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At least 595 records · Page 33Linked to original sources

PubMed1998

Effects of extracellular calcium on the proliferation and differentiation of porcine osteoblasts in vitro.

We examined the effects of various extracellular calcium concentrations on DNA content, procollagen type I carboxy-terminal propeptide (PICP) release (reflects type I collagen synthesis), and alkaline phosphatase activity of porcine osteoblasts. Osteoblasts seeded in control medium (2.2 mM calcium) were transferred to low (0. 5 or 1 mM) calcium medium or to high (3, 5, 7, or 10 mM) calcium medium at different stages of the culture period and for different incubation times. When osteoblasts were transferred to low or high (3 or 5 mM) calcium medium 1 or 2 days after plating and kept in that medium until the end of the culture period, PICP release was inhibited, but DNA content and alkaline phosphatase activity were unchanged, except in 5 mM calcium, which inhibited alkaline phosphatase activity. Short-term culture of subconfluent and near-confluent osteoblasts in 7 or 10 mM calcium for 48 h inhibited DNA content. DNA content returned to normal levels when cells were transferred back to control medium, whereas alkaline phosphatase inhibition induced by 5, 7, or 10 mM calcium was not reversible. Short-term culture in high calcium media did not affect PICP release. Thus, in porcine osteoblasts, low and high extracellular calcium concentrations affect DNA content, PICP release, and the expression of osteoblastic phenotype markers (alkaline phosphatase activity). These effects are dependent on the duration of calcium treatment and the state of differentiation of the osteoblasts.

Alkaline Phosphatase↗

PubMed2005

[Osteoblast reaction on SLA and microgrooved implant surfaces].

The osseointegration process of dental implants depends on the tissue reaction at the tissue-implant interface. Osteoblasts are the main cells responsible for the regulation of osteoinduction. The manner and kinetics of the tissue reaction crucially depend on the interaction between osteoblasts and the morphology of the implant surface. The aim of this study was to investigate osteoblast behaviour on different implant surfaces (smooth, microgrooved, SLA) under standardized conditions. For this in vitro investigation we used primary bovine osteoblasts. Attachment kinetics, proliferation rate and synthesis of bone-associated proteins were used as parameters for cell reaction. The results demonstrate that both attachment and adhesion strength of the primary cell surface interaction was higher on the microgrooved surfaces than on SLA surfaces. The proliferation rate of cells and the synthesis of bone-specific proteins were higher on microgrooved surfaces in contrast to SLA surfaces. Ultrastructural analysis revealed phenotypic osteoblast-like cells on smooth and microgrooved surfaces, whereas cells on SLA surfaces showed a more fibroblastic appearance. This study demonstrates that the morphology of the implant surface determined the subsequent osteoblast reaction. An optimal cell reaction was found at surfaces which are smooth in the microenvironment of osteoblasts.

Animals↗

PubMed1992

Behaviour of fetal rat osteoblasts cultured in vitro on bioactive glass and nonreactive glasses.

We examined the behaviour of fetal rat osteoblasts cultured upon bioactive glass and nonreactive glasses, and the supposed stimulatory effects of bioactive glass on osteoblasts. Nonreactive glass cultures showed flattened cells with almost no dorsal ruffles. Bioactive glass cultures showed compact cells with dorsal ruffles and filapodia resulting in the formation of a denser cell layer. For confluent nonreactive glass cultures the osteoblast expression was mainly concentrated in the clustered cells which were formed upon the monolayer, whereas for confluent bioactive glass cultures the osteoblast expression was more generally distributed. The production of type I collagen, osteocalcin and an osteoblast-specific antigen was shown by immunocytochemistry for all cultures, although differences in distribution were observed. The bioactive layer of bioactive glass is responsible for a better osteoblast-like morphology, a higher proliferation rate and generally a better osteoblast expression.

Alkaline Phosphatase↗

PubMed1994

Better histology and biochemistry for osteoblasts cultured on titanium-doped bioactive glass: bioglass 45S5 compared with iron-, titanium-, fluorine- and boron-containing bioactive glasses.

In the present study we used an established cell culture model to compare Bioglass 45S5 with four other bioactive glasses. Small substitutions or additions of certain ions like iron, titanium, fluorine or boron modified the basic 45S5 glass network. We used several histological and biochemical parameters to interpret the results found in terms of the used model. Regarding 45S5 as a reference, we found that osteoblasts cultured on iron-doped bioactive glass showed a more flattened morphology, and both lower proliferation rate and osteoblast expression. Osteoblasts cultured on titanium-doped glasses also showed a flattened morphology, but higher proliferation and remarkably higher osteoblast expression. On fluorine- and boron-containing glasses the osteoblasts showed a rather compact morphology, a normal proliferation but only moderate osteoblast expression. With microprobe analysis it was shown that the formation of calcium and phosphorus on titanium-doped glass was relatively lower and the release of sodium slower when compared with 45S5. Osteoblasts cultured on titanium-doped bioactive glasses demonstrated superior histological and biochemical parameters when compared with the other glass types. Further research into the physico-chemical properties and the in vivo behaviour of doped bioactive glasses is recommended.

Alkaline Phosphatase↗

PubMed1989

Bradykinin stimulates production of prostaglandin E2 and prostacyclin in murine osteoblasts.

The effect of bradykinin on prostaglandin production in mouse calvarial bones and in isolated osteoblasts has been examined. Bradykinin (1 mumol/l) stimulated prostaglandin formation in neonatal mouse calvarial bones incubated for 30 min. In isolated osteoblast-like cells from neonatal mice calvarial bones and in a cloned mouse calvarial osteoblastic cell lineage (MC3T3-E1) bradykinin stimulated the production of prostaglandin E2 (PGE2) and 6-keto-prostaglandin F1 alpha (the stable breakdown product of prostacyclin). The stimulation of PGE2 production occurred rapidly (30 s) and reached its maximum after 5-10 min. The stimulatory effect of bradykinin on PGE2 production in isolated osteoblast-like cells and in MC3T3-E1 cells was dose dependent with apparent half maximal stimulation seen at 10 and 3 nmol/l, respectively. Bradykinin-induced prostaglandin production was totally reversible after withdrawal of the agonist. Pretreatment with bradykinin (1 mumol/l) resulted in desensitization to a subsequent challenge with bradykinin (1 mumol/l), while pretreatment with bradykinin had no effect upon arachidonic acid (30 mumol/l) induced prostaglandin formation. Bradykinin-induced production of PGE2 was abolished by several structurally unrelated, competitive and non-competitive inhibitors of arachidonic acid metabolism as well as by corticosteroids. The mouse calvarial osteoblast-like cells also showed a PGE2 and 6-keto-PGF1 alpha response to thrombin, but not to parathyroid hormone (PTH), calcitonin and 1 alpha(OH)D3. The formation of cyclic AMP in mouse calvarial osteoblasts was enhanced by PTH, bradykinin, thrombin and arachidonic acid but not by calcitonin and 1 alpha(OH)D3. The cyclic AMP response to bradykinin, thrombin and arachidonic acid, but not that to PTH, was abolished by indomethacin. The degree of confluency of the cell cultures greatly influenced the amount of prostaglandins being produced. At higher cell density the amount of prostanoids synthesized per cell was substantially decreased in untreated control cultures as well as in bradykinin- and arachidonic acid-treated cells. These data suggest that osteoblasts are equipped with receptors for bradykinin coupled to prostaglandin production.

6-Ketoprostaglandin F1 alpha↗

PubMed1993

Insulin-like growth factors-I and -II stimulate chemotaxis of osteoblasts isolated from fetal rat calvaria.

Repair and regeneration of damaged bone is believed to be regulated in part by growth factors stored in the bone matrix. These growth factors are synthesized and secreted by osteoblasts and are incorporated into the developing bone. This pool of stored growth factors is then released into the immediate area following resorption of the matrix. One of the initial steps in bone repair is the recruitment of osteoblasts to the repair site. Growth factors, such as TGF-beta and PDGF, which are present in bone matrix, have been shown to be chemotactic for osteoblasts. In this study, primary cultures of osteoblasts isolated from fetal rat calvaria were examined for chemotaxis in response to IGF-I and IGF-II. IGF-I stimulated a dose-dependent increase in osteoblast chemotaxis, while IGF-II stimulated chemotaxis maximally at the lowest concentration studied (0.1 ng/ml), and had no effect at the highest concentration studied (100 ng/ml). IGF-I and -II had no effect on osteoblast proliferation at any of the concentrations examined. These results indicate that IGFs may be playing an important role in the early stages of bone repair by stimulating osteoblast chemotaxis to the repair site.

Animals↗

PubMed1988

Characterization of endosteal osteoblastic cells isolated from mouse caudal vertebrae.

We have developed a reliable procedure for isolating endosteal osteoblasts from mouse trabecular bone. Endosteal osteoblasts were obtained by migration and proliferation of the cells from the metaphyseal bone surface of caudal vertebrae onto nylon meshes. The isolated cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The cell population consisted of 95% alkaline-phosphatase-positive cells. The cell level of alkaline phosphatase was elevated (1.19 +/- 0.26 (SD) mumol PNP/mn/mg protein) and the enzyme activity was heat-inhibitable, indicating its skeletal origin. Light and electron microscopic observation revealed that cells have morphologic and ultrastructural appearance of typical osteoblasts with high protein synthesis activity. Osteoblasts grown in multilayers in the presence of 50 micrograms/ml ascorbic acid produced within 4 days an abundant fibrous intercellular collagenous matrix forming nodules in which osteocyte-like cells were embedded. Immunolabeling revealed synthesis of type I collagen but no detectable type III collagen. In presence of 7 mM beta-glycerophosphate the matrix became mineralized after 14-21 days of culture. Mineralization could not be induced by mouse skin fibroblasts cultured under similar conditions. The mineral deposits were closely associated with the collagen matrix, consisted of EDTA-removable, Von Kossa and alizarin red S stainable material and were composed of hydroxyapatite crystals identified by X-ray electron probe microanalysis. The isolated endosteal osteoblasts also displayed an intense (+457%) increase in intracellular cAMP production in response to human (1-34) PTH (2 x 10(-8) M) stimulation. The confluent cells responded to 20 nM 1,25(OH)2D3 by a significant 45% reduction in heat labile alkaline phosphatase activity. This procedure allowed us to isolate from trabecular bone a cell population that differentiates into osteoblasts in vitro, respond to calcitropic hormones and that retains its capacity to form a calcified bone tissue in culture. This method provided us a culture system for investigating the differentiation and metabolism of endosteal osteoblastic bone forming cells.

Animals↗

PubMed1988

Comparison of bone formed intramuscularly after transplantation of scapular and calvarial osteoblasts.

Previous work suggested that osteoblasts determine the size of the bone marrow area within the bone and that calvarial osteoblasts differ from those induced intramuscularly by cartilage formed by transplanted epiphyseal chondrocytes. This study reports morphological observations of bone formed by transplanted scapular and calvarial osteoblasts isolated from bones of young rats. In intact scapulas of 28-day-old rats the percentage area occupied by bone tissue in relation to bone marrow was 6 times larger than in parietal bones of comparable age. Isolated syngeneic scapular osteoblasts usually produced an ossicle with similar general structure and ratio bone tissue/bone marrow area as in intact scapulas. In transplants of calvarial osteoblasts numerous islands of bone tissue with a small amount of bone marrow appeared. Bone formed in allogenic transplants was rejected. These results suggest that osteoblasts from endochondral scapular bone may have different properties than those from intramembranous calvarial bones. Alternatively, the large amount of medullary space in bone produced by transplanted scapular osteoblasts could result from their contamination with bone marrow stromal cells.

Animals↗

PubMed1996

Bisphosphonates act on osteoblastic cells and inhibit osteoclast formation in mouse marrow cultures.

We examined the mode of action of bisphosphonates on osteoclastic cell recruitment using mouse marrow cultures with or without osteoblastic cells. Tartrate-resistant acid phosphatase-positive multinucleated cells [TRAP(+)MNC] formed in cultures were determined to be osteoclastic cells. In marrow cultures, TRAP(+) MNC formation in the presence of 10(-8) mol/L 1,25(OH)2D3 was not affected by the addition of 10(-6) mol/L dihydrogen (cycloheptylamino)-methylenebisphosphonate monohydrate (YM175). However, it was inhibited in cocultures of marrow cells with osteoblastic cells. The inhibitory effect was evident throughout the entire culture period. YM175 dose dependently inhibited TRAP(+) MNC formation, and other bisphosphonates--pamidronate and alendronate--also inhibited TRAP(+) MNC formation in the coculture. Similar observations were also made in the coculture of spleen cells with osteoblastic cells. The conditioned media of osteoblastic cells treated with 10(-6) mol/L YM175 inhibited TRAP(+) MNC formation in marrow cultures. The presence of YM175 in methylcellulose cultures affected neither the colony formation of monocyte-macrophage lineage, nor TRAP(+) MNC formation in the succeeding cocultures of recovered cells with osteoblastic cells. These results indicate that YM175 and probably other bisphosphonates as well preferentially inhibit the later stage of osteoclastogenesis through its action on osteoblastic cells. Our findings suggest that part of the inhibitory action by osteoblastic cells in the presence of bisphosphonates is mediated through soluble factor(s).

Acid Phosphatase↗

PubMed2006

Myricetin induces human osteoblast differentiation through bone morphogenetic protein-2/p38 mitogen-activated protein kinase pathway.

Myricetin (3,3',4',5,5',7-hexahydroxyflavone), a flavonoid compound, is present in vegetables and fruits. By means of alkaline phosphatase (ALP) activity, osteocalcin, and type I collagen enzyme-linked immunosorbent assay (ELISA), we have shown that myricetin exhibits a significant induction of differentiation in MG-63 and hFOB human osteoblasts. Alkaline phosphatase and osteocalcin are phenotypic markers for early-stage differentiated osteoblasts and terminally differentiated osteoblasts, respectively. Our results indicate that myricetin stimulates osteoblast differentiation at various stages, from maturation to terminally differentiated osteoblasts. Induction of differentiation by myricetin is associated with increased bone morphogenetic protein-2 (BMP-2) production. The BMP-2 antagonist noggin blocked myricetin-mediated ALP activity and osteocalcin secretion enhancement, indicating that BMP-2 production is required in myricetin-mediated osteoblast maturation and differentiation. Induction of differentiation by myricetin is associated with increased activation of SMAD1/5/8 and p38 mitogen-activated protein kinases. Cotreatment of p38 inhibitor SB203580 inhibited myricetin-mediated ALP upregulation and osteocalcin production. In conclusion, myricetin increased BMP-2 synthesis, and subsequently activated SMAD1/5/8 and p38 MAPK, and this effect may contribute to its action on the induction of osteoblast maturation and differentiation, followed by an increase of bone mass.

Alkaline Phosphatase↗

PubMed2005

Studies on the receptors mediating responses of osteoblasts to thrombin.

The serine protease thrombin stimulates proliferation in osteoblasts, but decreases alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation. Three thrombin receptors have been identified, protease activated receptor (PAR)-1, PAR-3 and PAR-4; we have previously demonstrated that mouse osteoblasts express PAR-1 and PAR-4. The effect of thrombin on osteoblast proliferation and differentiation was studied to determine which of the thrombin receptors is responsible for the primary effects of thrombin. Primary mouse calvarial osteoblasts from PAR-1-null and wild-type mice, and synthetic peptides that specifically activate PAR-1 (TFFLR-NH2) and PAR-4 (AYPGKF-NH2) were used. Both the PAR-1-activating peptide and thrombin stimulated incorporation of 5-bromo-2'-deoxyuridine (two to four-fold, P < 0.001) and reduced alkaline phosphatase activity (approximately three-fold, P < 0.05) in cells from wild-type mice. The PAR-4-activating peptide, however, had no effect on either alkaline phosphatase activity or proliferation in these cells. Neither thrombin nor PAR-4-activating peptide was able to affect osteoblast proliferation or alkaline phosphatase activity in cells isolated from PAR-1-null mice. The results demonstrate that thrombin stimulates proliferation and inhibits differentiation of osteoblasts through activation of PAR-1. No other thrombin receptor appears to be involved in these effects.

Animals↗

PubMed2005

PGE2 induces the gene expression of bone matrix metalloproteinase-1 in mouse osteoblasts by cAMP-PKA signaling pathway.

Prostaglandin E2 (PGE2), an abundant eicosanoid in bone, has been implicated in a number of pathological states associated with bone loss, and is also known to stimulate matrix metalloproteinase (MMP)-1 synthesis and secretion in rat and human osteoblast cells, although the nature of the intracellular reaction remains unclear. Although MMP-1 plays a critical role in bone-remodeling, it would be of interest to examine whether PGE2 regulates MMP-1 expression by mouse osteoblasts or not. Here we demonstrate that PGE2 is a potent inducer of MMP-1 production in fetal osteoblasts and show that PGE2 stimulates the activity of the MMP-1 promoter in osteoblasts, suggesting that PGE2 controls MMP-1 gene expression at least at the transcriptional level. PGE2 induced MMP-1 messenger RNA (mRNA) expression in the cells within 4 h, and this expression was maintained for 36 h. The increase in MMP-1 production with 0.1-2.0 microM PGE2 was dose-dependent. We also found that PGE2 (1.5 microM) up-regulated MMP-1 protein levels in cultured mouse osteoblasts, as evidenced by ELISA. To examine whether PGE2 mediated response and signal pathway are involved in the intracellular action, the PGE2-mediated expression of the MMP-1 gene was investigated in mouse osteoblast cells. A Northern blot analysis showed that PGE2 and PGE1 were potent stimulators of MMP-1 transcription, and the presence of thromboxane B2 had no effect. The increase in MMP-1 transcript after PGE2 treatment was observed at 4h, reaching a maximum at 6h, and persisted for 24h. This response was dose-dependent. Cycloheximide, an inhibitor of protein synthesis, completely blocked this effect by PGE2, indicating that the expression of other genes is also required. The second messenger analog, 8-bromo-cAMP, mimicked the effects of PGE2 by stimulating a dose-dependent increase in MMP-1 mRNA levels, with a maximal effect that was quantitatively similar to that observed with PGE2. Thus, the present results strongly suggest that the PGE2 stimulation of MMP-1 synthesis is due to the activation of MMP-1 gene transcription and a subsequent marked increase in MMP-1 transcription. This effect is dependent on de novo protein synthesis and is mimicked by protein kinase A activation. The findings suggest that PGE2 is involved in the cAMP-PKA signaling pathway in regulating MMP-1 gene expression in osteoblasts.

Animals↗

PubMed2004

Chitosan supports the initial attachment and spreading of osteoblasts preferentially over fibroblasts.

The aim of this study was to determine chitosan's effect on osteoblast and fibroblast cell attachment. Mouse MC3T3-E1 osteoblasts and 3T3 fibroblasts were grown in the presence of serum on two commercially available chitosans, Chitosan-H (CH) and Protasan CL212 (PR). Cell attachment and immunofluorescent analysis at various time points were done to analyze initial phenotypic profiles. At 1h, significantly (P<0.05) fewer fibroblasts attached to CH or PR than serum-coated substrates. Osteoblast attachment to the same biopolymers at 1h was significantly greater than those seen with fibroblasts. At 24 h, levels of cell attachment for fibroblasts to both CH and PR significantly increased and were similar to levels seen in osteoblast cultures at both 1 and 24 h. Morphologically, immunofluorescent analysis showed that osteoblasts plated on the biopolymers were attached and beginning to spread at 1h, whereas fibroblasts appeared more rounded. At 24 h, fibroblasts plated on CH or PR revealed a heterogeneous population of round and semi-spread cells. In comparison, osteoblasts displayed phenotypes that were well spread with a developed cytoskeleton. These results suggest that CH and PR support the initial attachment and spreading of osteoblasts preferentially over fibroblasts, and that manipulation of the biopolymer can alter the level of attachment and spreading.

3T3 Cells↗

PubMed2004

Microcellular polyHIPE polymer supports osteoblast growth and bone formation in vitro.

A novel micro-cellular polymer with a well-defined and uniform micro-architecture has been developed as a three-dimensional support matrix for in vitro tissue engineering applications. This material is manufactured through a high internal phase emulsion (HIPE) polymerization route and may be modified with hydroxyapatite. The generic form of the support is known as PolyHIPE Polymer (PHP). By changing the chemical composition of the emulsion and the processing conditions, the pore size can be altered from sub-micron range to a few hundred microns and the porosity varied from 70% to 97%. Our work has investigated the use of this micro-porous polymer as a biomaterial to support the growth of osteoblasts, the bone forming cells in vitro. Three groups of polymers were used that had pore sizes of 40, 60 and 100 microm. Results demonstrated in vitro cell-polymer compatibility, with osteoblasts forming multicellular layers on the polymer surface and also migrating to a maximum depth of 1.4mm inside the scaffold after 35 days in culture. PHP was also able to support the differentiation of osteoblasts and the production of a bone-like matrix. The effect of modifying the polymer with hydroxyapatite was also studied and showed that there was a significant increase in osteoblast numbers penetrating into the polymer. There were few differences, between the pore sizes studied, on the overall penetration of osteoblasts into the polymer but the rate of movement into 100 microm PHP was significantly higher compared to the other sizes investigated. This study shows that osteoblasts seeded onto PHP demonstrate cellular attachment, proliferation and ingrowth leading to the support of an osteoblastic phenotype. Therefore this highly porous scaffold has a potential for bone tissue engineering.

Animals↗

PubMed2004

Osteoblast proliferation and maturation by bisphosphonates.

Aseptic loosening and osteolysis are currently the most common causes of failure of total joint replacements. Osteolysis is initiated by a macrophage response to wear debris, resulting in localized, osteoclastic peri-implant bone loss. We have previously inhibited osteoclast-mediated bone resorption in a canine total hip arthroplasty model using oral bisphosphonate therapy. Based on serendipitous observations from our canine study, we hypothesized that bisphosphonates have an anabolic effect on osteoblasts, in a manner distinct from their inhibitory effect on osteoclastic bone resorption. We studied the anabolic effects of two FDA-approved bisphosphonates (alendronate and risedronate) on two in vitro models: a primary human trabecular bone cell culture and the MG-63 osteoblast-like cell line. Following treatment with bisphosphonates at varying concentrations and time periods, cells were assayed for proliferation effects and results were quantified using the methods of direct cell count, and the colorimetric MTT (3-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay at 24, 48, and 72 h. The effect of bisphosphonates on the maturation of osteoblasts were tested with alkaline phosphatase bioassay and reverse transcription-polymerase chain reaction for markers of osteoblast differentiation. Results from both the primary human trabecular bone cell culture and the MG-63 osteoblast-like cell line showed that both bisphosphonates significantly increased the cell number over controls, attaining peak levels at a concentration of 10(-8)M. Alkaline phosphatase activity was also increased, representing earlier commitment of osteoprogenitor cells towards the osteoblastic phenotype. Bisphosphonates also enhanced gene expression of BMP-2, Type I collagen and osteocalcin. In summary, bisphosphonates, aside from their role as inhibitors of osteoclastic bone resorption, are promoters of osteoblast proliferation and maturation.

Adaptation, Physiological↗

PubMed2004

Increased osteoblast adhesion on nanophase metals: Ti, Ti6Al4V, and CoCrMo.

Previous studies have demonstrated increased functions of osteoblasts (bone-forming cells) on nanophase compared to conventional ceramics (specifically, alumina, titania, and hydroxyapatite), polymers (such as poly lactic-glycolic acid and polyurethane), carbon nanofibers/nanotubes, and composites thereof. Nanophase materials are unique materials that simulate dimensions of constituent components of bone since they possess particle or grain sizes less than 100 nm. However, to date, interactions of osteoblasts on nanophase compared to conventional metals remain to be elucidated. For this reason, the objective of the present in vitro study was to synthesize, characterize, and evaluate osteoblast adhesion on nanophase metals (specifically, Ti, Ti6Al4V, and CoCrMo alloys). Such metals in conventional form are widely used in orthopedic applications. Results of this study provided the first evidence of increased osteoblast adhesion on nanophase compared to conventional metals. Interestingly, osteoblast adhesion occurred preferentially at surface particle boundaries for both nanophase and conventional metals. Since more particle boundaries are present on the surface of nanophase compared to conventional metals, this may be an explanation for the measured increased osteoblast adhesion. Lastly, material characterization studies revealed that nanometal surfaces possessed similar chemistry and only altered in degree of nanometer surface roughness when compared to their respective conventional counterparts. Because osteoblast adhesion is a necessary prerequisite for subsequent functions (such as deposition of calcium-containing mineral), the present study suggests that nanophase metals should be further considered for orthopedic implant applications.

Alloys↗

PubMed2006

Integrin beta1 silencing in osteoblasts alters substrate-dependent responses to 1,25-dihydroxy vitamin D3.

Surface microroughness increases osteoblast differentiation and enhances responses of osteoblasts to 1,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. The observations that beta1 integrin expression is increased in osteoblasts grown on Ti substrates with rough microarchitecture, and that it is regulated by 1alpha,25(OH)2D3 in a surface-dependent manner, suggest that beta1 may play a role in mediating osteoblast response. To test this hypothesis, we silenced beta1 expression in MG63 human osteoblast-like cells using small interfering RNA (siRNA) and examined the responses of the beta1-silenced osteoblasts to surface microtopography and 1alpha,25(OH)2D3. To better understand the role of beta1, MG63 cells were also treated with two different monoclonal antibodies to human beta1 to block ligand binding. beta1-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor beta1, prostaglandin E2, and osteoprotegerin in comparison with control cells. Moreover, beta1-silencing inhibited the effects of surface roughness on these parameters and partially inhibited effects of 1alpha,25(OH)2D3. Anti beta1 antibody AIIB2 had no significant effect on cell number and osteocalcin, but decreased alkaline phosphatase; MAB2253Z caused dose-dependent decreases in cell number and alkaline phosphatase and an increase in osteocalcin. Effects of 1alpha,25(OH)2D3 on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased. These findings indicate that beta1 plays a major and complex role in osteoblastic differentiation modulated by either surface microarchitecture or 1alpha,25(OH)2D3. The results also show that beta1 mediates, in part, the synergistic effects of surface roughness and 1alpha,25(OH)2D3.

Animals↗

PubMed2004

Extracellular signal-regulated kinase-1 and -2 are both essential for the shear stress-induced human osteoblast proliferation.

Extracellular signal-regulated kinases (Erk)-1 and -2 are key mediators of various mitogenic signaling pathways, including mechanical stress-induced osteoblast proliferation. Mechanical stimuli, such as flow shear stress, simultaneously activate both Erk-1 and -2 in osteoblasts, resulting in stimulation of osteoblast proliferation. This study sought to test whether Erk-1, -2, or both are essential for the fluid flow shear stress-induced osteoblast proliferation. Moloney leukemia virus (MLV)-based vectors expressing wild-type (wt)- or kinase-deficient (kd) Erk-1 and Erk-2, respectively, were constructed and used to transduce human TE85 osteosarcoma cells with an MOI of 30. An MLV-red fluorescent protein (RFP) vector was included as a control. Effects of Erk-1 and -2 overexpression on cell proliferation in response to a 30-min constant fluid flow shear stress at 20 dynes/cm2 were determined with [3H]thymidine incorporation 24 h after the shear stress. The MLV-Erk vector-transduced TE85 cells showed a >10- and approximately 2-fold overexpression of Erk-1 and -2 protein, respectively. The RFP expressing control cells and the parental TE85 cells each showed an approximately twofold increase (P < 0.01) in [3H]thymidine incorporation in response to the shear stress. Cells overexpressing wt-Erk-1 or -2 showed small enhancing effects on the response to the shear stress in the increases in [3H]thymidine incorporation and cell number. Conversely, overexpression of kd-Erk-1 or -2 each alone completely abolished the shear stress-induced osteoblast proliferation. Overexpression of either kd-Erk-1 or kd-Erk-2 alone did not have a significant effect on basal osteoblast proliferation, suggesting that the Erk signaling pathway may not be essential for basal cell proliferation. In summary, this study demonstrates for the first time that Erk-1 and -2 are both required for the mitogenic response to fluid flow shear stress in human osteoblasts and that blocking Erk-1 or -2 each alone is sufficient to completely block the mitogenic response to shear stress-induced proliferation.

Amino Acid Sequence↗