cell proliferation of BMSCs. Scratch test and Transwell chamber test showed that the degree of scratch healing in MSC-LATS2 group was significantly lower than that in MSC-eGFP group [(22.11±3.02)% vs. (45.99±6.58)%], while the number of cells migrating to the subventricular layer of Transwell was significantly reduced (cells/MP: 20.82±3.05 vs. 111.33±13.28, both P < 0.05); the degree of scratch healing in MSC-shLATS2 group was significantly higher than that in MSC-shcontrol group [(70.32±7.17)% vs. (39.28±2.98)%], the number of cells migrating to the subventricular layer of Transwell was increased significantly (cells/MP: 206.19±30.58 vs. 120.10±25.10, both P < 0.05). It suggested that high and low expression of LATS2 could inhibit or promote the horizontal and vertical migration of BMSCs. LATS2-mediated alteration of Hippo pathway could modulate differentiation, proliferation, and migration of mesenchymal stem cells in vitro.

Mechanism and research progress of long non-coding RNA in osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells

With the continuous development of high throughput transcriptome analysis and further research of epigenetics, more and more studies have shown that long non-coding RNA (lncRNA) have involved in the osteogenesis and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), which have also become a hot spot in the field of orthopedic research. This review summarizes the research progress in the role and mechanism of lncRNA in regulating osteogenesis and adipogenic differentiation of BMSCs. Key words: Long non-coding RNA; Bone marrow mesenchymal stem cells; Osteogenic differentiation; Adipogenic differentiation