Stimulated Alkaline Phosphatase Activity Research Articles

Osteogenic Activity of Yellow Flag Iris (Iris pseudacorus) Extract Modulating Differentiation of Osteoblasts and Osteoclasts

Bone integrity is maintained through a balance between bone formation by osteoblasts and bone resorption by osteoclasts. Imbalance of the process results in metabolic bone diseases such as osteoporosis. This study investigated the yellow flag iris extract (YFIE) and revealed its anti-osteoporotic effects in osteoblastic MC3T3-E1 mouse cells and RAW 264.7 murine macrophages. When osteoblasts were treated with 1-20 μg/ml YFIE in an osteogenic medium, the bone nodule formation by calcium deposits was markedly enhanced during differentiation. Consistently, YFIE stimulated alkaline phosphatase activity and collagen type I secretion with a substantial effect on osteoblast proliferation. On the other hand, RAW 264.7 macrophages were pre-incubated with 1-20 μg/ml YFIE for 5 days in the presence of receptor activator of nuclear factor-κB ligand (RANKL). Non-toxic YFIE markedly attenuated the differentiation of macrophages to multi-nucleated osteoclasts. YFIE diminished RANKL-elevated tartrate-resistant acid phosphatase activity and bone resorption. In addition, the YFIE treatment retarded RANKL-induced cathepsin K production and carbonic anhydrase II expression, both of which are involved in bone resorption. Therefore, YFIE potentially posesses therapeutic agents that may prevent osteoporosis through promoting bone formation and reducing bone resorption.

Adenosine receptor subtype expression and activation influence the differentiation of mesenchymal stem cells to osteoblasts and adipocytes

Osteoblasts and adipocytes differentiate from a common precursor cell, the mesenchymal stem cell (MSC). Adenosine is known to signal via four adenosine receptor subtypes, and significantly, recent findings indicate that these may play a role in MSC differentiation. We therefore investigated adenosine receptor expression and activation during the differentiation of MSCs to osteoblasts and adipocytes. The A(2B) R was dominant in MSCs, and its expression and activity were transiently upregulated at early stages of osteoblastic differentiation. Both activation and overexpression of A(2B) R induced the expression of osteoblast-related genes [Runx2 and alkaline phosphatase (ALP)], as well as ALP activity, and stimulation increased osteoblast mineralization. The expression of A(2A) R was upregulated during later stages of osteoblastic differentiation, when its activation stimulated ALP activity. Differentiation of MSCs to adipocytes was accompanied by significant increases in A(1) R and A(2A) R expression, and their activation was associated with increased adipogenesis. Enhanced A(2A) R expression was sufficient to promote expression of adipocyte-related genes (PPARγ and C/EBPα), and its activation resulted in increased adipocytic differentiation and lipid accumulation. In contrast, the A(1) R was involved mainly in lipogenic activity of adipocytes rather than in their differentiation. These results show that adenosine receptors are differentially expressed and involved in lineage-specific differentiation of MSCs. We conclude, therefore, that fruitful strategies for treating diseases associated with an imbalance in the differentiation and function of these lineages should include targeting adenosine receptor signal pathways. Specifically, these research avenues will be useful in preventing or treating conditions with insufficient bone or excessive adipocyte formation.

TGF-β regulates β-catenin signaling and osteoblast differentiation in human mesenchymal stem cells

Human adult bone marrow-derived skeletal stem cells a.k.a mesenchymal stem cells (hMSCs) have been shown to be precursors of several different cellular lineages, including osteoblast, chondrocyte, myoblast, adipocyte, and fibroblast. Several studies have shown that cooperation between transforming growth factor β (TGF-β) and Wnt/β-catenin signaling pathways plays a role in controlling certain developmental events and diseases. Our previous data showed that agents like TGF-β, cooperation with Wnt signaling, promote chondrocyte differentiation at the expense of adipocyte differentiation in hMSCs. In this study, we tested mechanisms by which TGF-β activation of β-catenin signaling pathway and whether these pathways interact during osteoblast differentiation of hMSCs. With selective small chemical kinase inhibitors, we demonstrated that TGF-β1 requires TGF-β type I receptor ALK-5, Smad3, phosphoinositide 3-kinases (PI3K), and protein kinase A (PKA) to stabilize β-catenin, and needs ALK-5, PKA, and JNK to inhibit osteoblastogenesis in hMSCs. Knockdown of β-catenin with siRNA stimulated alkaline phosphatase activity and antagonized the inhibitory effects of TGF-β1 on bone sialoprotein (BSP) expression, suggested that TGF-β1 cooperated with β-catenin signaling in inhibitory of osteoblastogenesis in hMSCs. In summary, TGF-β1 activates β-catenin signaling pathway via ALK-5, Smad3, PKA, and PI3K pathways, and modulates osteoblastogenesis via ALK5, PKA, and JNK pathways in hMSCs; the interaction between TGF-β and β-catenin signaling supports the view that β-catenin signaling is a mediator of TGF-β's effects on osteoblast differentiation of hMSCs.

Role of diabetes- and obesity-related protein in the regulation of osteoblast differentiation

Although thyroid hormone (TH) is known to exert important effects on the skeleton, the nuclear factors constituting the TH receptor coactivator complex and the molecular pathways by which TH mediates its effects on target gene expression in osteoblasts remain poorly understood. A recent study demonstrated that the actions of TH on myoblast differentiation are dependent on diabetes- and obesity-related protein (DOR). However, the role of DOR in osteoblast differentiation is unknown. We found DOR expression increased during in vitro differentiation of bone marrow stromal cells into osteoblasts and also in MC3T3-E1 cells treated with TH. However, DOR expression decreased during cellular proliferation. To determine whether DOR acts as a modulator of TH action during osteoblast differentiation, we examined whether overexpression or knockdown of DOR in MC3T3-E1 cells affects the ability of TH to induce osteoblast differentiation by evaluating alkaline phosphatase (ALP) activity. ALP activity was markedly increased in DOR-overexpressing cells treated with TH. In contrast, loss of DOR dramatically reduced TH stimulation of ALP activity in MC3T3-E1 cells and primary calvaria osteoblasts transduced with lentiviral DOR shRNA. Consistent with reduced ALP activity, mRNA levels of osteocalcin, ALP, and Runx2 were decreased significantly in DOR shRNA cells. In addition, a common single nucleotide polymorphism (SNP), DOR1 found on the promoter of human DOR gene, was associated with circulating osteocalcin levels in nondiabetic subjects. Based on these data, we conclude that DOR plays an important role in TH-mediated osteoblast differentiation, and a DOR SNP associates with plasma osteocalcin in men.

Effects of the immobilization of heparin and rhPDGF-BB to titanium surfaces for the enhancement of osteoblastic functions and anti-inflammation

PURPOSEThis study was to investigate the effects of recombinant human platelet-derived growth factor (rhPDGF-BB) and heparin to titanium surfaces for enhancement of osteoblastic functions and inhibition of inflammation activity.MATERIALS AND METHODSThe anodized titanium discs, not coated with any material, were used as a control group. In heparinized- Ti group, dopamine was anchored to the surface of Ti substrates, and coated with heparin. In PDGF-Ti group, rhPDGF-BB was immobilized onto heparinized Ti surface. The surface morphologies were investigated by the scanning electron microscope in each group. The release kinetics of rhPDGF-BB were analyzed, and cytotoxicity tests for each group were conducted. The biocompatibilities were characterized by measuring cell proliferation, alkaline phosphatase activity, and calcium deposition using MG-63 cells. Statistical comparisons were carried out by one-way ANOVA tests. Differences were considered statistically significant at *P<.05 and **P<.001.RESULTSThe combination of rhPDGF-BB and heparin stimulated alkaline phosphatase activity and OCN mRNA expression in osteoblastic cells (*P<.05 and **P<.001). MG-63 cells grown on PDGF-Ti had significantly higher amounts of calcium deposition than those grown on anodized Ti (**P<.001). Heparinized Ti was more anti-inflammatory compared to anodized Ti, when exposed to lipopolysaccharide using the transcript levels of TNF-α and IL-6 of proinflammatory cytokine (*P<.05 and **P<.001).CONCLUSIONThe result of this study demonstrated that the incorporation of rhPDGF-BB and heparin onto Ti surface enhanced osteoblastic functions and inhibited inflammation.

Panax Notoginseng Saponins Promote Osteogenic Differentiation of Bone Marrow Stromal Cells Through the ERK and P38 MAPK Signaling Pathways

The Chinese medicinal herb, Panax notoginseng, has long been used to treat bone fractures and Panax notoginseng saponins (PNS) could promote bone formation. Here, we investigated whether PNS could promote osteogenesis of bone marrow stromal cells (BMSCs) through modulating the MAPK signaling pathways, which are implicated in BMSC osteogenesis. We found that PNS markedly increased the mineralization of BMSCs by alizarin red S assays and stimulate alkaline phosphatase activity of these cells. Additionally, PNS significantly increased the mRNA levels of alkaline phosphatase, core-binding factor a1, and bone sialoprotein while decreasing PPARγ2 mRNA levels. Furthermore, inhibitors of ERK, PD98059, and p38, SB203580 inhibited the osteogenesis-potentiating effects by PNS. PNS stimulated the activation of ERK and p38 as evidenced by increased phosphorylation of these proteins, which was inhibited by PD98059 and SB203580. Our findings indicate that PNS could promote BMSC osteogenesis by activating the ERK and p38 signaling pathways.

In vitro estrogenic potency of phytoestrogen-glycosides and some plant flavanoids

Many plants produce chemicals that mimic or interact with hormonal signals in animals. Their presence in human diet is the object of many studies concerned with prevention of breast and prostate cancer, osteoporosis and other hormone-dependent diseases. Isoflavones, daidzein and genistein occurring either free or bound in glycosides are the main phytoestrogens in soya. The present study investigated the estrogenic activities of two isoflavones, two glucosylated isoflavones, one isoflavandiol, two flavanoids and some other plant-derived phenolic compounds. The bioassay used in this study was an estrogen responsive cell line Ishikawa Var-1. Besides, this study also used the recombinant yeast cells bearing the human estrogen receptor for confirming the actual estrogenic nature of the plant chemicals. There was a marked stimulation of alkaline phosphatase activity in Ishikawa cells and β-galactosidase activity in yeast cells by glucosylated isoflavones, genistin and daidzin. Considering the potency of body’s endogenous estrogen (17β-estradiol) as 100, in the Ishikawa assay the relative estrogenic potency of these compounds were found to be genistein (0.11), genistin (0.06), daidzein (0.08) and daidzin (0.07). In the yeast cell assay, the relative estrogenic potencies were genistein (0.1), genistin (0.01), daidzein (0.02) and daidzin (0.002). Estrogenic potency of equol was almost equal (0.18 & 0.16) in both Ishikawa and yeast cell assays, respectively. While kaempferol and resveratrol were weakly estrogenic, quercetin, rutin, catechin, hesperetin and luteolin did not show any estrogenic activity.Keywords: Phytoestrogen, flavanoids, estrogenic activity, glycosides.

Synthetic ameloblastin peptide stimulates differentiation of human periodontal ligament cells

ObjectiveThis study investigates the effect of the N-terminal region of a synthetic porcine ameloblastin peptide on the proliferation and differentiation of human periodontal ligament cells (PDLC). DesignWe used a cell counter to assess the effect of ameloblastin peptides on the proliferation of PDLC. To investigate the effect of ameloblastin peptides on the differentiation of PDLC, we examined quantitative analysis of alkaline phosphatase (ALP) activity by the Bessey–Lowry enzymological method, mineral nodule formation by Dahl's method, and expression of mineralization-related genes by RT-PCR. We used an anti-ameloblastin antibody to determine whether stimulation of ALP activity was caused by the peptide. ResultsAt all concentrations examined, the effect of the ameloblastin peptide on cell proliferation was not significantly different compared with the control. However, the peptide significantly stimulated ALP activity in a dose-dependent manner. ALP activity was significantly inhibited by an anti-ameloblastin antibody, which caused ALP levels to revert to their approximate levels in the untreated condition. At concentrations greater than 1ng/ml, the peptide promoted mineralized nodule formation of PDLC. And the peptide induced higher expressions of ALP and bone sialoprotein (BSP) than the control. ConclusionOur results show that the ameloblastin peptide upregulate ALP and BSP levels and can enhance calcification of PDLC. Thus, we suggest that the N-terminal synthetic ameloblastin peptide promotes the differentiation activity of PDLC.

P38 Regulates Expression of Osteoblast-specific Genes by Phosphorylation of Osterix

Osterix, a zinc finger transcription factor, is specifically expressed in osteoblasts and osteocytes of all developing bones. Because no bone formation occurs in Osx-null mice, Osterix is thought to be an essential regulator of osteoblast differentiation. We report that, in several mesenchymal and osteoblastic cell types, BMP-2 induces an increase in expression of the two isoforms of Osterix arising from two alternative promoters. We identified a consensus Sp1 sequence (GGGCGG) as Osterix binding regions in the fibromodulin and the bone sialoprotein promoters in vitro and in vivo. Furthermore, we show that Osterix is a novel substrate for p38 MAPK in vitro and in vivo and that Ser-73 and Ser-77 are the regulatory sites phosphorylated by p38. Our data also demonstrate that Osterix is able to increase recruitment of p300 and Brg1 to the promoters of its target genes fibromodulin and bone sialoprotein in vivo and that it directly associates with these cofactors through protein-protein interactions. Phosphorylation of Osterix at Ser-73/77 increased its ability to recruit p300 and SWI/SNF to either fibromodulin or bone sialoprotein promoters. We therefore propose that Osterix binds to Sp1 sequences on target gene promoters and that its phosphorylation by p38 enhances recruitment of coactivators to form transcriptionally active complexes.

Synergism between Wnt3a and Heparin Enhances Osteogenesis via a Phosphoinositide 3-Kinase/Akt/RUNX2 Pathway

A new strategy has emerged to improve healing of bone defects using exogenous glycosaminoglycans by increasing the effectiveness of bone-anabolic growth factors. Wnt ligands play an important role in bone formation. However, their functional interactions with heparan sulfate/heparin have only been investigated in non-osseous tissues. Our study now shows that the osteogenic activity of Wnt3a is cooperatively stimulated through physical interactions with exogenous heparin. N-Sulfation and to a lesser extent O-sulfation of heparin contribute to the physical binding and optimal co-stimulation of Wnt3a. Wnt3a-heparin signaling synergistically increases osteoblast differentiation with minimal effects on cell proliferation. Thus, heparin selectively reduces the effective dose of Wnt3a needed to elicit osteogenic, but not mitogenic responses. Mechanistically, Wnt3a-heparin signaling strongly activates the phosphoinositide 3-kinase/Akt pathway and requires the bone-related transcription factor RUNX2 to stimulate alkaline phosphatase activity, which parallels canonical beta-catenin signaling. Collectively, our findings establish the osteo-inductive potential of a heparin-mediated Wnt3a-phosphoinositide 3-kinase/Akt-RUNX2 signaling network and suggest that heparan sulfate supplementation may selectively reduce the therapeutic doses of peptide factors required to promote bone formation.

Src family kinase/abl inhibitor dasatinib suppresses proliferation and enhances differentiation of osteoblasts

Dasatinib, a dual Src family kinase and Abl inhibitor, is being tested clinically for the treatment of prostate cancer bone metastasis. Bidirectional interactions between osteoblasts and prostate cancer cells are critical in the progression of prostate cancer in bone, but the effect of dasatinib on osteoblasts is unknown. We found that dasatinib inhibited proliferation of primary mouse osteoblasts isolated from mouse calvaria and the immortalized MC3T3-E1 cell line. In calvarial osteoblasts from Col-luc transgenic mice carrying osteoblast-specific Col1alpha1 promoter reporter, luciferase activity was inhibited. Dasatinib also inhibited fibroblast growth factor-2-induced osteoblast proliferation, but strongly promoted osteoblast differentiation, as reflected by stimulation of alkaline phosphatase activity, osteocalcin secretion and osteoblast mineralization. To determine how dasatinib blocks proliferative signaling in osteoblasts, we analyzed the expression of a panel of tyrosine kinases, including Src, Lyn, Fyn, Yes and Abl, in osteoblasts. In the Src family kinases, only Src was activated at a high level. Abl was expressed at a low level in osteoblasts. Phosphorylation of Src-Y419 or Abl-Y245 was inhibited by dasatinib treatment. Knockdown of either Src or Abl by lenti-shRNA in osteoblasts enhances osteoblast differentiation, suggesting that dasatinib enhances osteoblast differentiation through inhibition of both Src and Abl.

Modulation of osteogenic properties of biodegradable polymer/extracellular matrix scaffolds generated with a flow perfusion bioreactor

In this study, composite scaffolds consisting of both synthetic and natural components with controllable properties were generated by incorporating mineralized extracellular matrix (ECM) and electrospun poly(ε-caprolactone) (PCL) microfiber scaffolds. Mesenchymal stem cells (MSCs) were cultured on PCL scaffolds under flow perfusion conditions with culture medium supplemented with dexamethasone to investigate the effect of culture duration on mineralized extracellular matrix deposition. MSCs differentiated down the osteogenic lineage and produced extracellular matrix with different compositions of mineral, collagen, and glycosaminoglycan with distinct morphologies at various stages of osteogenesis. To determine whether the presence and maturity of mineralized extracellular matrix influences osteogenic differentiation in vitro, PCL/ECM constructs were decellularized to yield PCL/ECM composite scaffolds that were subsequently seeded with MSCs and cultured in the absence of dexamethasone. The presence of mineralized matrix reduced cellular proliferation while stimulating alkaline phosphatase activity with increasing amounts of calcium deposition over time. PCL/ECM composite scaffolds containing the most mature mineralized matrix resulted in the most rapid increase and highest levels of alkaline phosphatase activity and calcium deposition compared to all other scaffold groups. Therefore, we demonstrate that mineralized extracellular matrix generated under controlled flow perfusion conditions can impart osteogenic properties to an osteoconductive polymer scaffold, and that the maturity of this matrix influences osteogenic differentiation in vitro, even in the absence of dexamethasone.

Ultrasonic-assisted extraction, chemical characterization of polysaccharides from Yunzhi mushroom and its effect on osteoblast cells

The objective of the current study was to determine the optimum ultrasonic-assisted extraction conditions for polysaccharides from Yunzhi mushroom. The optimum conditions were evaluated with fractional factorial design and optimized using response surface methodology. The ultrasonic power and the ratio of material to solvent were the most significant parameters on polysaccharides extraction. The extraction conditions were further investigated with Box–Behnken design. The fitted second-order model revealed that the optimal conditions consisted of ultrasonic power 30 W, 1:40 the ratio of raw material to solvent and extraction time 8 h. Under the optimized condition, the maximum productivity of Yunzhi polysaccharides predicted is 5.95%. The extraction productivity and purity of Yunzhi polysaccharides under the optimized extraction conditions were great higher than that of the non-optimized condition. In this study, Yunzhi polysaccharides could enhance alkaline phosphatase (ALP) activity in osteoblast cell. The present study still demonstrates that Yunzhi polysaccharides affects osteoblast cell growth in a dose-dependent manner by stimulating ALP activity.

Panax notoginseng Saponins Potentiate Osteogenesis of Bone Marrow Stromal Cells by Modulating Gap Junction Intercellular Communication Activities

Aims: The Chinese medicinal herb, Panax notoginseng, has long been used to treat bone fractures and Panax notoginseng saponins (PNS) could promote bone formation. We investigated the effects of PNS on gap junction intercellular communication (GJIC) and osteogenesis-associated genes in rat bone marrow stromal cells (BMSCs). Methods and Results:Our MTT assays demonstrated that PNS enhanced BMSC proliferation under basal medium culture in vitro. Alkaline phosphatase (ALP) assays and alizarin Red staining showed that PNS stimulated ALP activity and calcium deposition by BMSCs. Measurement of the traversing of Lucifer yellow through intercellular junctions revealed that PNS significantly stimulated GJIC activities. RT-PCR assays further showed that PNS augmented the increase in the mRNA levels of ALP, core-binding factor a1, and bone sialoprotein while decreasing the mRNA level of PPARΓ2. PNS also reduced RANKL levels and increased osteoprotegerin levels. Gap junction inhibitor, 18a-glycyrrhetinic acid, could partially reverse the actions of PNS on BMSCs. Conclusions: Our findings indicate that PNS could promote osteogenesis of BMSCs by targeting osteogenesis-associated genes, which could be mediated by their actions on GJIC.

Fasudil hydrochloride induces osteoblastic differentiation of stromal cell lines, C3H10T1/2 and ST2, via bone morphogenetic protein-2 expression

Rho-kinase (ROK), downstream of the mevalonate pathway, is detrimental to vessels, and suppressing its activity is a target for the treatment of human disease such as coronary artery disease and pulmonary hypertension. Recent studies have shown that ROK has a crucial role in bone metabolism. However, the role of ROK in stromal cells is still unclear. The present study was undertaken to investigate the effect of a ROK inhibitor, fasudil hydrochloride, on stromal cell lines, C3H10T1/2 and ST2. In both cells, Fasudil significantly stimulated alkaline phosphatase activity and enhanced cell mineralization. Moreover, fasudil significantly increased the mRNA expression of collagen-I, osteocalcin, and bone morphogenetic protein-2 (BMP-2). Supplementation of noggin, a BMP-2 antagonist, significantly reversed the fasudil-induced collagen-I and osteocalcin mRNA expression in both cells. These findings suggest that fasudil induces the osteoblastic differentiation of stromal cells via enhancing BMP-2 expression, and that this drug might be beneficial for not only atherosclerosis but also osteoporosis by promoting bone formation.

Schlafen-3: A novel regulator of intestinal differentiation

Schlafen-3 (Slfn-3), a novel gene, has been shown to be a negative regulator of proliferation. The current investigation was undertaken to determine whether Slfn-3 might play a role in regulating cellular differentiation. Butyric acid, a short chain fatty acid, which induced differentiation of intestinal cells as evidenced by increased alkaline phosphatase (ALP) activity in the rat small intestinal IEC-6 cells, also produced a marked increase in Slfn-3 expression. Furthermore, overexpression of Slfn-3 caused stimulation of ALP activity in IEC-6 cells, which was exacerbated by butyrate. On the other hand, downregulation of Slfn-3 by slfn-3-si-RNA greatly attenuated the butyrate-mediated induction of differentiation of IEC-6 cells. Additionally, we observed that increased expression of Slfn-3 in colon cancer HCT-116 cells stimulated TGF-β expression and modulated expression of its downstream effectors as evidenced by increased expression of p27kip1 and downregulation of CDK-2. In addition, Slfn-3 increases E-cadherin expression but downregulates β-catenin. In conclusion, our data show that Slfn-3 plays a critical role in regulating intestinal mucosal differentiation. Furthermore our data also show that TGF-β signaling pathway plays an important role in mediating slfn-3 induced differentiation.

Effects of Tea Catechins, Epigallocatechin, Gallocatechin, and Gallocatechin Gallate, on Bone Metabolism

In this study, three tea catechins, epigallocatechin (EGC), gallocatechin (GC), and gallocatechin gallate (GCG), were investigated for their effects on bone metabolism. The effects of the tea catechins on bone formation were evaluated using cultured rat osteoblast-like osteosarcoma cell line UMR-106. EGC stimulated alkaline phosphatase activity significantly at concentrations of 10 and 20 microM. The amount of mineralization also increased significantly with EGC. On another cell culture platform, EGC significantly inhibited osteoclast formations from RAW 264.7 cells upon receptor activation of nuclear factor-kappaB ligand induction on the fourth day of treatment, at a concentration of 10 microM. EGC also dose-dependently inhibited the mRNA expression of tatrate-resistant acid phosphatase. GC and GCG could decrease osteoclastogenesis at 20 microM. The present study illustrated that the tea catechins, EGC in particular, had positive effects on bone metabolism through a double process of promoting osteoblastic activity and inhibiting osteoclast differentiations.

Effect of chitosan particles and dexamethasone on human bone marrow stromal cell osteogenesis and angiogenic factor secretion

Chitosan is a polysaccharide scaffold used to enhance cartilage repair during treatments involving bone marrow stimulation, and it is reported to increase angiogenesis and osteogenesis in vivo. Here, we tested the hypotheses that addition of chitosan particles to the media of human bone marrow stromal cell (BMSC) cultures stimulates osteogenesis by promoting osteoblastic differentiation and by favoring the release of angiogenic factors in vitro. Confluent BMSCs were cultured for 3 weeks with 16% fetal bovine serum, ascorbate-2-phosphate and disodium β-glycerol phosphate, in the absence or presence of dexamethasone, an anti-inflammatory glucocorticoid commonly used as an inducer of BMSC osteoblast differentiation in vitro. As expected, dexamethasone slowed cell division, stimulated alkaline phosphatase activity and enhanced matrix mineralization. Added chitosan particles accumulated intra- and extracellularly and, while not affecting most osteogenic features, they inhibited osteocalcin release to the media at day 14 and interfered with mineralized matrix deposition. Interestingly, dexamethasone promoted cell attachment and suppressed the release and activation of matrix metalloprotease-2 (MMP-2). While chitosan particles had no effect on the release of angiogenic factors, dexamethasone significantly inhibited ( p < 0.05 to p < 0.0001) the release of vascular endothelial growth factor (VEGF), granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-α), interleukins 1β, 4, 6, and 10 (IL-1β, IL-4, IL-6, IL-10), and a host of other inflammatory factors that were constitutively secreted by BMSCs. These results demonstrate that chitosan particles alone are not sufficient to promote osteoblast differentiation of BMSCs in vitro, and suggest that chitosan promotes osteogenesis in vivo through indirect mechanisms. Our data further show that continuous addition of dexamethasone promotes osteoblastic differentiation in vitro partly by inhibiting gelatinase activity and by suppressing inflammatory cytokines which result in increased cell attachment and cell cycle exit.

P.1.10 Acute hypoxia of early organogenesis: influence on stress-realising system of descendants

The effect of a low Ca environment on the alkaline phosphatase (ALP) activity in bone cells was examined. Bone cells were isolated from the calvaria of 18 to 21-day-old fetal rats and cultured in BGJb or α-minimum essential medium (α-MEM) with calcium concentrations of 1.87 mM (control group), 1.20 mM (middle group) or 0.34 mM (low-Ca group). The control and low-Ca groups grew and reached a confluent state at 10 days of culture and there were no significant differences in the number of cells, total protein, or DNA content between the two groups. The specific ALP activity measured at 10 days of culture of the cells with 0.34, 1.20 and 1.87 mM calcium was 106.3 ± 11.9, 73.3 ± 10.4,and38.8 ± 7.3 unit per mg protein (n = 9), respectively. When the low-Ca medium was replaced with the control medium during 10 days of culture, the activity decreased to the level of the control group. On the other hand, when control medium was replaced with low-Ca medium, the activity increased to the level of the low-Ca group. After confluence, cell growth in the low-Ca group was poorer than that in the control group with time. The ALP activity markedly increased and the mineralized nodular structures were observed in the control group of α-MEM, but not in the low-Ca group. These findings suggest that stimulation of ALP activity is a compensation mechanism to maintain normal cell functions in a low-Ca condition and that the role of ALP in the low-Ca environment may be different from that on mineralization.

Transient dynamic actin cytoskeletal change stimulates the osteoblastic differentiation

Dynamic cytoskeletal changes appear to be one of intracellular signals that control cell differentiation. To test this hypothesis, we examined the effects of short-term actin cytoskeletal changes on osteoblastic differentiation. We found an actin polymerization interfering reagent, cytochalasin D, promoted osteoblastic differentiation in mouse preosteoblastic MC3T3-E1 cells. We also found that these effects were mediated by the protein kinase D (PKD) pathway. Short-term cytochalasin D treatment increased alkaline phosphatase (ALP) activity, osteocalcin (OCN) secretion, and mineralization of the extracellular matrix in MC3T3-E1 cells, with temporary changes in actin cytoskeleton. Furthermore, the disruption of actin cytoskeleton induced phosphorylation of 744/748 serine within the activation loop of PKD in a dose-dependent manner. The protein kinase C (PKC)/PKD inhibitor Go6976 suppressed cytochalasin D-induced acceleration of osteoblastic differentiation, whereas Go6983, a specific inhibitor of conventional PKCs, did not. Involvement of PKD signaling was confirmed by using small interfering RNA to knock down PKD. In addition, another actin polymerization interfering reagent, latrunculin B, also stimulated ALP activity and OCN secretion with PKD activation. On the other hand, the present data suggested that transient dynamic actin cytoskeletal reorganization could be a novel cellular signal that directly stimulated osteoblastic differentiation.