ALP-Mimetic Cyclic Peptide Nanotubes: A Multifunctional Strategy for Osteogenesis and Bone Regeneration.

Mesenchymal stem cells (MSCs) can differentiate into multiple cell lineages, including osteoblasts and adipocytes. We reported previously that glucocorticoid-induced leucine zipper (GILZ) inhibits peroxisome proliferator-activated receptor gamma-2 (Ppargamma2) expression and blocks adipocyte differentiation. Here we show that overexpression of GILZ in mouse MSCs, but not MC3T3-E1 osteoblasts, increases alkaline phosphatase activity and enhances mineralized bone nodule formation, whereas knockdown of Gilz reduces MSC osteogenic differentiation capacity. Consistent with these observations, real-time reverse transcription-PCR analysis showed that both basal and differentiation-induced transcripts of the lineage commitment gene Runx2/Cbfa1, as well as osteoblast differentiation marker genes including alkaline phosphatase, type I collagen, and osteocalcin, were all increased in GILZ-expressing cells. In contrast, the mRNA levels of adipogenic Ppargamma2 and C/ebpalpha were significantly reduced in GILZ-expressing cells under both osteogenic and adipogenic conditions. Together, our results demonstrate that GILZ functions as a modulator of MSCs and that overexpression of GILZ shifts the balance between osteogenic and adipogenic differentiation of MSCs toward the osteogenic pathway. These data suggest that GILZ may have therapeutic value for stem cell-based therapies of metabolic bone diseases, such as fracture repair.

The purpose of this study was to determine whether metformin (MF) could alleviate the expresssion of reactive oxygen species (ROS) and improve the osteogenic ability of bone marrow mesenchymal stem cells derived from diabetic rats (drBMSCs) in vitro, and to evaluate the effect of MF on the ectopic osteogenesis of drBMSCs in a nude mouse model in vivo. BMSCs were extracted from normal and diabetic rats. In vitro, a cell viability assay (Cell Counting Kit-8), tests of alkaline phosphatase (ALP) activity, and western blot analysis were first used to determine the cell proliferation and osteogenic differentiation of drBMSCs that were subjected to treatment with different concentrations of MF (0, 50, 100, 200, 500 μM). The cells were then divided into 5 groups: (1) normal rat BMSCs (the BMSCs derived from normal rats group), (2) the drBMSCs group, (3) the drBMSCs + Mito-TEMPO (10 μM, ROS scavenger) group, (4) the drBMSCs + MF (200 μM) group, and (5) the drBMSCs + MF (200 μM) + H2O2 (50 μM, ROS activator) group. Intracellular ROS detection, a senescence-associated β-galactosidase assay, ALP staining, alizarin red staining, western blotting, and immunofluorescence assays were performed to determine the effects of MF on oxidative stress and osteogenic differentiation in drBMSCs. In vivo, the effect of MF on the ectopic osteogenesis of drBMSCs was evaluated in a nude mouse model. MF effectively reduced ROS levels in drBMSCs. The cell proliferation, ALP activity, mineral deposition, and osteogenic-related protein expression of drBMSCs were demonstrably higher in the MF-treated group than in the non-MF-treated group. H2O2 inhibited the effects of MF. In addition, ectopic osteogenesis was significantly increased in drBMSCs treated with MF. MF promoted the proliferation and osteogenic differentiation of drBMSCs by inhibiting the oxidative stress induced by diabetes and enhenced the ectopic bone formation of drBMSCs in nude mice.

Inorganic pyrophosphate is a potent inhibitor of bone mineralization by preventing the seeding of calcium-phosphate complexes. Plasma cell membrane glycoprotein-1 and tissue nonspecific alkaline phosphatase were reported to be antagonistic regulators of mineralization toward inorganic pyrophosphate formation (by plasma cell membrane glycoprotein-1) and degradation (by tissue nonspecific alkaline phosphatase) under physiological conditions. In addition, they possess broad overlapping enzymatic functions. Therefore, we examined the roles of tissue nonspecific alkaline phosphatase within matrix vesicles isolated from femurs of 17-day-old chick embryos, under conditions where these both antagonistic and overlapping functions could be evidenced. Addition of 25 microM ATP significantly increased duration of mineralization process mediated by matrix vesicles, while supplementation of mineralization medium with levamisole, an alkaline phosphatase inhibitor, reduces the ATP-induced retardation of mineral formation. Phosphodiesterase activity of tissue nonspecific alkaline phosphatase for bis-p-nitrophenyl phosphate was confirmed, the rate of this phosphodiesterase activity is in the same range as that of phosphomonoesterase activity for p-nitrophenyl phosphate under physiological pH. In addition, tissue nonspecific alkaline phosphatase at pH 7.4 can hydrolyze ADPR. On the basis of these observations, it can be concluded that tissue nonspecific alkaline phosphatase, acting as a phosphomonoesterase, could hydrolyze free phosphate esters such as pyrophosphate and ATP, while as phosphodiesterase could contribute, together with plasma cell membrane glycoprotein-1, in the production of pyrophosphate from ATP.

Human adipose-derived stem cells (hASCs) are a promising cell type for bone tissue engineering, given their potential to differentiate into osteoblast-like cells. Interactions among biochemical and mechanical signals result in bone formation and repair. In this process stem cells have a crucial role. Extracorporeal shockwaves (ESWs) are acoustic waves capable of enhancing bone regeneration, suggesting that ESWs may induce some signals for mesenchymal progenitor maturation. The aim of the present work is to investigate the effects of ESW treatment on the differentiation of hASCs into osteoblast-like cells and to better clarify the mechanisms involved. The hASCs were treated with ESWs and osteogenic medium, and the effects in terms of gene expression, alkaline phosphatase (ALP) activity and calcium deposition were then evaluated. Moreover, to investigate the mechanisms of ESW action, reactive oxygen species (ROS) production, extracellular-signal-regulated kinase (ERK) and small 'mothers against' decapentaplegic (Smad) phosphorylation, and bone morphogenetic protein 2 (BMP2) expression were assessed. The ESW treatment increased Runt-related transcription factor 2 (Runx2), ALP and BMP2 expression, as well as ALP activity and calcium deposits with respect to untreated cells. Moreover ESWs induced ROS formation, and both ERK and Smad phosphorylation. The present study shows the effects of ESWs on osteogenic differentiation in an in vitro model using hASCs and defines the mechanisms involved in this process. The observations suggest that the combination of autologous hASCs and ESW treatment may improve bone tissue repair in tissue engineering procedures. Copyright © 2014 John Wiley & Sons, Ltd.

Differential effects of mixed lymphocyte reaction supernatant on human mesenchymal stromal cells

Partial breakdown of glycated alkaline phosphatases mediated by reactive oxygen species

Although bone morphogenetic proteins (BMPs) are clinically useful for bone regeneration, large amounts are required to induce new bone formation in monkeys and humans. We found recently that heparin stimulates BMP activity in vitro (Takada, T., Katagiri, T., Ifuku, M., Morimura, N., Kobayashi, M., Hasegawa, K., Ogamo, A., and Kamijo, R. (2003) J. Biol. Chem. 278, 43229-43235). In the present study, we examined whether heparin enhances bone formation induced by BMPs in vivo and attempted to determine the molecular mechanism by which heparin stimulates BMP activity using C2C12 myoblasts. Heparin enhanced BMP-2-induced gene expression and Smad1/5/8 phosphorylation at 24 h and thereafter, although not within 12 h. Heparitinase treatment did not affect the response of cells to BMP-2. In the presence of heparin, degradation of BMP-2 was blocked, and the half-life of BMP-2 in the culture medium was prolonged by nearly 20-fold. Although noggin mRNA was induced by BMP-2 within 1 h regardless of the presence of heparin, noggin failed to inhibit BMP-2 activity in the presence of heparin. Furthermore, simultaneous administration of BMP-2 and heparin in vivo dose-dependently induced larger amounts of mineralized bone tissue compared with BMP-2 alone. These findings clearly indicate that heparin enhances BMP-induced osteoblast differentiation not only in vitro but also in vivo. This study indicates that heparin enhances BMP-induced osteoblast differentiation in vitro and in vivo by protecting BMPs from degradation and inhibition by BMP antagonists.

Osteoclast-derived small extracellular vesicles induce osteogenic differentiation via inhibiting ARHGAP1

Objective To investigate different expression levels between young and old bone marrow mesenchymal stem cells in microRNAs (miRNAs) that are significantly conserved between humans and mice. Additional studies have been conducted to discover changes in miRNA expression in old mice relative to that in young adults and discussed the roles of miRNAs in primary osteoporosis. Methods MiRNAs that are highly conserved between human and mice, and are expressed at significantly different levels in the bone marrow mesenchymal stem cells of young and old people were identified by searching the Gene Expression Omnibus (GEO) database. Human bone mesenchymal stem cells (hBMSCs) were transfected with miRNA mimics, and their relative alkaline phosphatase (ALP) activity levels were then determined. Micro-CT scanning was employed to quantitatively characterize cortical and cancellous bones of young and old mice, and to confirm that these mice accurately modeled natural aging osteoporosis. Simultaneously, we investigated differences in expression levels of miRNAs that influence ALP activity in hBMSCs in the two groups of mice. Correlations between miRNA expression levels, and parameters of bone mass and bone strength were studied. Results 28 miRNAs were found to be more than 2 fold up-regulated (down-regulated) with statistical significance (P<0.05) in the GEO database. We also found that ALP activity was lower in hBMSCs transfected with 4 miRNAs (mir-124-3p, mir-126-3p, mir-128-3p, mir-424-5p, P<0.05 or P<0.01). The micro-CT scans indicated that the mice are accurately modeled natural aging osteoporosis. Expression of mir-124-3p increased significantly in older mice. This upregulation correlated positively with trabecular separation, and negatively with trabecular pattern factor in trabecular bone. However, in cortical bone, its expression correlated positively with trabecular separation, and negatively with bone volume fraction, trabecular number, and bone mineral density (P<0.05). Conclusion Hsa-mir-124-3p, which is expressed differently in young and old bone marrow stromal cells, inhibited the osteogenic differentiation of hBMSCs. Upregulation of this miRNA in the bone tissue of aged mice may be related to the development of osteoporosis. Key words: Human bone marrow mesenchymal stem cells; mir-124-3p; Osteogenic differentiation

Mitochondrial DNA (mtDNA)-encoded proteins function in eukaryotes as subunits of respiratory complexes that also contain nuclear DNA (nDNA)-encoded subunits. The importance of functional interactions between mtDNA- and nDNA-encoded proteins was previously demonstrated by testing the survivability of cybrid cells or individuals containing nDNA and mtDNA from different populations or species. This report focuses on the multisubunit respiratory complex cytochrome c oxidase (COX), made up of both mtDNA-encoded and nDNA-encoded subunits. A combination of evolutionary and crystallographic data is employed to determine whether rates of nonsynonymous substitutions have been higher, the same, or lower for residues in close proximity that are encoded by a different genome (nDNA or mtDNA). This determination is performed by simply taking the ratio, called the interaction ratio i, of the nonsynonymous substitution rate of the close-contact residues to the nonsynonymous substitution rate of the noncontact residues. We assume that the close-contact residues (which are more likely to interact) are functionally important and that, therefore, amino acid replacements among these residues cannot escape the scrutiny of natural selection. i = 1 indicates that the close-contact residues have been under neither greater purifying selection nor greater positive selection than the noncontact residues as a specific consequence of their being encoded by separate genomes. i < 1 indicates that the close-contact residues have been under greater purifying selection but less positive selection than have the noncontact residues. Conversely, i > 1 indicates that the close-contact residues have been under less purifying but greater positive selection than have the noncontact residues. i < 1 may be referred to as a constraining interaction; i.e., the close-contact residues compared with the noncontact residues appear to be under greater structural-functional constraints. On the other hand, i > 1 may be referred to as an optimizing interaction; i.e., apparently many different amino acid replacements are required to optimize this subunit's interaction with the other subunit. A major finding is that the nDNA-encoded residues in close physical proximity to mtDNA-encoded residues evolve more slowly than the other nuclear-encoded residues (and thus display a constraining interaction), whereas the mtDNA-encoded residues in close physical proximity to nDNA-encoded residues evolve more rapidly than the other mitochondrial-encoded residues (and thus display an optimizing interaction). A possible reason for this striking difference between the nuclear- and mitochondrial-encoded COX subunits in how their functional interaction evolves is discussed.

To explore the interaction between reactive oxygen species (ROS) and ferroptosis in methylglyoxalinduced injury of mouse embryonic osteoblasts (MC3T3-E1 cells). MC3T3-E1 cells were treated with methylglyoxal to establish a cell model of diabetic osteoporosis. CCK-8 assay was used to detect the viability of MC3T3-E1 cells. Rhodamine 123 staining followed by photofluorography was used to examine mitochondrial membrane potential (MMP). The intracellular ROS level was detected by 2', 7'-dichlorodihydrofluorescein diacetate staining with photofluorograph. Alkaline phosphatase (ALP) activity in the cells was detected using an ALP kit, the number of mineralized nodules was determined with alizarin red S staining, and the level of iron ions was detected using a detection kit. The expression level of glutathione peroxidase 4 (GPX4, a marker protein that inhibits ferroptosis) in the osteoblasts was determined using Western blotting. Treatment of MC3T3-E1 cells with 0.6 mmol/L methylglyoxal for 24 h significantly inhibited the expression level of GPX4 (P < 0.001), increased intracellular iron ion concentration, decreased the cell viability, increased the loss of MMP and intracellular ROS level, decreased both ALP activity and the number of mineralized nodules in the cells (P < 0.001). Co-treatment of MC3T3-E1 cells with 2 mmol/L N-acetylcysteine (NAC, a ROS scavenger) and methylglyoxal significantly increased the expression level of GPX4 (P < 0.01); co-treatment with 4 mmo/L FER-1 (a ferroptosis inhibitor) and methylglyoxal obviously decreased the intracellular ROS level (P < 0.001). Co-treatment of the cells either with NAC and methylglyoxal or with FER-1 and methylglyoxal attenuated methylglyoxal-induced injuries in the osteoblasts (P < 0.001). The interaction between ROS and ferroptosis pathway plays an important role in methylglyoxal-induced injury of mouse embryonic osteoblasts.

The responses of the immortalized rat preosteoblast UMR-201-10B to ascorbic acid (AA), 1,25(OH)2D3 (calcitriol), and retinoic acid (RA) were examined. UMR-201-10B cells have an undetectable basal alkaline phosphatase (ALP) activity that is induced after 24 h of treatment with 10(-6) M RA (4.64 +/- 0.06 mumol/h/mg of protein). The addition of 10(-8) M calcitriol resulted in a slight induction of ALP activity after 72 h (0.43 +/- 0.07 mumol/h/mg of protein). When calcitriol was added to RA, however, over the same period ALP activity was enhanced significantly compared with treatment with RA alone (RA and calcitriol, 12.29 +/- 0.86 mumol/h/mg of protein). Treatment with AA (50 micrograms/ml) alone had no effect on ALP activity but increased RA-induced ALP activity to 6.78 +/- 0.28 mumol/h/mg of protein at 24 h. In contrast, AA inhibited calcitriol-induced ALP activity after 7 days of combined treatment with calcitriol (calcitriol, 7.73 +/- 0.16 mumol/h/mg of protein; AA and calcitriol, 1.44 +/- 0.06 mumol/h/mg of protein). Individually, RA and calcitriol induced mRNA expression for ALP, matrix-gla protein (MGP), and osteopontin (OP). The steady state level of pro-alpha 1(I) collagen mRNA also was increased significantly by treatment with RA and AA individually. The combination of RA and calcitriol had a synergistic effect on ALP, OP, and especially MGP mRNA expression but significantly reduced the expression of pro-alpha 1(I) collagen mRNA. AA enhanced the effect of RA on the expression of pro-alpha 1(I) collagen, MGP, and ALP mRNAs as well as the effect of calcitriol on OP and MGP. The addition of AA to RA resulted in a decrease in the steady state level of OP, whereas its cotreatment with calcitriol caused a decrease in pro-alpha 1(I) collagen and ALP mRNA. In conclusion, these studies identify RA, calcitriol, and AA as regulators of differentiated osteoblast function.

Vascular calcification is strongly linked with increased morbidity and mortality from cardiovascular disease. Vascular calcification is an active cell-mediated process that involves the differentiation of vascular smooth muscle cells (VSMCs) to an osteoblast-like phenotype. Several inhibitors of this process have been identified, including insulin-like growth factor-I (IGF-I). In this study, we examined the role of the IGF receptor (IGFR) and the importance of IGFR glycosylation in the maintenance of the VSMC phenotype in the face of factors known to promote osteogenic conversion. IGF-I (25 ng/ml) significantly protected VSMCs from β-glycerophosphate-induced osteogenic differentiation (p < 0.005) and mineral deposition (p < 0.01). Mevalonic acid depletion (induced by 100 nm cerivastatin) significantly inhibited these IGF protective effects (p < 0.01). Mevalonic acid depletion impaired IGFR processing, decreased the expression of mature IGFRs at the cell surface, and inhibited the downstream activation of Akt and MAPK. Inhibitors of N-linked glycosylation (tunicamycin, deoxymannojirimycin, and deoxynojirimycin) also markedly attenuated the inhibitory effect of IGF-I on β-glycerophosphate-induced mineralization (p < 0.05) and activation of Akt and MAPK. These results demonstrate that alterations in the glycosylation of the IGFR disrupt the ability of IGF-I to protect against the osteogenic differentiation and mineralization of VSMCs by several interrelated mechanisms: decreased IGFR processing, reduced IGFR cell-surface expression, and reduced downstream signaling via the Akt and MAPK pathways. IGF-I thus occupies a critical position in the maintenance of normal VSMC phenotype and protection from factors known to stimulate vascular calcification.

The majority of experiments show that tumor necrosis factor-alpha (TNF-α) inhibits osteogenic differentiation of mesenchymal stem cells and pre-osteoblasts by activated nuclear factor-kappaB (NF-κB) signaling. However, the underlying mechanisms by which NF-κB signaling inhibits osteogenic differentiation are not fully understood. The aim of the present study was to investigate whether EphB4 signaling inhibition mediates the effects of TNF-α-activated NF-κB signaling on osteogenic differentiation of pre-osteoblasts. Murine MC3T3-E1 pre-osteoblasts were treated with 10ng/mL of TNF-α. NF-κB inhibitor, pyrrolidine dithiocarbamate, was used to achieve NF-κB signaling inhibition. EphB4 signaling was activated using ephrinB2-fc. The mRNA expressions of runt related transcription factor 2 (Runx2), bone sialoprotein (BSP) and EphB4 were determined using reverse transcription-polymerase chain reaction. The protein levels of Runx2, BSP, Col Ia1, osteopontin, EphB4, p-NF-κB p65 and NF-κB p65 were evaluated using western blot assays. Alkaline phosphatase (ALP) activity in MC3T3-E1 cells was evaluated by ALP activity kit, and mineral nodule formation was evaluated by Alizarin Red S staining. TNF-α inhibited EphB4 expression, while it suppressed Runx2, BSP expression from gene and protein levels as well as ALP activity and mineral nodule formation in MC3T3-E1 cells. Activation of EphB4 signaling by ephrinB2-fc promoted osteogenic differentiation of MC3T3-E1 cells, whereas TNF-α impaired the osteogenic differentiation enhanced by ephrinB2-fc. Pyrrolidine dithiocarbamate blocked the activation of NF-κB signaling induced by TNF-α, while it prevented the downregulation of Runx2, BSP and EphB4, induced by TNF-α. TNF-α inhibits osteogenic differentiation of pre-osteoblasts by downregulation of EphB4 signaling via activated NF-κB signaling pathway.

MicroRNA-34a and microRNA-146a target CELF3 and suppress the osteogenic differentiation of periodontal ligament stem cells under cyclic mechanical stretch