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PubMed2006

Establishment of a novel method for enriching osteoblast progenitors from adipose tissues using a difference in cell adhesive properties.

In the clinical field, cell-based therapies are used to treat bone defects. Adipose tissues contain many osteoblast progenitors, among other cell types. We separated mouse adipose tissue-derived stromal cells (ATSCs) according to their cell adhesive properties. Cells in a fraction adherent to the culture dishes 0.5h after inoculation (AF-0.5) had a potent ability to differentiate into both osteoblasts and adipocytes in vitro. Their differentiation pathways depended on the culture conditions. In these cells, the expression of marker genes for osteoblast differentiation was induced in osteogenic medium. Moreover, the AF-0.5 cells, which were induced to differentiate into osteoblasts in vitro, formed abundant bone tissues in vivo. These results suggest that the AF-0.5 cells have been enriched with bi-potential progenitor cells destined for either osteoblasts or adipocytes. This simple and efficient method for preparing osteoblast progenitor cells from ATSCs may be utilized for bone defect treatment clinically.

Adipose Tissue↗

PubMed2006

Effects of different magnitudes of mechanical strain on Osteoblasts in vitro.

In addition to systemic and local factors, mechanical strain plays a crucial role in bone remodeling during growth, development, and fracture healing, and especially in orthodontic tooth movement. Although many papers have been published on the effects of mechanical stress on osteoblasts or osteoblastic cells, little is known about the effects of different magnitudes of mechanical strain on such cells. In the present study, we investigated how different magnitudes of cyclic tensile strain affected osteoblasts. MC3T3-E1 osteoblastic cells were subjected to 0%, 6%, 12% or 18% elongation for 24h using a Flexercell Strain Unit, and then the mRNA and protein expressions of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand (RANKL) were examined. The results showed that cyclic tensile strain induced a magnitude-dependent increase (0%, 6%, 12%, and 18%) in OPG synthesis and a concomitant decrease in RANKL mRNA expression and sRANKL release from the osteoblasts. Furthermore, the induction of OPG mRNA expression by stretching was inhibited by indomethacin or genistein, and the stretch-induced reduction of RANKL mRNA was inhibited by PD098059. These results indicate that different magnitudes of cyclic tensile strain influence the biological behavior of osteoblasts, which profoundly affects bone remodeling.

Animals↗

PubMed2007

Phosphodiesterase 4 inhibitor regulates the TRANCE/OPG ratio via COX-2 expression in a manner similar to PTH in osteoblasts.

Phosphodiesterase 4 (PDE4) inhibitors stimulate osteoclast formation by increasing the TRANCE/OPG mRNA ratio via cAMP-mediated pathways in a manner similar to parathyroid hormone (PTH) in osteoblasts. We investigated the role of cyclooxygenase-2 (COX-2) in osteoclast formation induced by the PDE4 inhibitor rolipram. Rolipram induced COX-2 expression in mRNA and protein levels, followed by increased prostaglandin E(2) production in osteoblasts. PKA, ERK, and p38 MAPK pathways regulate COX-2 mRNA expression induced by rolipram, in which PKA is a central regulator of the ERK and p38 MAPK pathways. A COX-2 inhibitor reversed the up-regulation of the TRANCE/OPG mRNA ratio induced by rolipram in osteoblasts, resulting in decreased osteoclast formation. These data suggest that COX-2 mediates rolipram induced osteoclast formation by regulating the TRANCE/OPG mRNA ratio in osteoblasts. Furthermore, the effects of the PDE4 inhibitor on osteoblasts were very similar to those of PTH, indicating that the PDE4 inhibitor largely shares the biological actions of PTH in osteoblasts.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2005

The licorice root derived isoflavan glabridin increases the function of osteoblastic MC3T3-E1 cells.

Glabridin, an isoflavan purified from licorice root, exhibits diverse biological activities, including estrogen-like activity. To investigate the bioactivities of glabridin, which act on bone metabolism, the effects of glabridin on the function of mouse osteoblastic cell line (MC3T3-E1) and the production of local factors in osteoblasts were studied. Glabridin (1-10microM) significantly increased the growth of MC3T3-E1 cells and caused a significant elevation of alkaline phosphatase (ALP) activity, collagen content and osteocalcin secretion in the cells (P<0.05). The effect of glabridin (10microM) in increasing ALP activity and collagen content was completely prevented by the presence of 10(-6)M cycloheximide and 10(-6)M tamoxifen, suggesting that glabridin's effect results from a newly synthesized protein component and might be partly involved in estrogen action. Then, the effects of glabridin on the TNF-alpha-induced apoptosis and production of prostaglandin E2 (PGE2) and nitric oxide (NO) in osteoblasts were examined. Treatment with glabridin (1-10microM) prevented apoptosis induced by TNF-alpha (10(-10)M) in osteoblastic cells. Moreover, glabridin (50microM) decreased the 10(-10)M TNF-alpha-induced production of PGE2 and NO in osteoblasts. Our data indicate that the enhancement of osteoblast function by glabridin may result in the prevention for osteoporosis and inflammatory bone diseases.

Animals↗

PubMed2004

Advanced glycation endproducts interefere with integrin-mediated osteoblastic attachment to a type-I collagen matrix.

The adhesion of osteoblasts to bone extracellular matrix, of which type-I collagen constitutes >85%, can modulate diverse aspects of their physiology such as growth, differentiation and mineralisation. In this study we examined the adhesion of UMR106 rat osteoblast-like cells either to a control (Col) or advanced-glycation-endproduct-modified (AGEs-Col) type I collagen matrix. We investigated the possible role of different integrin receptors in osteoblastic adhesion, by co-incubating these cells either with beta-peptide (conserved sequence 113-125 of the beta subunit of integrins) or with two other peptides, RGD (Arg-Gly-Asp) and DGEA (Asp-Gly-Glu-Ala), which are recognition sequences for the alpha-subunits of alpha(1,5)beta(1) and alpha(2)beta(1) integrins. Collagen glycation inhibited the adhesion of UMR106 osteoblasts to the matrix (40% reduction versus Col, P > 0.001). beta-Peptide showed a dose- and glycation-dependent inhibitory effect on adhesion, and at a concentration of 100 microM decreased the attachment of UMR106 cells to both matrices (42% to Col, P<0.001and 25% to AGEs-Col, P<0.01). The synthetic peptides RGD (1mM) and DGEA (5mM) inhibited the attachment of UMR106 cells to Col (30 and 20%, P > 0.01 and P< 0.001, respectively), but not to AGEs-Col. beta-Peptide induced an increase in UMR106 cell clumping and a decrease in cellular spreading, while DGEA increased spreading with cellular extensions in multiple directions. These results indicate that both alpha and beta integrin subunits participate in osteoblastic attachment to type-I collagen, probably through the alpha(1,5)beta(1) and alpha(2)beta(1) integrins. AGEs-modification of type-I collagen impairs the integrin-mediated adhesion of osteoblastic cells to the matrix, and could thus contribute to the pathogenesis of diabetic osteopenia.

Animals↗

PubMed2004

Cellular biocompatibility and stimulatory effects of calcium metaphosphate on osteoblastic differentiation of human bone marrow-derived stromal cells.

In the present study, the in vitro biocompatibility of calcium metaphosphate (CMP) with human bone marrow stromal cells (HBMSCs) and its effect on osteoblastic differentiation have been investigated. Powder and disk forms of CMP do not exert a cytotoxic effect on the HBMSCs undergoing osteoblastic differentiation. In addition, the HBMSCs adhere to the surface of the CMP disk as successfully as to the culture plate or hydroxyapatite (HA) disk. The HBMSCs adhered to either the HA or CMP disk display an undistinguishable actin arrangement and cellular phenotypes, indicating that the CMP does not disrupt normal cellular responses. An analysis of the differentiation of the HBMSCs cultured on culture plate, the HA and the CMP disk shows that three matrices are capable of supporting osteoblastic differentiation of the HBMSCs as accessed by alkaline phosphatase (ALP) staining. Further molecular analysis of osteoblastic differentiation of HBMSCs reveals that the CMP disk has a better ability than the HA disk to induce an expression of osteoblast-related genes, including ALP, osteoprotegerin (OPG), a decoy receptor for RANK ligand, and osteopontin (OPN), a non-collagenous bone matrix protein. The results demonstrate that, in addition to favorable biocompatibility, the CMP can stimulate osteoblastic differentiation of the HBMSCs in vitro.

Biocompatible Materials↗

PubMed2004

Characterization of titanium surfaces with calcium and phosphate and osteoblast adhesion.

The titanium surfaces containing calcium, phosphate ions and the carbonate apatite were characterized. The effect of surface chemistry on the initial rabbit osteoblast response on these surfaces was investigated. The cell count and alkaline phosphatase (ALP) specific activity assay were used for biochemical analyses. Scanning electron microscopy was used for morphology observation and in particular X-ray photoelectron spectroscopy (XPS) for surface chemistry characterization. The number of cells adhering to the apatite coating surface was the maximum, the number of cells on the surface containing calcium without phosphate ions was higher than that containing phosphate without calcium, and the number on the unmodified titanium surface was the least. The osteoblasts cultured on the apatite surface exhibited the highest ALP specific activity, next were the ones on the surface containing solely calcium, the lowest were on the unmodified titanium surface. On the substrate surfaces removed of adhered cells, the order of nitrogen amounts detected by XPS was consistent with ones of ALP specific activity and cell number, except for the unmodified titanium surface. For the substrate surfaces removed of adhered osteoblasts, XPS analysis showed that calcium and phosphorous amounts decreased during cell adhesion. After cell culture the Ca2p binding energy (BE) values for apatite coating and the surface containing solely calcium were similar to those of the two surfaces adsorbed bovine serum albumin (BSA). The P2p BE values for the surfaces containing phosphate ions, including the apatite coating and the surface containing solely phosphate ions, showed the same change. But after cell culture the decrease of the P2p BE value for the coating surface was larger than the one for the surface containing solely phosphate ions. Considering the bovine serum albumin adsorption on the same samples, these results indicated that calcium ions on titanium surfaces play a more important role than phosphate ions in initial interactions among culture medium, osteoblasts and titanium surfaces. On the apatite coating surface, calcium ions are active sites for osteoblast adhesion, while calcium and phosphate ions co-exist on titanium surfaces, the former promotes the osteoblast adhesion onto the phosphate sites on titanium surfaces. The cell adhesion was a complicated biological and chemical process relating to surface several elements similar to protein adsorption.

Animals↗

PubMed2004

The effect of rhBMP-2 on canine osteoblasts seeded onto 3D bioactive polycaprolactone scaffolds.

Our strategy entails investigating the influence of varied concentrations (0, 10, 100 and 1000ng/ml) of human recombinant bone morphogenetic protein-2 (rhBMP-2) on the osteogenic expression of canine osteoblasts, seeded onto poly-caprolactone 20% tricalcium phosphate (PCL-TCP) scaffolds in vitro. Biochemical assay revealed that groups with rhBMP-2 displayed an initial burst in cell growth that was not dose-dependent. However, after 13 days, cell growth declined to a value similar to control. Significantly less cell growth was observed for construct with 1000ng/ml of rhBMP-2 from 20 days onwards. Confocal microscopy confirmed viability of osteoblasts and at day 20, groups seeded with rhBMP-2 displayed heightened cell death as compared to control. Phase contrast and scanning electron microscopy revealed that osteoblasts heavily colonized surfaces, rods and pores of the PCL-TCP scaffolds. This was consistent for all groups. Finally, Von Kossa and osteocalcin assays demonstrated that cells from all groups maintained their osteogenic phenotype throughout the experiment. Calcification was observed as early as four days after stimulation for groups seeded with rhBMP-2. In conclusion, rhBMP-2 seems to enhance the differentiated function of canine osteoblasts in a non-dose dependent manner. This resulted in accelerated mineralization, followed by death of osteoblasts as they underwent terminal differentiation. Notably, PCL-TCP scaffolds seeded only with canine osteoblasts could sustain excellent osteogenic expression in vitro. Hence, the synergy of PCL with bioactive TCP and rhBMP-2 in a novel composite scaffold, could offer an exciting approach for bone regeneration.

Animals↗

PubMed2005

The effect of irradiation modification and RGD sequence adsorption on the response of human osteoblasts to polycaprolactone.

Using techniques of tissue engineering, synthetic substitutes can be applied for the repair and regeneration of damaged bone. It has been found that material surface properties are crucial for cell adhesion and spreading, i.e. cell activities that are related directly to the ability of osteoblasts to proliferate. This fact has promoted the strategy of creating an ECM-like layer onto materials, so as to influence the cell response. In this study human bone-derived osteoblasts have been used to test the effects of surface modification by low energy ion beams of a poly epsilon-caprolactone (PCL) substrate and subsequent RGD adsorption. Osteoblasts were seeded and grown onto untreated and irradiated poly epsilon-caprolactone films, with or without RGD-adsorption step, and viability, morphology, and spreading of the osteoblasts were studied at different time endpoints. Differences were observed in the organization of cytoskeleton within cells: stress fibers were more evident in irradiated samples vs. untreated and total cell adhesion was higher. Surface characterization by X-ray Photoelectron Spectroscopy, Atomic Force Microscopy, and surface free energy measurements showed that the polar character of PCL, i.e., the acid-base term, was increased following irradiation treatment. Moreover the irradiated PCL had a nano-sized topography, which also could improve osteoblasts adhesion. We found that the treatment of the surface with ion beam is per se improving osteoblasts adhesion and spreading onto PCL. Furthermore, also if a significant RGD adsorption was obtained for irradiated PCL surfaces, it was found that in the investigated conditions it seems to have only a minor effect on the cell response. This study suggests that new strategies involving irradiation-based treatments can be adopted to promote the initial steps of bone deposition onto synthetic surfaces, exploiting the surface-induced reorganization of the ECM matrix.

Adsorption↗

PubMed2005

The enhancement of osteoblast growth and differentiation in vitro on a peptide hydrogel-polyHIPE polymer hybrid material.

The objective of this study was to investigate the effect of combining two biomaterials on osteoblast proliferation, differentiation and mineralised matrix formation in vitro. The first biomaterial has a well-defined architecture and is known as PolyHIPE polymer (PHP). The second biomaterial is a biologically inspired self-assembling peptide hydrogel (RAD16-I, also called PuraMatrix) that produces a nanoscale environment similar to native extracellular matrix (ECM). Our work investigates the effect of combining RAD16-I with two types of PHP (HA (Hydroxyapatite)-PHP and H (Hydrophobic)-PHP) and evaluates effects on osteoblast growth and differentiation. Results demonstrated successful incorporation of RAD16-I into both types of PHP. Osteoblasts were observed to form multicellular layers on the combined biomaterial surface and also within the scaffold. Dynamic cell seeding and culturing techniques were compared to static seeding methods and produced a more even distribution of cells throughout the constructs. Cells were found to penetrate the scaffold to a maximum depth of 3 mm after 35 days in culture. There was a significant increase in cell number in H-PHP constructs coated with RAD16-I compared to H-PHP alone. Our results show that RAD16-I enhances osteoblast differentiation and indicates that the incorporation of this peptide provides a more permissive environment for osteoblast growth. We have developed a microcellular polymer containing a nanoscale environment to enhance cell: biomaterial interactions and promote osteoblast growth in vitro.

Alkaline Phosphatase↗

PubMed2006

Osteoblast functions on functionalized PMMA-based polymers exhibiting Staphylococcus aureus adhesion inhibition.

Staphylococcus aureus adhesion and osteoblast functions were assessed on functionalized poly(methyl methacrylate)-based terpolymers bearing randomly distributed carboxylate and sulfonate groups. These terpolymers were synthesized by radical polymerization, characterized by nuclear resonance spectroscopy and classified by the ratio R=[COO(-)/COO(-)+SO(3)(-)] in the range 0.5-0.8. Bacterial adhesion study showed that fibronectin-coated terpolymers with R varying from 0.5 to 0.8 exhibited inhibition rate of S. aureus adhesion from 90% to 98% as compared to the adhesion on unfunctionalized poly(methyl methacrylate). In contrast, the adhesion of osteoblasts onto the same functionalized terpolymers was decreased by 20% when compared to the results obtained on poly(methyl methacrylate). While the amount of attached osteoblasts are similar onto all the functionalized terpolymers whatever its R value, the cell proliferation was different and was found to vary with R in the range 0.5-0.8. Osteoblast proliferation, alkaline phosphatase activity and accumulation of calcium in the extracellular matrix of these cells, cultured on the functionalized terpolymers with R equal to 0.7-0.8 were similar to that observed onto non-functionalized poly(methyl methacrylate). In contrast, osteoblast proliferation was inhibited on terpolymers with an R value around 0.6. These results provide evidence that functionalized poly(methyl methacrylate)-based terpolymers with R ratio equaling 0.7-0.8 simultaneously inhibit bacteria adhesion and support osteoblast functions pertinent to new bone formation. These functionalized polymers could, therefore, be used as coating or grafted on orthopedic and dental implants to render them both bone compatible and able to prevent bacterial infection.

Animals↗

PubMed2004

Nitric oxide mediates ultrasound-induced hypoxia-inducible factor-1alpha activation and vascular endothelial growth factor-A expression in human osteoblasts.

Vascular endothelial growth factor (VEGF) is an important regulator for angiogenesis and endochondral bone formation. Although low-intensity pulsed ultrasound (US) has been recently used for accelerating fracture healing, the effect of US stimulation on angiogenic factor production by osteoblasts remains undetermined. Here, we found that US elevation of VEGF-A expression in human osteoblasts to be mediated by nitric oxide (NO) and hypoxia-inducible factor-1alpha (HIF-1alpha). Human osteoblasts were treated with or without US stimulation (200 micros pulse, 1 kHz at 30 mW/cm2) for 20 min. Cells were subjected to assessment of VEGF-A expression, NO production, nitric oxide synthase (NOS) catalytic activities, and HIF-1alpha transactivation. Results showed that US significantly increased VEGF-A mRNA and protein levels in 6 h. US augmentation of VEGF level was transcriptionally mediated. Early inhibition of NO production, but not calcium or prostaglandin E2, significantly reduced US-enhanced VEGF-A levels. Osteoblasts responded to US treatment by increasing NO production, NOS catalytic activities, iNOS immunoexpression, nuclear HIF-1alpha activation, and binding to the VEGF-A promoter. Inhibition of NOS activity by N-nitro-L-arginine methyl ester (L-NAME) or blockade of guanylate cyclase activity by ODQ reduced US-augmented HIF-1alpha transactivation and VEGF-A levels. Conditioned medium harvested from US-treated osteoblasts promoted tube formation of human umbilical vein endothelial cells (HUVEC). Monoclonal VEGF-A antibody neutralization or L-NAME pretreatment reduced the promoting effect of conditioned medium on angiogenesis of HUVEC. Together, these findings show that NO plays an important role in mediating extracellular stimuli released by US and triggering intracellular response of osteoblasts to produce angiogenic factor after US treatment.

Calcium↗

PubMed2004

Vitamin D inhibits Fas ligand-induced apoptosis in human osteoblasts by regulating components of both the mitochondrial and Fas-related pathways.

Apoptosis plays an important role in the regulation of bone turnover. Previously, we showed that 1,25(OH)2D3, the active form of vitamin D, may increase osteoblast survival by inhibiting apoptosis induced by serum deprivation. Human osteoblasts express the Fas receptor on their surface and its interaction with Fas ligand has been closely associated with human osteoblast apoptosis. To investigate the mechanism of 1,25(OH)2D3 inhibition of apoptosis in osteoblasts isolated from human calvaria, cells were exposed to Fas antibody. Visualization of apoptotic cells using annexin V revealed a significant decrease in apoptosis at 48 h in the presence of 1,25(OH)2D3 (14 +/- 4%, P < 0.04) compared with non-treated cells (52 +/- 4%). Furthermore, flow cytometric analysis of TUNEL-labeled osteoblasts showed a significant decrease in apoptotic cells in 1,25(OH)2D3-treated cultures (12 +/- 2%) at 48 h compared with non-treated cultures (44 +/- 3%, P < 0.04). Additionally, cells treated with 1,25(OH)2D3 survived longer as found by MTS analysis. To further explore the mechanism of 1,25(OH)2D3-mediated inhibition of apoptosis, we examined the changes in activation of death domain proteins, cleavage of caspases and mitochondrial regulators of apoptosis by Western blot analysis. A significant inhibition of caspase-8 cleavage and activity in 1,25(OH)2D3-treated cells was observed in conjunction with a decrease in the expression of the proapoptotic protein Bax with a significant increase in the expression of antiapoptotic protein Bcl-2. Furthermore, the levels of p21Cip1/WAF1, which inhibits the cleavage of caspase-8, was found to be highly induced in 1,25(OH)2D3-treated cells. In summary, these results demonstrate that the anti-apoptotic effect of 1,25(OH)2D3 in human osteoblasts after the activation of Fas-ligand is mediated by the regulation of components of both the mitochondrial and Fas-related pathways.

Annexin A5↗

PubMed2004

Long-term effects of neridronate on human osteoblastic cell cultures.

UNLABELLED: Bisphosphonates (BPs) are widely used in the treatment of a variety of bone-related diseases, particularly where the bone turnover is skewed in favor of osteolysis. The mechanisms by which BPs reduce bone resorption directly acting on osteoclasts are now largely clarified even at molecular level. Researches concerning the BP's effects on osteoblast have instead shown variable results. Many in vitro studies have reported positive effects on osteoblasts proliferation and mineralization for several BPs; however, the observed effects differ, depending on the variety of different model system that has been used. OBJECTIVES: We have investigated if neridronate, an aminobisphosphonate suitable for pulsatory parenteral administration, could have an effect on human osteoblastic proliferation and differentiation in vitro. METHODS: We have investigated whether prolonged addition of neridronate (from 10(-3) to 10(-11) M) to different human osteoblasts cultures, obtained from 14 different bone specimens, could affect the cells number, the endogenous cellular alkaline phosphatase (ALKP) activity, and the formation of mineralized nodules. RESULTS: Our results show that neridronate does not negatively affect in vitro the viability, proliferation, and cellular activity of normal human osteoblasts even after a long period addition of the drug (20 days) at concentrations equal or lower than 10(-5) mol/l (therapeutic dose). In addition, neridronate seems to enhance the differentiation of cultured osteoblasts in mature bone-forming cells. A maximum increase of alkaline phosphatase activity (+50% after 10 days; P < 0.01) and mineralized nodules (+48% after 20 days; P < 0.05) was observed in cultures treated with neridronate 10(-8) M. CONCLUSIONS: These results encourage the use of neridronate in long-term therapy of demineralizing metabolic bone disorders.

Aged↗

PubMed2005

The hyalectan degrading ADAMTS-1 enzyme is expressed by osteoblasts and up-regulated at regions of new bone formation.

During bone formation, there are numerous pivotal changes in the interrelationships between osteoblasts and molecules of the extracellular matrix (ECM). Consequently, the mechanisms that underlie the temporal and spatial distribution of ECM molecules in bone are of considerable interest in understanding its formation. A subfamily of a disintegrin and metalloproteinase (ADAMs) has been identified, which contain thrombospondin-like motifs (ADAMTS), and can break down several ECM molecules. Using reversed transcribed PCR, we identified ADAMTS-1, -4 and -5 mRNA expression in cultures of rat osteoblasts treated with ascorbic acid, beta-glycerophosphate and dexamethasone, molecules known to drive osteoblast differentiation. Of these, ADAMTS-1 followed most closely the osteogenic marker osteocalcin during in vitro mineralisation. Consequently, we studied, in detail, protein expression of ADAMTS-1 during in vitro osteogenesis together with ADAMTS-1 immunohistochemistry staining of sections from 2- and 10-day-old rat femur. Western analysis of osteoblast proteins showed ADAMTS-1 products that correspond well with both full-length and furin-processed species. In the ECM laid down by osteoblasts, only the mature secreted protein (approximately 90 kDa) and its accumulation during the later stages of osteogenesis in vitro were noticed. Furthermore, immunostaining with an antibody recognising ADAMTS-1 demonstrated strong expression around mineralised nodules and intense focal staining of putative new areas of nodule formation in vitro. Finally, immunohistochemistry of 2- and 10-day-old rat femur localised ADAMTS-1 protein to regions associated with osteogenesis. These data show that ADAMTS-1 protein accumulates in osteoblast ECM during differentiation. Furthermore, the focalised expression of ADAMTS-1 in regions of osteogenesis, both in vitro and in vivo, implicates this multifunctional protein to be involved in mineralised nodule and bone formation.

ADAM Proteins↗

PubMed2005

Ghrelin stimulates proliferation and differentiation and inhibits apoptosis in osteoblastic MC3T3-E1 cells.

Ghrelin is a 28-amino-acid peptide identified in the stomach as an endogenous ligand of the growth hormone secretagogue receptor (GHS-R) that strongly stimulates the release of growth hormone at the hypothalamus and pituitary level. Although GHS-Rs are expressed in a variety of peripheral tissues, little is known about its effect on bone independent of GH/IGF-1 axis. This study was undertaken to investigate whether ghrelin exerts a direct effect on osteoblasts. We identified mRNA and protein expression of GHS-R in primary osteoblasts as well as a number of osteoblastic cell lines, including MC3T3-E1, ROS 17/2.8, UMR-106, MG63, and SaOS2 cells. Treatment of ghrelin (10(-11) to 10(-7) M) to MC3T3-E1 cells showed dose-dependent stimulation of proliferation, which was abrogated by treatment with [d-Lys]-GHRP-6 (10(-3) M), a selective antagonist of the ghrelin receptor. Ghrelin activated ERK1/2 MAPK and pretreatment with MAPK kinase inhibitors, PD98059 attenuated the ghrelin-induced cell proliferation. Ghrelin also inhibited TNFalpha-induced apoptosis and suppressed caspase-3 activation that occurs in response to TNFalpha as well as during in vitro differentiation process. Moreover, ghrelin treatment enhanced in vitro osteoblast differentiation as evidenced by matrix mineralization, alkaline phosphatase activity, and osteoblast-specific gene expression. These results suggest that ghrelin promotes proliferation and differentiation and inhibits apoptosis of osteoblasts.

Animals↗

PubMed2005

Osteoblasts produce monocyte chemoattractant protein-1 in a murine model of Staphylococcus aureus osteomyelitis and infected human bone tissue.

Incidences of osteomyelitis caused by Staphylococcus aureus have increased dramatically in recent years, in part, due to the appearance of community-acquired antibiotic-resistant strains. Therefore, understanding the pathogenesis of this organism has become imperative. Recently, we have described the surprising ability of bone-forming osteoblasts to secrete a number of important immune mediators when exposed to S. aureus in vitro. In the present study, we provide the first evidence for the in vivo production of the pivotal inflammatory chemokine, monocyte chemoattractant protein-1 (MCP-1), by osteoblasts during S. aureus-associated bone infection. Quantitative real-time PCR was employed to determine levels of mRNA encoding MCP-1 in vivo using a mouse model that closely resembles the pathology of trauma-induced staphylococcal osteomyelitis. Expression of this inflammatory chemokine and osteoblast-specific markers was investigated by confocal laser scanning microscopy in bone tissue from organ cultures of neonatal mouse calvaria and from the in vivo mouse model. Furthermore, the clinical relevancy of these findings was investigated by performing similar studies on infected human bone tissue from patients with S. aureus-associated osteomyelitis. Here, we confirm that expression of mRNA encoding MCP-1 is elevated in bacterially infected murine bone tissue. Importantly, we show that these increases translate into marked elevations in the expression of MCP-1 protein that co-localizes with osteoblast markers in infected bone tissue. Such increases could not be attributed solely to mechanical damage as a similar response was observed in infected but otherwise undamaged organ cultures. Finally, we have demonstrated the in vivo production of MCP-1 by osteoblasts in bone specimens from patients with S. aureus-associated osteomyelitis. As such, these studies demonstrate that bacterial challenge of osteoblasts during bone diseases such as staphylococcal osteomyelitis induces cells to produce a key inflammatory chemokine that can direct appropriate host responses or may contribute to progressive inflammatory damage.

Animals↗

PubMed2006

Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells.

Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis of osteoblast migration after wounds were introduced into cell monolayers indicated that LPA stimulated both migration velocity and the average migration distance per cell. LPA also elicited substantial changes in cell shape and actin cytoskeletal structure; lipid-treated cells contained fewer stress fibers and displayed long membrane processes that were enriched in F-actin. Quantitative RT-PCR analysis showed that MC3T3-E1 cells express all four known LPA-specific G-protein-coupled receptors (LPA1-LPA4) with a relative mRNA abundance of LPA1>LPA4>LPA2>>LPA3. LPA-induced changes in osteoblast motility and morphology were antagonized by both pertussis toxin and Ki16425, a subtype-specific blocker of LPA1 and LPA3 receptor function. Cell migration in many cell types is linked to changes in intracellular Ca2+. Ki16425 also inhibited LPA-induced Ca2+ signaling in a dose-dependent manner, suggesting a link between LPA-induced Ca2+ transients and osteoblast chemotaxis. Our data show that LPA stimulates MC3T3-E1 osteoblast motility via a mechanism that is linked primarily to the G-protein-coupled receptor LPA1.

3T3 Cells↗