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Osteoblast Cultures (osteoblast + culture)
Selected AbstractsTGF-,1 alone and in combination with calcium hydroxide is synergistic to TGF-,1 production by osteoblasts in vitroINTERNATIONAL ENDODONTIC JOURNAL, Issue 5 2000A. Jaunberzins Abstract Aim To examine the effects of calcium hydroxide (Ca(OH)2), transforming growth factor-beta (TGF-,1), and Ca(OH)2/TGF-,1 coadministration on TGF-,1 and interleukin-6 (IL-6) synthesis by early (subculture 1) and late (subculture 5) osteoblast cultures. Methodology Early and late cultures were established using bone cells harvested from 21-day-old fetal rat calvaria. Cell cultures of both early and late osteoblasts were divided into four groups: group 1, control; group 2, cells challenged with Ca(OH)2; group 3, cells challenged with TGF-,1; and group 4, cells challenged with Ca(OH)2 and TGF-,1 in combination. TGF-,1 and IL-6 levels for all groups were determined using ELISA methodology. Results anova and Tukey HS analyses revealed that osteoblasts of groups 3 and 4 significantly increased (P < 0.001) TGF-,1 synthesis in both early and late cultures of osteoblasts. IL-6 was not detected in any of the groups considered in this study. Conclusions Exogenous TGF-,1 has an autocrine effect on cell cultures of osteoblasts. Administration of TGF-,1 alone or in combination with Ca(OH)2 increases the synthesis of TGF-,1 in osteoblast cultures. Ca(OH)2 and TGF-,1 are compatible when placed in a culture of osteoblasts. Ca(OH)2 provides a favourable environment for the anabolic effects of TGF-,1. [source] Effect of Osteoblast-Targeted Expression of Bcl-2 in Bone: Differential Response in Male and Female Mice,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2005Alexander G Pantschenko Abstract Transgenic mice (Col2.3Bcl-2) with osteoblast-targeted human Bcl-2 expression were established. Phenotypically, these mice were smaller than their wildtype littermates and showed differential effects of the transgene on bone parameters and osteoblast activity dependent on sex. The net effect was an abrogation of sex differences normally observed in wildtype mice and an inhibition of bone loss with age. Ex vivo osteoblast cultures showed that the transgene had no effect on osteoblast proliferation, but decreased bone formation. Estrogen was shown to stimulate endogenous Bcl-2 message levels. These studies suggest a link between Bcl-2 and sex regulation of bone development and age-related bone loss. Introduction: Whereas Bcl-2 has been shown to be an important regulator of apoptosis in development, differentiation, and disease, its role in bone homeostasis and development is not well understood. We have previously showed that the induction of glucocorticoid-induced apoptosis occurred through a dose-dependent decrease in Bcl-2. Estrogen prevented glucocorticoid-induced osteoblast apoptosis in vivo and in vitro by preventing the decrease in Bcl-2 in osteoblasts. Therefore, Bcl-2 may be an important regulator of bone growth through mechanisms that control osteoblast longevity and function. Materials and Methods: Col2.3Bcl-2 mice were developed carrying a 2.3-kb region of the type I collagen promoter driving 1.8 kb of human Bcl-2 (hBcl-2). Tissue specific expression of hBcl-2 in immunoassays validated the transgenic animal model. Histomorphometry and DXA were performed. Proliferation, mineralization, and glucocorticoid-induced apoptosis were examined in ex vivo cultures of osteoblasts. The effect of estrogen on mouse Bcl-2 in ex vivo osteoblast cultures was assayed by RT-PCR and Q-PCR. Results and Conclusions: Two Col2.3Bcl-2 (tg/+) founder lines were established and appeared normal except that they were smaller than their nontransgenic wildtype (+/+) littermates at 1, 2, and 6 months of age, with the greatest differences at 2 months. Immunohistochemistry showed hBcl-2 in osteoblasts at the growth plate and cortical surfaces. Nontransgenic littermates were negative. Western blots revealed hBcl-2 only in type I collagen-expressing tissues. Histomorphometry of 2-month-old mice showed a significant decrease in tg/+ calvaria width with no significant differences in femoral trabecular area or cortical width compared with +/+. However, tg/+ males had significantly more trabecular bone than tg/+ females. Female +/+ mice showed increased bone turnover with elevated osteoblast and osteoclast parameters compared with +/+ males. Col2.3Bcl-2 mice did not show such significant differences between sexes. Male tg/+ mice had a 76.5 ± 1.5% increase in ObS/BS with no significant differences in bone formation rate (BFR) or mineral apposition rate (MAR) compared with male +/+ mice. Transgenic females had a significant 48.4 ± 0.1% and 20.1 ± 5.8% decrease in BFR and MAR, respectively, compared with +/+ females. Osteoclast and osteocyte parameters were unchanged. By 6 months, femurs from female and male +/+ mice had lost a significant amount of their percent of trabecular bone compared with 2-month-old mice. There was little to no change in femoral bone in the tg/+ mice with age. Ex vivo cultures of osteoblasts from +/+ and Col2.3Bcl-2 mice showed a decrease in mineralization, no effect on proliferation, and an inhibition of glucocorticoid-induced apoptosis in Col2.3Bcl-2 cultures. Estrogen was shown to increase mouse Bcl-2 transcript levels in osteoblast cultures of wildtype mice, supporting a role for Bcl-2 in the sex-related differences in bone phenotype regulated by estrogen. Therefore, Bcl-2 differentially affected bone phenotype in male and female transgenic mice, altered bone cell activity associated with sex-related differences, and decreased bone formation, suggesting that apoptosis is necessary for mineralization. In addition, Bcl-2 targeted to mature osteoblasts seemed to delay bone development, producing a smaller transgenic mouse compared with wildtype littermates. These studies suggest that expression of Bcl-2 in osteoblasts is important in regulating bone mass in development and in the normal aging process of bone. [source] Activation of Protease-Activated Receptor-2 Leads to Inhibition of Osteoclast Differentiation,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2004Rosealee Smith Abstract PAR-2 is expressed by osteoblasts and activated by proteases present during inflammation. PAR-2 activation inhibited osteoclast differentiation induced by hormones and cytokines in mouse bone marrow cultures and may protect bone from uncontrolled resorption. Introduction: Protease-activated receptor-2 (PAR-2), which is expressed by osteoblasts, is activated specifically by a small number of proteases, including mast cell tryptase and factor Xa. PAR-2 is also activated by a peptide (RAP) that corresponds to the "tethered ligand" created by cleavage of the receptor's extracellular domain. The effect of activating PAR-2 on osteoclast differentiation was investigated. Materials and Methods: Mouse bone marrow cultures have been used to investigate the effect of PAR-2 activation on osteoclast differentiation induced by parathyroid hormone (PTH), 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], and interleukin-11 (IL-11). Expression of PAR-2 by mouse bone marrow, mouse bone marrow stromal cell-enriched cultures, and the RAW264.7 osteoclastogenic cell line was demonstrated by RT-PCR. Results: RAP was shown to inhibit osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL-11. Semiquantitative RT-PCR was used to investigate expression of mediators of osteoclast differentiation induced by PTH, 1,25(OH)2D3, or IL-11 in mouse bone marrow cultures and primary calvarial osteoblast cultures treated simultaneously with RAP. In bone marrow and osteoblast cultures treated with PTH, 1,25(OH)2D3, or IL-11, RAP inhibited expression of RANKL and significantly suppressed the ratio of RANKL:osteoprotegerin expression. Activation of PAR-2 led to reduced expression of prostaglandin G/H synthase-2 in bone marrow cultures treated with PTH, 1,25(OH)2D3, or IL-11. RAP inhibited PTH- or 1,25(OH)2D3 -induced expression of IL-6 in bone marrow cultures. RAP had no effect on osteoclast differentiation in RANKL-treated RAW264.7 cells. Conclusion: These observations indicate that PAR-2 activation inhibits osteoclast differentiation by acting on cells of the osteoblast lineage to modulate multiple mediators of the effects of PTH, 1,25(OH)2D3, and IL-11. Therefore, the role of PAR-2 in bone may be to protect it from uncontrolled resorption by limiting levels of osteoclast differentiation. [source] Increased Bone Formation in Mice Lacking Plasminogen Activators,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2003E Daci Abstract Plasminogen activators tPA and uPA are involved in tissue remodeling, but their role in bone growth is undefined. Mice lacking tPA and uPA show increased bone formation and bone mass. The noncollagenous components of bone matrix are also increased, probably from defective degradation. This study underlines the importance of controlled bone matrix remodeling for normal endochondral ossification. Introduction: Proteolytic pathways are suggested to play a role in endochondral ossification. To elucidate the involvement of the plasminogen activators tPA and uPA in this process, we characterized the long bone phenotype in mice deficient in both tPA and uPA (tPA,/,:uPA,/,). Materials and Methods: Bones of 2- to 7-day-old tPA,/,:uPA,/, and wild-type (WT) mice were studied using bone histomorphometry, electron microscopy analysis, and biochemical assessment of bone matrix components. Cell-mediated degradation of metabolically labeled bone matrix, osteoblast proliferation, and osteoblast differentiation, both at the gene and protein level, were studied in vitro using cells derived from both genotypes. Results: Deficiency of the plasminogen activators led to elongation of the bones and to increased bone mass (25% more trabecular bone in the proximal tibial metaphysis), without altering the morphology of the growth plate. In addition, the composition of bone matrix was modified in plasminogen activator deficient mice, because an increased amount of proteoglycans (2×), osteocalcin (+45%), and fibronectin (+36%) was detected. Matrix degradation assays showed that plasminogen activators, by generating plasmin, participate in osteoblast-mediated degradation of the noncollagenous components of bone matrix. In addition, proliferation of primary osteoblasts derived from plasminogen activator-deficient mice was increased by 35%. Finally, osteoblast differentiation and formation of a mineralized bone matrix were enhanced in osteoblast cultures derived from tPA,/,:uPA,/, mice. Conclusions: The data presented indicate the importance of the plasminogen system in degradation of the noncollagenous components of bone matrix and suggest that the accumulation of these proteins in bone matrix,as occurs during plasminogen activator deficiency,may in turn stimulate osteoblast function, resulting in increased bone formation. [source] Cytokines, Osteoprotegerin, and RANKL In Vitro and Histomorphometric Indices of Bone Turnover in Patients With Different Bone Diseases,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2003Heide Siggelkow Abstract Cytokines are supposed to play an essential role in the regulation of the bone metabolic unit. However, information on cytokine production of primary human osteoblasts from patients with metabolic bone disease is scarce, and few attempts have been made to correlate such data to histomorphometric parameters of individual patients. We investigated 11 patients with metabolic bone disease referred to our outpatient department for bone biopsy and analyzed interleukin (IL)-1, IL-6, and TNF-, protein release and gene expression in primary osteoblast cultures. Compared with four controls, five patients showed normal cytokine protein release, whereas six patients showed much higher levels of interleukin-6 (26-fold) and TNF-, (84-fold). All three cytokines were strongly correlated concerning gene expression and/or protein levels (r = 0.72,0.96). Histomorphometric analysis of the bone samples showed that eroded surface (ES/BS) as a parameter of bone resorption was significantly associated with TNF-,. In addition, RANKL gene expression was positively associated with ES/BS and osteoclast surface (Oc.S/BS). Finally, the formation parameters osteoid volume and osteoid surface were negatively associated with TNF-,. In conclusion, in an in vitro-ex vivo model of bone cells obtained from a group of 11 patients with different forms of metabolic bone disease, cytokine release in conditioned medium was significantly associated with bone resorption and bone formation, as quantified by histomorphometry. TNF-, seemed to be the more important cytokine; its effect on bone resorption could be mediated by RANKL. [source] Role of the Latent Transforming Growth Factor ,,Binding Protein 1 in Fibrillin-Containing Microfibrils in Bone Cells In Vitro and In VivoJOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2000Sarah L. Dallas Abstract Latent transforming growth factor ,,binding proteins (LTBPs) are extracellular matrix (ECM) proteins that bind latent transforming growth factor , (TGF-,) and influence its availability in bone and other connective tissues. LTBPs have homology with fibrillins and may have related functions as microfibrillar proteins. However, at present little is known about their structural arrangement in the ECM. By using antibodies against purified LTBP1, against a short peptide in LTBP1, and against epitope-tagged LTBP1 constructs, we have shown colocalization of LTBP1 and fibrillin 1 in microfibrillar structures in the ECM of cultured primary osteoblasts. Immunoelectron microscopy confirmed localization of LTBP1 to 10- to 12-nm microfibrils and suggested an ordered aggregation of LTBP1 into these structures. Early colocalization of LTBP1 with fibronectin suggested a role for fibronectin in the initial assembly of LTBP1 into the matrix; however, in more differentiated osteoblast cultures, LTBP1 and fibronectin 1 were found in distinct fibrillar networks. Overexpression of LTBP1 deletion constructs in osteoblast-like cells showed that N-terminal amino acids 67,467 were sufficient for incorporation into fibrillin-containing microfibrils and suggested that LTBP1 can be produced by cells distant from the site of fibril formation. In embryonic long bones in vivo, LTBP1 and fibrillin 1 colocalized at the surface of newly forming osteoid and bone. However, LTBP1-positive fibrils, which did not contain fibrillin 1, were present in cartilage matrix. These studies show that in addition to regulating TGF,1, LTBP1 may function as a structural component of connective tissue microfibrils. LTBP1 may therefore be a candidate gene for Marfan-related connective tissue disorders in which linkage to fibrillins has been excluded. [source] Inhibition of osteoblast function in vitro by aminobisphosphonatesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2009Isabel R. Orriss Abstract Bisphosphonates are analogues of pyrophosphate, a key physicochemical inhibitor of mineralisation. We examined the direct actions of bisphosphonates on the function of cultured osteoblasts derived from rat calvariae. Treatment with zoledronate, the most potent bisphosphonate studied, reduced osteoblast number at concentrations ,100 nM and was strongly toxic at 10 µM, causing a threefold decrease in osteoblast viability after 2 days and a 90% decrease in cell numbers after 14 days. In control osteoblast cultures on plastic, abundant formation of ,trabecular' mineralised bone matrix nodules began after 10 days. Continuous exposure to zoledronate inhibited bone mineralisation at concentrations as low as 10 nM. Pamidronate and clodronate exerted similar effects but at higher doses (,1 and ,10 µM, respectively). Short-term or intermittent exposure of osteoblasts to zoledronate and pamidronate (1,10 µM) was sufficient to inhibit bone mineralisation by ,85%. Zoledronate but not pamidronate or clodronate also strongly inhibited osteoblast alkaline phosphatase activity at concentrations ,100 nM and soluble collagen production at concentrations ,1 µM. We additionally studied the effects of zoledronate on osteoblasts cultured on dentine, a bone-like mineralised substrate, observing similar inhibitory effects, although at concentrations 10,100-fold higher; this shift presumably reflected adsorption of zoledronate to dentine mineral. Thus, zoledronate blocked bone formation in two ways: first, a relatively non-toxic, selective inhibition of mineralisation at concentrations in the low nanomolar range and second, a cytotoxic inhibition of osteoblast growth and function at concentrations ,1 µM. Although no data are available on the bisphosphonate concentrations that osteoblasts could be exposed to in vivo, our results are consistent with earlier observations that bisphosphonates may inhibit bone formation. J. Cell. Biochem. 106: 109,118, 2009. © 2008 Wiley-Liss, Inc. [source] Opposing effects of glucocorticoids and Wnt signaling on Krox20 and mineral deposition in osteoblast culturesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2008Nathalie Leclerc Abstract Krox20 is expressed in osteoblasts and chondrocytes, and is required for trabecular bone formation during embryogenesis. Here we show by RT-qPCR and Western blot analysis that Krox20 is up-regulated during late stages of osteoblast differentiation in culture. Glucocorticoids (GCs) rapidly inhibit the expression of Krox20 as well its co-activator, HCF-1, resulting in inhibition of the Osteocalcin Krox20-binding Enhancer (OKE). GCs also inhibit expression of EGR1, EGR3, and EGR4. OKE activity, which is dependent on the presence of Runx2, was independent of the osteocalcin promoter Runx2 binding site. In contrast to GCs, activation of the Wnt, but not the BMP or the PTH signaling pathways, stimulated Krox20 expression as well as activity of the OKE. GC-mediated suppression of Krox20 expression was compromised, albeit not completely, in the presence of DKK1, suggesting that the inhibition occurs in both Wnt-dependent and Wnt-independent manners. Furthermore, Wnt3A partially rescued Krox20 expression in GC-arrested osteoblast cultures and this was accompanied by rescue of mineralization. These findings are consistent with a role for Krox20 in osteoblast function and suggest that this transcription factor may contribute to the opposing effects of GCs and Wnt signaling on bone formation. J. Cell. Biochem. 103: 1938,1951, 2007. © 2007 Wiley-Liss, Inc. [source] Role of TNF alpha and PLF in bone remodeling in a rat model of repetitive reaching and grasping,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2010Shobha Rani We have previously developed a voluntary rat model of highly repetitive reaching that provides an opportunity to study effects of non-weight bearing muscular loads on bone and mechanisms of naturally occurring inflammation on upper limb tissues in vivo. In this study, we investigated the relationship between inflammatory cytokines and matricellular proteins (Periostin-like-factor, PLF, and connective tissue growth factor, CTGF) using our model. We also examined the relationship between inflammatory cytokines, PLF and bone formation processes. Rats underwent initial training for 5 weeks, and then performed a high repetition high force (HRHF) task (12,reaches/min, 60% maximum grip force, 2,h/day, 3 days/week) for 6 weeks. We then examined the effect of training or task performance with or without treatment with a rat specific TNF, antibody on inflammatory cytokines, osteocalcin (a bone formation marker), PLF, CTGF, and behavioral indicators of pain or discomfort. The HRHF task decreased grip strength and induced forepaw mechanical hypersensitivity in both trained control and 6-week HRHF animals. Two weeks of anti-TNF, treatment improved grip strength in both groups, but did not ameliorate forepaw hypersensitivity. Moreover, anti-TNF, treatment attenuated task-induced increases in inflammatory cytokines (TNF,, IL-1,, and MIP2 in serum; TNF, in forelimb bone and muscles) and serum osteocalcin in 6-week HRHF animals. PLF levels in forelimb bones and flexor digitorum muscles increased significantly in 6-week HRHF animals, increases attenuated by anti-TNF, treatment. CTGF levels were unaffected by task performance or anti-TNF, treatment in 6-week HRHF muscles. In primary osteoblast cultures, TNF,, MIP2 and MIP3a treatment increased PLF levels in a dose dependent manner. Also in primary osteoblast cultures, increased PLF promoted proliferation and differentiation, the latter assessed by measuring Runx2, alkaline phosphatase (ALP) and osteocalcin mRNA levels; ALP activity; as well as calcium deposition and mineralization. Increased PLF also promoted cell adhesion in MC3T3-E1 osteoblast-like cell cultures. Thus, tissue loading in vivo resulted in increased TNF,, which increased PLF, which then induced anabolic bone formation, the latter results confirmed in vitro. J. Cell. Physiol. 225: 152,167, 2010. © 2010 Wiley-Liss, Inc. [source] Bone-specific heparan sulfates induce osteoblast growth arrest and downregulation of retinoblastoma proteinJOURNAL OF CELLULAR PHYSIOLOGY, Issue 1 2006Kerry J. Manton The heparan sulfate (HSs) sugars of the extracellular matrix (ECM) play a key role during both development and wound repair in regulating the flow of growth and adhesive factors across their cell surface receptors. The aim of this study was to assess the structural and functional differences of HS chains extracted from the conditioned media (soluble), cell surface, and ECM of primary human osteoblast cultures, and to analyze their effects on osteoblast cell growth. HS chains from these compartments were characterized through a combination of enzymatic degradation, anion exchange chromatography, and molecular sieving. Although the chains were all approximately the same size, they varied systematically in their sulfate content, suggesting differences in their protein-binding domains. When added to pre-confluent hFOB1.19 osteoblast cultures, HS doses exceeding 500 ng/ml inhibited proliferation, without affecting viability, irrespective of their origin. Furthermore, HS doses of 500 ng/ml also downregulated retinoblastoma, Cyclin A and CDK1 protein expression, indicating that high doses of osteoblast HS negatively regulate cell cycle, resulting in growth arrest; when high doses of HS were withdrawn after a prolonged period, linear cell growth was reestablished. Thus, despite differences in sulfation, HS from either the soluble, cell surface, or matrix compartments of primary human osteoblast cultures are functionally similar with respect to their effects on growth. Binding assays revealed that the HS chains bound TGF,1, a known inhibitor of osteoprogenitor growth, at higher affinity than a suite of other bone-related, heparin-binding growth factors. Overcoming such sugar-mediated inhibition may prove important for wound repair. J. Cell. Physiol. 209: 219,229, 2006. © 2006 Wiley-Liss, Inc. [source] Intracellular Staphylococcus aureus and antibiotic resistance: Implications for treatment of staphylococcal osteomyelitisJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2006J. Kent Ellington Abstract Staphylococcus aureus is responsible for 80% of human osteomyelitis. It can invade and persist within osteoblasts. Antibiotic resistant strains of S. aureus make successful treatment of osteomyelitis difficult. Null Hypothesis: antibiotic sensitivities of S. aureus do not change after exposure to the osteoblast intracellular environment. Human and mouse osteoblast cultures were infected and S. aureus cells were allowed to invade. Following times 0, 12, 24, and 48 h (,± the addition of erythromycin, clindamycin, and rifampin at times 0 or 12 h), the osteoblasts were lysed and intracellular bacteria enumerated. Transmission electron microscopy was performed on extracellular and intracellular S. aureus cells. In mouse osteoblasts, administration of bacteriostatic antibiotics at time 0 prevented the increase in intracellular S. aureus. If the antibiotics were delayed 12 h, this did not occur. When rifampin (bactericidal) was introduced at time 0 to human and mouse osteoblasts, there was a significant decrease in number of intracellular S. aureus within osteoblasts compared to control. If rifampin was delayed 12 h, this did not occur. Significant time-dependent S. aureus structural changes were observed after exposure to the osteoblast intracellular environment. These studies demonstrate that once S. aureus is established intracellularly for 12 h, the bacteria are less sensitive to antibiotics capable of eukaryotic cell penetration (statistically significant). These antibiotic sensitivity changes could be due in part to the observed structural changes. This leads to the rejection of our null hypotheses that the antibiotic sensitivities of S. aureus are unaltered by their location. © 2005 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res [source] | |||||||||||||