Cilomilast enhances osteoblast differentiation of mesenchymal stem cells and bone formation induced by bone morphogenetic protein 2

A rapid and efficient method to stimulate bone regeneration would be useful in orthopaedic stem cell therapies. Rolipram is an inhibitor of phosphodiesterase 4 (PDE4), which mediates cyclic adenosine monophosphate (cAMP) degradation. Systemic injection of rolipram enhances osteogenesis induced by bone morphogenetic protein 2 (BMP-2) in mice. However, there is little data on the precise mechanism, by which the PDE4 inhibitor regulates osteoblast gene expression. In this study, we investigated the combined ability of BMP-2 and cilomilast, a second-generation PDE4 inhibitor, to enhance the osteoblastic differentiation of mesenchymal stem cells (MSCs). The alkaline phosphatase (ALP) activity of MSCs treated with PDE4 inhibitor (cilomilast or rolipram), BMP-2, and/or H89 was compared with the ALP activity of MSCs differentiated only by osteogenic medium (OM). Moreover, expression of Runx2, osterix, and osteocalcin was quantified using real-time polymerase chain reaction (RT-PCR). It was found that cilomilast enhances the osteoblastic differentiation of MSCs equally well as rolipram in primary cultured MSCs. Moreover, according to the H89 inhibition experiments, Smad pathway was found to be an important signal transduction pathway in mediating the osteogenic effect of BMP-2, and this effect is intensified by an increase in cAMP levels induced by PDE4 inhibitor.     

Cathepsin K deficiency promotes alveolar bone regeneration by promoting jaw bone marrow mesenchymal stem cells proliferation and differentiation via glycolysis pathway

ObjectivesTo clarify the possible role and mechanism of Cathepsin K (CTSK) in alveolar bone regeneration mediated by jaw bone marrow mesenchymal stem cells (JBMMSC).Materials and MethodsTooth extraction models of Ctsk knockout mice (Ctsk ^‐/‐) and their wildtype (WT) littermates were used to investigate the effect of CTSK on alveolar bone regeneration. The influences of deletion or inhibition of CTSK by odanacatib (ODN) on proliferation and osteogenic differentiation of JBMMSC were assessed by CCK‐8, Western blot and alizarin red staining. To explore the differently expressed genes, RNA from WT and Ctsk^‐/‐ JBMMSC was sent to RNA‐seq. ECAR, glucose consumption and lactate production were measured to identify the effect of Ctsk deficiency or inhibition on glycolysis. At last, we explored whether Ctsk deficiency or inhibition promoted JBMMSC proliferation and osteogenic differentiation through glycolysis.ResultsWe found out that Ctsk knockout could promote alveolar bone regeneration in vivo. In vitro, we confirmed that both Ctsk knockout and inhibition by ODN could promote proliferation of JBMMSC, up‐regulate expression of Runx2 and ALP, and enhance matrix mineralization. RNA‐seq results showed that coding genes of key enzymes in glycolysis were significantly up‐regulated in Ctsk^‐/‐ JBMMSC, and Ctsk deficiency or inhibition could promote glycolysis in JBMMSC. After blocking glycolysis by 3PO, the effect of Ctsk deficiency or inhibition on JBMMSC’s regeneration was blocked subsequently.ConclusionsOur findings revealed that Ctsk knockout or inhibition could promote alveolar bone regeneration by enhancing JBMMSC regeneration via glycolysis. These results shed new lights on the regulatory mechanism of CTSK on bone regeneration.     

Connective tissue growth factor (CTGF) is regulated by Wnt and bone morphogenetic proteins signaling in osteoblast differentiation of mesenchymal stem cells

Osteoblast lineage-specific differentiation of mesenchymal stem cells is a well regulated but poorly understood process. Both bone morphogenetic proteins (BMPs) and Wnt signaling are implicated in regulating osteoblast differentiation and bone formation. Here we analyzed the expression profiles of mesenchymal stem cells stimulated with Wnt3A and osteogenic BMPs, and we identified connective tissue growth factor (CTGF) as a potential target of Wnt and BMP signaling. We confirmed the microarray results, and we demonstrated that CTGF was up-regulated at the early stage of BMP-9 and Wnt3A stimulations and that Wnt3A-regulated CTGF expression was beta-catenin-dependent. RNA interference-mediated knockdown of CTGF expression significantly diminished BMP-9-induced, but not Wnt3A-induced, osteogenic differentiation, suggesting that Wnt3A may also regulate osteoblast differentiation in a CTGF-independent fashion. However, constitutive expression of CTGF was shown to inhibit both BMP-9- and Wnt3A-induced osteogenic differentiation. Exogenous expression of CTGF was shown to promote cell migration and recruitment of mesenchymal stem cells. Our findings demonstrate that CTGF is up-regulated by Wnt3A and BMP-9 at the early stage of osteogenic differentiation, which may regulate the proliferation and recruitment of osteoprogenitor cells; however, CTGF is down-regulated as the differentiation potential of committed pre-osteoblasts increases, strongly suggesting that tight regulation of CTGF expression may be essential for normal osteoblast differentiation of mesenchymal stem cells.     

生长因子对骨髓间充质干细胞增殖、分化的影响

骨髓间充质干细胞(bone mesenchymal stemcell,BMSC)是骨髓基质细胞的重要组成部分,由于其不但能与其他细胞一起支持造血干细胞造血,而且还具有较强的增殖功能及多向分化潜能,在一定诱导因素下可定向分化成骨细胞、软骨细胞和脂肪细胞等,近年来已成为生物学和医学的研究热点。本文简要介绍了不同生长因子如血管内皮生长因子、碱性成纤维细胞生长因子、转化生长因子-β等对BMSC增殖、分化的影响。     

Antagonistic and synergistic effects of bone morphogenetic protein 2/7 and all‐trans retinoic acid on the osteogenic differentiation of rat bone marrow stromal cells

The osteogenesis of bone marrow stromal cells (BMSCs) is of paramount importance for the repair of large‐size bone defects, which may be compromised by the dietary‐accumulated all‐trans retinoic acid (ATRA). We have shown that heterodimeric bone morphogenetic protein 2/7 (BMP2/7) could induce bone regeneration in a significantly higher dose‐efficiency in comparison with homodimeric BMPs. In this study, we evaluated the effects of ATRA and BMP2/7 on the proliferation, differentiation, mineralization and osteogenic genes. ATRA and BMP2/7 exhibited both antagonistic and synergistic effects on the osteogenesis of BMSCs. ATRA significantly inhibited proliferation and expression of osteocalcin but enhanced the activity of alkaline phosphatase of BMSCs. On day 21, 50 ng/mL BMP2/7 could antagonize the inhibitive effects of ATRA and significantly enhance osteogenesis of BMSCs. These findings suggested a promising application potential of heterodimeric BMP2/7 in clinic to promote bone regeneration for the cases with dietary accumulated ATRA.     

Insulin-like growth factor 1 (IGF-I) improves hepatic differentiation of human bone marrow-derived mesenchymal stem cells

The ability of MSCs (mesenchymal stem cells) to differentiate between other cell types makes these cells an attractive therapeutic tool for cell transplantation. This project was designed to improve transdifferentiation of human MSCs into liver cells using IGF-I (insulin-like growth factor 1) which, despite its important role in liver development, has not been used for in vitro hepatic differentiation. In the present study, the MSCs derived from healthy human bone marrow samples were cultured and characterized by immunophenotyping and differentiation potential into osteoblast and adipocytes. Transdifferentiation into hepatocyte-like cells was performed in the presence/absence of IGF-I in combination with predefined hepatic differentiation cocktail. To evaluate transdifferentiation, morphological features, immuno-cytochemical staining of specific biological markers and hepatic functions were assessed. Morphological assessment and evaluation of glycogen content, albumin and AFP (α-feto protein) expression as well as albumin and urea secretion revealed statistically significant difference between experimental groups compared with the control. Morphology and function (albumin secretion) of IGF-I-treated cells were significantly better than IGF-I-free experimental group. To the best of our knowledge, our study is the first to demonstrate that the combination of IGF-I with the predefined hepatic differentiation cocktail will significantly improve the morphological features of the differentiated cells and albumin secretion.     

Identification of receptors and signaling pathways for orphan bone morphogenetic protein/growth differentiation factor ligands based on genomic analyses

There are more than 30 human transforming growth factor beta/bone morphogenetic protein/growth differentiation factor (TGFbeta/BMP/GDF)-related ligands known to be important during embryonic development, organogenesis, bone formation, reproduction, and other physiological processes. Although select TGFbeta/BMP/GDF proteins were found to interact with type II and type I serine/threonine receptors to activate downstream Smad and other proteins, the receptors and signaling pathways for one-third of these TGFbeta/BMP/GDF paralogs are still unclear. Based on a genomic analysis of the entire repertoire of TGFbeta/BMP/GDF ligands and serine/threonine kinase receptors, we tested the ability of three orphan BMP/GDF ligands to activate a limited number of phylogenetically related receptors. We characterized the dimeric nature of recombinant GDF6 (also known as BMP13), GDF7 (also known as BMP12), and BMP10. We demonstrated their bioactivities based on the activation of Smad1/5/8-, but not Smad2/3-, responsive promoter constructs in the MC3T3 cell line. Furthermore, we showed their ability to induce the phosphorylation of Smad1, but not Smad2, in these cells. In COS7 cells transfected with the seven known type I receptors, overexpression of ALK3 or ALK6 conferred ligand signaling by GDF6, GDF7, and BMP10. In contrast, transfection of MC3T3 cells with ALK3 small hairpin RNA suppressed Smad signaling induced by all three ligands. Based on the coevolution of ligands and receptors, we also tested the role of BMPRII and ActRIIA as the type II receptor candidates for the three orphan ligands. We found that transfection of small hairpin RNA for BMPRII and ActRIIA in MC3T3 cells suppressed the signaling of GDF6, GDF7, and BMP10. Thus, the present approach provides a genomic paradigm for matching paralogous polypeptide ligands with a limited number of evolutionarily related receptors capable of activating specific downstream Smad proteins.     

Advances of tooth‐derived stem cells in neural diseases treatments and nerve tissue regeneration

Nerous system diseases, both central and peripheral, bring an incredible burden onto patients and enormously reduce their quality of life. Currently, there are still no effective treatments to repair nerve lesions that do not have side effects. Stem cell–based therapies, especially those using dental stem cells, bring new hope to neural diseases. Dental stem cells, derived from the neural crest, have many characteristics that are similar to neural cells, indicating that they can be an ideal source of cells for neural regeneration and repair. This review summarizes the neural traits of all the dental cell types, including DPSCs, PDLCs, DFCs, APSCs and their potential applications in nervous system diseases. We have summed up the advantages of dental stem cells in neural repair, such as their neurotrophic and neuroprotective traits, easy harvest and low rejective reaction rate, among others. Taken together, dental stem cells are an ideal cell source for neural tissue regeneration and repair.     

Effect of connective tissue growth factor (CCN2/CTGF) on proliferation and differentiation of mouse periodontal ligament-derived cells

Background

CCN2/CTGF is known to be involved in tooth germ development and periodontal tissue remodeling, as well as in mesenchymal tissue development and regeneration. In this present study, we investigated the roles of CCN2/CTGF in the proliferation and differentiation of periodontal ligament cells (murine periodontal ligament-derived cell line: MPL) in vitro.

Results

In cell cultures of MPL, the mRNA expression of the CCN2/CTGF gene was stronger in sparse cultures than in confluent ones and was significantly enhanced by TGF-β. The addition of recombinant CCN2/CTGF (rCCN2) to MPL cultures stimulated DNA synthesis and cell growth in a dose-dependent manner. Moreover, rCCN2 addition also enhanced the mRNA expression of alkaline phosphatase (ALPase), type I collagen, and periostin, the latter of which is considered to be a specific marker of the periosteum and periodontium; whereas it showed little effect on the mRNA expression of typical osteoblastic markers, e.g., osteopontin and osteocalcin. Finally, rCCN2/CTGF also stimulated ALPase activity and collagen synthesis.

Conclusion

These results taken together suggest important roles of CCN2/CTGF in the development and regeneration of periodontal tissue including the periodontal ligament.     

Insulin‐like growth factor binding protein 4 inhibits proliferation of bone marrow mesenchymal stem cells and enhances growth of neurospheres derived from the stem cells

Insulin‐like growth factor binding protein 4 (IGFBP‐4) was reported to trigger cellular senescence and reduce cell growth of bone marrow mesenchymal stem cells (BMSCs), but its contribution to neurogenic differentiation of BMSCs remains unknown. In the present study, BMSCs were isolated from the femur and tibia of young rats to investigate effects of IGFBP‐4 on BMSC proliferation and growth of neurospheres derived from BMSCs. Bone marrow mesenchymal stem cell proliferation was assessed using CCK‐8 after treatment with IGFBP‐4 or blockers of IGF‐IR and β‐catenin. Phosphorylation levels of Akt, Erk, and p38 in BMSCs were analysed by Western blotting. Bone marrow mesenchymal stem cells were induced into neural lineages in NeuroCult medium; the number and the size of BMSC‐derived neurospheres were counted after treatment with IGFBP‐4 or the blockers. It was shown that addition of IGFBP‐4 inhibited BMSC proliferation and immunodepletion of IGFBP‐4 increased the proliferation. The blockade of IGF‐IR with AG1024 increased BMSC proliferation and reversed IGFBP‐4‐induced proliferation inhibition; however, blocking of β‐catenin with FH535 did not. p‐Erk was significantly decreased in IGFBP‐4‐treated BMSCs. IGFBP‐4 promoted the growth of neurospheres derived from BMSCs, as manifested by the increases in the number and the size of the derived neurospheres. Both AG1024 and FH535 inhibited the formation of NeuroCult‐induced neurospheres, but FH535 significantly inhibited the growth of neurospheres in NeuroCult medium with EGF, bFGF, and IGFBP‐4. The data suggested that IGFBP‐4 inhibits BMSC proliferation through IGF‐IR pathway and promotes growth of BMSC‐derived neurospheres via stabilizing β‐catenin.     

Growth factor gene expression profiles of bone morphogenetic protein-2-treated human adipose stem cells seeded on calcium phosphate scaffolds in vitro

The secretome of stem cells strongly determines the outcome of tissue engineering strategies. We investigated how the secretome of human adipose stem cells (hASCs) can be affected by substrate, BMP-2 treatment, and degree of differentiation. We hypothesized that as differentiation progresses, hASCs produce increasingly more gene products associated with processes such as angiogenesis and bone remodeling.     

Induction of Ym1/2 in mouse bone marrow-derived mast cells by IL-4 and identification of Ym1/2 in connective tissue type-like mast cells derived from bone marrow cells cultured with IL-4 and stem cell factor

Mast cells play an important role in allergic inflammation by releasing various bioactive mediators. The function of mast cells is enhanced by various stimuli, partly due to the induction of specific genes and their products. Although many inducible genes have been identified, a significant number of genes remain to be identified. Therefore, this study used PCR-selected cDNA subtraction to establish the profile of induced genes in the connective tissue (CT) type-like mast cells derived from bone marrow cells cultured in the presence of IL-4 and stem cell factor. Two hundred and fifty cDNA clones were obtained from the CT type-like mast cells by PCR-selected cDNA subtraction. Among them, Ym1/2, a chitinase-like protein, is one of the most abundantly induced genes. Ym1 is produced by activated macrophages in a parasitic infection, whereas its isotype, Ym2, is highly upregulated in allergic lung disease. In order to differentiate which isotype is expressed in bone marrow cells, specific primers for bone marrow-derived mast cells (BMMC), and CT type-like mast cells were used for RT-PCR. The results showed that Ym1 was constitutively expressed in bone marrow cells and gradually decreased in the presence of IL-3, whereas Ym2 was induced only in the presence of IL-4. CT type-like mast cells from bone marrow cells expressed Ym1 throughout the culture period and Ym2 was induced only by the addition of IL-4 into BMMC, indicating that IL-4 is essential for the expression of Ym1/2 genes.     

CREB/Wnt10b mediates the effect of COX-2 on promoting BMP9-induced osteogenic differentiation via reducing adipogenic differentiation in mesenchymal stem cells

Bone morphogenetic protein 9 (BMP9) is one of the most potent osteogenic factors, which may be a potential candidate for bone tissue engineering. However, the osteogenic capacity of BMP9 still need to be further enhanced. In this study, we determined the effect of Wnt10b on BMP9-induced osteogenic differentiation in mesenchymal stem cell (MSCs) and the possible mechanism underlying this process. We introduced the polymerase chain reaction (PCR), Western blot analysis, histochemical stain, ectopic bone formation, and microcomputed tomography analysis to evaluate the effect of Wnt10b on BMP9-induced osteogenic differentiation. Meanwhile, PCR, Western blot analysis, chromatin immunoprecipitation, and immunoprecipitation were used to analyze the possible relationship between BMP9 and Wnt10b. We found that BMP9 upregulates Wnt10b in C3H10T1/2 cells. Wnt10b increases the osteogenic markers and bone formation induced by BMP9 in C3H10T1/2 cells, and silencing Wnt10b decreases these effects of BMP9. Meanwhile, Wnt10b enhances the level of phosphorylated Smad1/5/8 (p-Smad1/5/8) induced by BMP9, which can be reduced by silencing Wnt10b. On the contrary, Wnt10b inhibits adipogenic markers induced by BMP9, which can be decreased by silencing Wnt10b. Further analysis indicated that BMP9 upregulates cyclooxygenase-2 (COX-2) and phosphorylation of cAMP-responsive element binding (p-CREB) simultaneously. COX-2 potentiates the effect of BMP9 on increasing p-CREB and Wnt10b, while silencing COX-2 decreases these effects. p-CREB interacts with p-Smad1/5/8 to bind the promoter of Wnt10b in C3H10T1/2 cells. Our findings suggested that Wnt10b can promote BMP9-induced osteogenic differentiation in MSCs, which may be mediated through enhancing BMP/Smad signal and reducing adipogenic differentiation; BMP9 may upregulate Wnt10b via the COX-2/p-CREB-dependent manner.     

Aspirin promotes tenogenic differentiation of tendon stem cells and facilitates tendinopathy healing through regulating the GDF7/Smad1/5 signaling pathway

Tendinopathy is a common musculoskeletal system disorder in sports medicine, but regeneration ability of injury tendon is limited. Tendon stem cells (TSCs) have shown the definitive treatment evidence for tendinopathy and tendon injuries due to their tenogenesis capacity. Aspirin, as the representative of nonsteroidal anti-inflammatory drugs for its anti-inflammatory and analgestic actions, has been commonly used in treating tendinopathy in clinical, but the effect of aspirin on tenogenesis of TSCs is unclear. We hypothesized that aspirin could promote injury tendon healing through inducing TSCs tenogenesis. The aim of the present study is to make clear the effect of aspirin on TSC tenogenesis and tendon healing in tendinopathy, and thus provide new treatment evidence and strategy of aspirin for clinical practice. First, TSCs were treated with aspirin under tenogenic medium for 3, 7, and 14 days. Sirius Red staining was performed to observe the TSC differentiation. Furthermore, RNA sequencing was utilized to screen out different genes between the induction group and aspirin treatment group. Then, we identified the filtrated molecules and compared their effect on tenogenesis and related signaling pathway. At last, we constructed the tendinopathy model and compared biomechanical changes after aspirin intake. From the results, we found that aspirin promoted tenogenesis of TSCs. RNA sequencing showed that growth differentiation factor 6 (GDF6), GDF7, and GDF11 were upregulated in induction medium with the aspirin group compared with the induction medium group. GDF7 increased tenogenesis and activated Smad1/5 signaling. In addition, aspirin increased the expression of TNC, TNMD, and Scx and biomechanical properties of the injured tendon. In conclusion, aspirin promoted TSC tenogenesis and tendinopathy healing through GDF7/Smad1/5 signaling, and this provided new treatment evidence of aspirin for tendinopathy and tendon injuries.