Osteoblast Collagen Synthesis Research Articles

Metabolic, but not respiratory, acidosis increases bone PGE(2) levels and calcium release.

A decrease in blood pH may be due to either a reduction in bicarbonate concentration ([HCO(3)(-)]; metabolic acidosis) or to an increase in PCO(2) (respiratory acidosis). In mammals, metabolic, but not respiratory, acidosis increases urine calcium excretion without altering intestinal calcium absorption, indicating that the additional urinary calcium is derived from bone. In cultured bone, chronic metabolic, but not respiratory, acidosis increases net calcium efflux (J(Ca)), decreases osteoblastic collagen synthesis, and increases osteoclastic bone resorption. Metabolic acidosis increases bone PGE(2) production, which is correlated with J(Ca), and inhibition of PGE(2) production inhibits this acid-induced J(Ca). Given the marked differences in the osseous response to metabolic and respiratory acidosis, we hypothesized that incubation of neonatal mouse calvariae in medium simulating respiratory acidosis would not increase medium PGE(2) levels, as observed during metabolic acidosis. To test this hypothesis, we determined medium PGE(2) levels and J(Ca) from calvariae incubated at pH approximately 7.1 to model either metabolic (Met; [HCO(3)(-)] approximately 11 mM) or respiratory (Resp; PCO(2) approximately 83 Torr) acidosis, or at pH approximately 7.5 as a control (Ntl). We found that after 24-48 and 48-51 h in culture, periods when cell-mediated J(Ca) predominates, medium PGE(2) levels and J(Ca) were increased with Met, but not Resp, compared with Ntl, and there was a direct correlation between medium PGE(2) levels and J(Ca). Thus metabolic, but not respiratory, acidosis induces the release of bone PGE(2), which mediates J(Ca) from bone.

Transcriptional regulation of alpha 2(I) collagen gene expression by fibroblast growth factor-2 in MC3T3-E1 osteoblast-like cells

Fibroblast growth factor-2 (FGF-2) stimulates proliferation and inhibits differentiated function of osteoblasts by suppressing synthesis of type I collagen and other proteins. However, little is known regarding the molecular mechanisms regulating the suppressive effects of FGF-2 on type I collagen synthesis in osteoblasts. The zinc finger transcription factor Egr-1 and the basic helix-loop-helix (bHLH) family of proteins have been implicated in the regulation of genes crucial to mesodermal cell growth and differentiation. The aim of this study was to determine whether Egr-1 and TWIST might be potential transcriptional regulators of the inhibitory effects of FGF-2 on alpha2(I) collagen expression in MC3T3-E1 osteoblasts which undergo a developmental sequence in vitro. Upon treatment of undifferentiated MC3T3-E1 cells with 1 nM FGF-2, Egr-1 mRNA increased with the effect maximal after 30-60 min. TWIST mRNA also increased with the effect maximal at 2 h. We analyzed the transcriptional control of alpha2(I) collagen gene expression by FGF-2 by transient transfection of an alpha2(I) collagen-luciferase construct (pH5) into undifferentiated MC3T3-E1 cells. The activity of the pH5 luciferase promoter decreased in a dose-dependent manner following treatment with.01 and 1 nM FGF-2. We identified putative Egr-1 and TWIST recognition sequences in the proximal region of the promoter for the murine alpha2(I) collagen gene and a putative Egr-1 site in the 5' region of the murine TWIST promoter. In gel mobility shift assays, potential Egr-1 response elements in the 5' region of the murine TWIST and alpha2(I) collagen genes demonstrated specific Egr-1 binding activity with bFGF-treated nuclear extracts obtained from MC3T3-E1 cells. These results indicate that Egr-1 and TWIST are expressed in undifferentiated MC3T3-E1 osteoblast-like cells following treatment with FGF-2 and they may be potential transcriptional regulators of FGF-2s negative effects on alpha2(I) collagen gene expression. J. Cell. Biochem. 80:550-559, 2001. Published 2001 Wiley-Liss, Inc.

The effect of carboxyl-terminal propeptide of type I collagen (c-propeptide) on collagen synthesis of preosteoblasts and osteoblasts.

Recently we found that the carboxyl-terminal propeptide of type I collagen (c-propeptide) is a major secretory protein of osteoblasts. Mature osteoblasts secreted 64 nM c-propeptide, and it was reported that 40 nM c-propeptide inhibited collagen synthesis at 80% of the control level. In this study, we investigated the effect of c-propeptide on collagen synthesis of preosteoblasts and osteoblasts, and found that preosteoblasts downregulated collagen synthesis by 40 nM c-propeptide, but osteoblasts were not affected by the same condition. When the binding activities of c-propeptide for preosteoblasts and osteoblasts were compared, osteoblasts showed weak affinity to c-propeptide compared with preosteoblasts, and the number of receptors for c-propeptide decreased in osteoblasts. These results imply that a decrease of receptors in osteoblasts might reduce the sensitivity of osteoblasts to c-propeptide.

Gene expression of transforming growth factor-beta 3 and tissue inhibitor of metalloproteinase type 1 during membranous bone healing in rats.

A number of growth factors have been implicated in fracture repair. Transforming growth factor-beta 3 (TGF-beta 3) is believed to be involved in osteoblast proliferation, chemotaxis, and collagen synthesis. The collagens act as the scaffolding for new bone matrix formation, whereas tissue inhibitors of metalloproteinases (TIMPs) may help regulate matrix remodeling in bone repair. Despite their hypothesized integral role in fracture repair, the temporal expression of these molecules in membranous bone fracture healing remains unknown. The objective of this study was to assess the temporal pattern of TGF-beta 3 and TIMP type 1 (TIMP-1) expression in rat mandibular fracture healing. Twenty-eight adult male Sprague-Dawley rats underwent a mandibular osteotomy, and the healing regenerate was harvested on postoperative days 3, 5, 7, 9, 23, and 37. Total cellular ribonucleic acid was isolated, and Northern analysis was performed. TGF-beta 3 expression was downregulated dramatically 3 days after the osteotomy and remained less than 20% of control levels throughout repair. In marked contrast, TIMP-1 gene expression, low during early repair, increased more than twofold over control at later time points. Understanding the temporal pattern of gene expression during membranous fracture healing has important clinical implications because elucidating these mechanisms may lead to appropriate biomolecular approaches to augment membranous bone fracture healing.

Particulate wear debris activates protein tyrosine kinases and nuclear factor kappaB, which down-regulates type I collagen synthesis in human osteoblasts.

Particulate wear debris generated mechanically from prosthetic materials is phagocytosed by a variety of cell types within the periprosthetic space including osteoblasts, which cells with an altered function may contribute to periprosthetic osteolysis. Exposure of osteoblast-like osteosarcoma cells or bone marrow-derived primary osteoblasts to either metallic or polymeric particles of phagocytosable sizes resulted in a marked decrease in the steady-state messenger RNA (mRNA) levels of procollagen alpha1[I] and procollagen alpha1[III]. In contrast, no significant effect was observed for the osteoblast-specific genes, such as osteonectin and osteocalcin (OC). In kinetic studies, particles once phagocytosed, maintained a significant suppressive effect on collagen gene expression and type I collagen synthesis for up to five passages. Large particles of a size that cannot be phagocytosed also down-regulated collagen gene expression suggesting that an initial contact between cells and particles can generate gene responsive signals independently of the phagocytosis process. Concerning such signaling, titanium particles rapidly increased protein tyrosine phosphorylation and nuclear transcription factor kappaB (NF-kappaB) binding activity before the phagocytosis of particles. Protein tyrosine kinase (PTK) inhibitors such as genistein and the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) significantly reduced the suppressive effect of titanium on collagen gene expression suggesting particles suppress collagen gene expression through the NF-kappaB signaling pathway. These results provide a mechanism by which particulate wear debris can antagonize the transcription of the procollagen alpha1[I] gene in osteoblasts, which may contribute to reduced bone formation and progressive periprosthetic osteolysis.

Hypoxia increases insulinlike growth factor gene expression in rat osteoblasts.

Vascular disruption secondary to fracture leads to a hypoxic zone of injury where the oxygen tension at the center of the wound is quite low. In this dynamic microenvironment, a number of growth factors are elaborated to stimulate the synthetic processes of fracture repair. Previously the authors have shown the hypoxia-induced increase of vascular endothelial growth factor expression in osteoblasts. The purpose of these experiments was to examine osteoblast expression of insulinlike growth factors (IGF) I and II--cytokines believed to play a role in increased collagen synthesis, chemotaxis, and proliferation of osteoblasts in response to hypoxia. Primary cell cultures of osteoblasts isolated from neonatal rat calvaria were subjected to hypoxia (PO2 = 35 mmHg) for 0, 3, 6, 24, and 48 hours. Northern blot analysis of ribonucleic acid (RNA) from resulting cultures demonstrated a more than 60% increase in IGF-II messenger RNA (mRNA) expression after 3 hours of hypoxia. IGF-II mRNA expression continued to increase through later time points to 200% and 260% of baseline at 24 and 48 hours respectively. In contrast, IGF-I demonstrated no significant change in mRNA expression compared with baseline control (normoxia) cultures. In these experiments the authors have demonstrated a hypoxia-induced increase in IGF-II but not IGF-I in primary osteoblasts. The differential expression of these two growth factors may underscore important differences in the behavior of osteoblasts in the hypoxic fracture microenvironment. Taken together, these data add additional support to the theory that hypoxia induces gene-specific changes in expression of molecules important to extracellular matrix formation for successful bone healing.

In vitro metabolic and respiratory acidosis selectively inhibit osteoblastic matrix gene expression.

Clinically, a decrease in blood pH may be due to either a reduction in bicarbonate concentration ([HCO(-)(3)], metabolic acidosis) or an increase in PCO(2) (respiratory acidosis). In mammals, metabolic acidosis induces a far greater increase in urine calcium excretion than respiratory acidosis. In cultured bone, metabolic acidosis induces a marked increase in calcium efflux and a decrease in osteoblastic collagen synthesis, whereas isohydric respiratory acidosis has little effect on either parameter. We have shown that metabolic acidosis prevents the normal developmental increase in the expression of RNA for matrix Gla protein and osteopontin in chronic cultures of primary murine calvarial bone cells (predominantly osteoblasts) but does not alter expression of osteonectin. To compare the effects of isohydric metabolic and respiratory acidosis on expression of these genes, bone cell cultures were incubated in medium at pH approximately 7.2 to model metabolic ([HCO(-)(3)], approximately 13 mM) or respiratory (PCO(2), approximately 80 mmHg) acidosis or at pH approximately 7.4 as a control. Cells were sampled at weeks 4, 5, and 6 to assess specific RNA content. At all time periods studied, both metabolic and respiratory acidosis inhibited the expression of RNA for matrix Gla protein and osteopontin to a similar extent, whereas there was no change in osteonectin expression. In contrast to the significant difference in the effects of metabolic and respiratory acidosis on bone calcium efflux and osteoblastic collagen synthesis, these two forms of acidosis have a similar effect on osteoblastic RNA expression of both matrix Gla protein and osteopontin. Thus, although several aspects of bone cell function are dependent on the type of acidosis, expression of these two matrix genes appears to be regulated by extracellular pH, independently of the type of acidosis.

Effect of diclofenac on human osteoblasts and their stromal precursors in vitro in relation to arthroplasty

Results of animal experiments have demonstrated that the osseous integration of non-cemented prostheses can, at the very least temporarily, be impaired by the application of non-steroidal antiphlogistic agents (such as diclofenac). It is the objective of this study to examine whether there is a direct influence of diclofenac used in usual clinical dosages (3 times 50 mg daily) on bone cells and their progenitor cells which would explain the observed slow integration of the prostheses. To investigate this, cultivated human in vitro osteoblasts and stromal bone marrow cells were incubated with increasing doses of the medications. Our study focused on the effect of diclofenac application on proliferation and functional metabolism in both cell lines. The measurable maximal plasma concentration 2 h after the application of one tablet Voltaren 50 reached 1.6 micrograms/ml. This correlated with diclofenac concentrations between 1 and 10 ml found in our experiments. The detected values were correlated to the control group (0 microgram/ml diclofenac). The drug effect upon osteoblasts was higher than on progenitor cells. The proliferation of in vitro stromal bone marrow cells, compared to untreated cells, was found to be decreased. We observed a decrease to 82% at a diclofenac concentration of 1 microgram/ml, Osteoblasts exhibited a decrease to 97.5% at the same concentration. The DNA synthesis increased to 118% in stromal bone marrow cells, in osteoblasts to 144%. In contrast, we detected a neglectible decrease to 92% in the collagen synthesis of osteoblasts compared to untreated cells. The synthesis of osteocalcin by osteoblasts increased to 119%. The alkaline phosphatase activity was found to be decreased to 88% in stromal bone marrow cells and increased in osteoblasts to 111%. Temporary inhibiting effects on osseous integration in non-cemented prosthesis by diclofenac could be caused by a disturbance in the anabolic bone metabolism, exhibited by an increase of osteoblastic osteocalcin expression. Osteocalcin as a known negative regulator of the osteoneogenesis is most likely inhibiting the collagen matrix deposition.

Leukemia inhibitory factor and oncostatin M stimulate collagenase-3 expression in osteoblasts.

Leukemia inhibitory factor (LIF) and oncostatin M (OSM) have multiple effects on skeletal remodeling. Although these cytokines modestly regulate collagen synthesis in osteoblasts, their effects on collagenase expression and collagen degradation are not known. We tested whether LIF and OSM regulate the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in osteoblast-enriched cells isolated from fetal rat calvariae. LIF and OSM increased collagenase-3 (MMP-13) mRNA and immunoreactive protein levels in a time- and dose-dependent manner. LIF and OSM enhanced the rate of transcription of the collagenase gene and stabilized collagenase mRNA in transcriptionally arrested cells. LIF and OSM failed to regulate the expression of gelatinase A (MMP-2) and B (MMP-9). LIF and OSM modestly stimulated the expression of TIMP-1 but did not alter the expression of TIMP-2 and -3. In conclusion, LIF and OSM stimulate collagenase-3 and TIMP-1 expression in osteoblasts, and these effects may be involved in mediating the bone remodeling actions of these cytokines.

Hydroxyapatite coated with insulin-like growth factor 1 (IGF1) stimulates human osteoblast activity in vitro.

We studied the effects of a hydroxyapatite-tricalcium phosphate material coated with Insulin-like Growth Factor 1 (IGF1) on cell growth, collagen synthesis and alkaline phosphatase activity (ALP) of human osteoblasts in vitro. Cell proliferation was stimulated when osteoblasts were incubated with untreated hydroxyapatite (HA) and it was further increased by exposure to IGF1-coated HA. 3H-Proline uptake was significantly increased by treatment with either HA or IGF1-coated HA but no significant differences were found between these two groups. ALP activity was enhanced by exposure to HA, with respect to the control, and further increased by treatment with IGF1-coated HA. Our findings suggest that HA is useful for promoting osteoblast activity and IGF1 may help to improve its biological characteristics.

Growth factor stimulation of bone healing

Bone tissue has been shown to contain numerous cell-to-cell signalling peptides called growth factors. These growth factors are thought to have important regulating effects for bone remodeling and bone healing, due to their potent effects on bone cell metabolism. In vivo studies over the last half decade have demonstrated that growth factors candidates for future clinical use in orthopedic surgery. In numerous clinical situations enhanced bone formation and bone healing could lead to improved results of surgical procedures. This thesis describes the most important bone growth factors and their actions in vitro and in vivo. In vitro investigations of growth factor effects on osteoblast chemotaxis and metabolism are described as well as in vivo studies with growth factor stimulation of fracture healing and bone healing to prosthetic-like implants. In vitro results: Several growth factors exhibited chemotactic effects towards human osteoblasts. TGF-beta 1 and PDGF-BB had the strongest chemotactic effects, whereas PDGF-AA, IGF-1, and IGF-2 had less but significant chemotactic effects towards human osteoblasts. TGF-beta 1 exhibited the highest chemotactic potency with maximal activity at 100 pg/mL, whereas the other growth factors had maximal effects at 10-100 ng/mL. BMP-2 was found to have chemotactic effects toward human osteoblasts, human bone marrow osteoprogenitor cells, and U2-OS osteosarcoma cells. BMP-4 and BMP-6 were without any chemotactic effects towards these celltypes. Human bone marrow osteoprogenitor cells were the most responsive celltype to BMP-2 stimulation. Growth factor combinations resulted in synergic stimulative effects on different metabolic functions on human osteoblasts. Combinations with TGF-beta 1 and PDGF-BB strongly stimulated proliferation and chemotaxis. Combinations with TGF-beta 1, PDGF-BB and BMP-2 strongly stimulated an osteoblast differentiation parameter (alkaline phosphatase activity). The different growth factor combinations had no effect on collagen synthesis in human osteoblasts. In vivo results: Continuous application of 1 and 10 micrograms natural TGF-beta to a plated tibial osteotomy in rabbits increased mechanical bending strength and callus formation at 6 weeks observation. Diaphyseal cortical bone remodeling was not affected by the local growth factor application. In a dog model with unloaded implants surrounded by a gap, 0.3 microgram rhTGF-beta 1 adsorbed to gritblasted tricalcium phosphate coated implants, was able to enhance mechanical fixation, bone ingrowth and gap bone formation. 3.0 micrograms rhTGF-beta 1 had less but significant stimulative effect. In a weight-loaded model, 0.3 microgram rhTGF-beta 1, adsorbed to gritblasted tricalcium phosphate coated implants, was able to enhance bone ingrowth, without enhancement of mechanical fixation. In the unloaded model, 0.3 microgram rhTGF-beta 1, adsorbed to gritblasted hydroxyapatite coated implants, was able to enhance bone ingrowth, without enhancement of mechanical fixation. 3.0 micrograms rhTGF-beta 1 had no stimulative effects. The establishment of a biological implant fixation concept with growth factor absorbed to ceramic coatings of implants was successful. These data are promising for a possible future clinical usage of growth factors, especially for enhancement of bone healing to cementless prosthetic components.

Decreased potassium stimulates bone resorption.

Metabolic acidosis induces net calcium efflux (JCa+) from cultured bone, in part, through an increase in osteoclastic resorption and a decrease in osteoblastic formation. In humans provision of base as potassium (K+) citrate, but not sodium (Na+) citrate, reduces urine Ca (UCa), and oral KHCO3 decreases bone resorption and UCa in postmenopausal women. Potassium deprivation alone leads to an increase in UCa. To determine whether decreased extracellular K+ concentration ([K+]) at a constant pH, PCO2, and [HCO-3] alters JCa+ and bone cell activity, we measured JCa+, osteoblastic collagen synthesis, and osteoclastic beta-glucuronidase release from neonatal mouse calvariae cultured for 48 h in medium of varying [K+]. Calvariae were cultured in control medium (approximately 4 mM [K+]) or medium with mildly low K+ (MLK, approximately 3 mM [K+]), very low K+ (VLK, approximately 2 mM [K+]), or extremely low K+ (ELK, approximately 1 mM [K+]) (n > or = 9 in each group). Compared with control, ELK, but not MLK or VLK, resulted in a marked increase in JCa+ and an increase in beta-glucuronidase release and a decrease in collagen synthesis. JCa+ was correlated directly with medium beta-glucuronidase activity and inversely with collagen synthesis. To determine whether the reduction in medium [K+] was associated with a decrease in intracellular pH (pHi), we measured pHi in MC3T3-E1 cells, a mouse osteoblastic cell line. Incubation in 1 mM [K+] led to a significant decrease in pHi compared with 3 mM [K+]. Thus incubation in a reduced [K+] medium stimulates JCa+ and osteoclastic enzyme release and inhibits osteoblastic collagen synthesis, which may be mediated by a reduction in bone cell pH.

Metabolic alkalosis decreases bone calcium efflux by suppressing osteoclasts and stimulating osteoblasts.

In vivo and in vitro evidence indicates that metabolic acidosis, which may occur prior to complete excretion of end products of metabolism, increases urinary calcium excretion. The additional urinary calcium is almost certainly derived from bone mineral. Neutralization of this daily acid load, through the provision of base, decreases calcium excretion, suggesting that alkali may influence bone calcium accretion. To determine whether metabolic alkalosis alters net calcium efflux (JCa+) from bone and bone cell function, we cultured neonatal mouse calvariae for 48 h in either control medium (pH approximately equal to 7.4, [HCO3-] approximately equal to 24), medium simulating mild alkalosis (pH approximately equal to 7.5, [HCO3-] approximately equal to 31), or severe alkalosis (pH approximately equal to 7.6, [HCO3-] approximately equal to 39) and measured JCa+ and the release of osteoclastic beta-glucuronidase and osteoblastic collagen synthesis. Compared with control, metabolic alkalosis caused a progressive decrease in JCa+, which was correlated inversely with initial medium pH (pHi). Alkalosis caused a decrease in osteoclastic beta-glucuronidase release, which was correlated inversely with pHi and directly with JCa+. Alkalosis also caused an increase in osteoblastic collagen synthesis, which was correlated directly with pHi and inversely with JCa+. There was a strong inverse correlation between the effects alkalosis on osteoclastic beta-glucuronidase release and osteoblastic collagen synthesis. Thus metabolic alkalosis decreases JCa+ from bone, at least in part, by decreasing osteoclastic resorption and increasing osteoblastic formation. These results suggest that the provision of base to neutralize endogenous acid production may improve bone mineral accretion.

Additive effects of acidosis and parathyroid hormone on mouse osteoblastic and osteoclastic function.

Patients with end-stage renal disease are acidotic and often develop secondary hyperparathyroidism. Whether acidosis contributes to the bone disease observed in these patients is not clear. To determine whether acidosis and parathyroid hormone (PTH) have additive effects on net calcium efflux (JCa+) from bone and on bone cell function, we measured JCa+, osteoblastic collagen synthesis, and osteoclastic beta-glucuronidase release from neonatal mouse calvariae cultured in control (Ctl, pH approximately 7.4) or acidified (Met, pH approximately 7.1) medium with or without a submaximal concentration of PTH (10(-10) M) for 48 h. Compared with Ctl, from 24 to 48 h JCa+ was increased with Met and with PTH, and the combination of Met + PTH increased JCa+ further. Compared with Ctl, collagen synthesis was decreased with Met and with PTH and decreased further with Met + PTH. There was an inverse correlation between percent collagen synthesis and JCa+. Compared with Ctl, beta-glucuronidase release into the medium was increased with Met and with PTH and increased further with Met + PTH. There was a direct correlation between medium beta-glucuronidase activity and JCa+. Osteoclastic beta-glucuronidase activity correlated inversely with osteoblastic collagen synthesis. During cultures to 96 h, there continued to be greater JCa+ from calvariae incubated with Met + PTH than from those with either treatment alone. Thus acidosis and PTH independently stimulated JCa+ from bone, inhibited osteoblastic collagen synthesis, and stimulated osteoclastic beta-glucuronidase secretion, whereas the combination had a greater effect on each of these parameters than either treatment alone. These findings indicate that acidosis and PTH can have an additive effect on bone cell function and suggest that uremic osteodystrophy may result from a combination of a low pH and an elevated PTH.

The potential role of fibroblasts in periprosthetic osteolysis: fibroblast response to titanium particles.

Periprosthetic osteolysis with or without aseptic loosening is a major clinical problem in total hip arthroplasty. While the macrophage response to prosthetic wear debris and its role in periprosthetic osteolysis has been extensively studied, information regarding other cell types (fibroblasts, osteoblasts) is limited. This study explored the response of fibroblasts to particulate wear debris. Fibroblasts isolated from interfacial membranes of patients with failed total hip replacements and normal synovial tissue, when challenged with small-sized ( < 3 microns) titanium (Ti) particles, responded with significantly enhanced expressions of collagenase, stromelysin and, to a much lesser extent, their tissue inhibitor of metalloproteinases (TIMP). These "regulated" expressions at both mRNA and protein levels were correlated with the size and composition of particles. De novo protein synthesis was required for the regulation of these mRNAs. A similar effect could be induced by the treatment of the cells with particle-free conditioned medium from Ti particle-stimulated fibroblasts. Furthermore, this conditioned medium significantly suppressed the mRNA levels of procollagen alpha 1 (I) and alpha 1 (III) in osteoblast-like MG-63 cells. It is concluded that fibroblasts stimulated with certain particle debris may play an important role in periprosthetic osteolysis by releasing bone-resorbing metalloproteinases and mediator(s) which resulted in suppressed collagen synthesis in osteoblasts.

Stimulated osteoclastic and suppressed osteoblastic activity in metabolic but not respiratory acidosis

When bone is cultured in acidic medium produced by a reduced bicarbonate concentration ([HCO(3-)]), a model of metabolic acidosis, there is greater net calcium efflux than when the same decrement in pH is produced by an increased partial pressure of carbon dioxide (PCO2), a model of respiratory acidosis. To determine the effects of metabolic and respiratory acidosis on bone cell function we cultured neonatal mouse calvariae for 48 h under control conditions (pH approximately 7.40, PCO2 approximately 41 mmHg, [HCO(3-)] approximately 25 meq/l) or under isohydric acidic conditions simulating metabolic (pH approximately 7.09, [HCO(3-)] approximately 12) or respiratory (pH approximately 7.10, PCO2 approximately 86) acidosis and measured osteoblastic collagen synthesis and alkaline phosphatase activity and osteoclastic beta-glucuronidase activity. Collagen synthesis was inhibited by metabolic (23.2 +/- 1.3 vs. 30.3 +/- 1.0% in control) but was not altered by respiratory (32.3 +/- 0.6) acidosis. Alkaline phosphatase activity was inhibited by metabolic (402 +/- 16 vs. 471 +/- 15 nmol P.min-1.mg protein-1 in control) but not altered by respiratory (437 +/- 25) acidosis. beta-Glucuronidase activity was stimulated by metabolic (1.02 +/- 0.06 vs. 0.78 +/- 0.05 micrograms phenolphthalein released.bone-1.h-1 in control) but not altered by respiratory (0.73 +/- 0.06) acidosis. Net calcium efflux in control was increased by metabolic (783 +/- 57 vs. 20 +/- 57 nmol.bone-1.48 h-1 in control) and by respiratory (213 +/- 45) acidosis; however, calcium efflux with metabolic was greater than with respiratory acidosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of osteoclasts and osteoclast-like cells on osteoblast alkaline phosphatase activity and collagen synthesis

Osteoblasts have been shown to modulate osteoclast activity, but the reverse process has not been investigated. In the current study conditioned medium (CM) was collected from osteoclasts and osteoclast-like cells and its effects on osteoblast alkaline phosphatase (ALPase) activity and collagen synthesis ([3H]proline hydroxylation) were determined. In primary chick osteoblasts, cultured chick embryo frontal bones, and UMR-106-01 cells, collagen synthesis and ALPase activity, but not [3H]thymidine incorporation, were inhibited by CM from chick marrow-derived giant cells, which possess some of the phenotypic characteristics of osteoclasts. However, collagen synthesis in chick embryo fibroblasts was not affected by giant cell CM. CM collected from cultures of chicken osteoclasts and human osteoclastoma cells and marrow-derived giant cells inhibited collagen synthesis in UMR-106-01 cells, but the effects of ALPase activity varied with the cell type. In contrast, mononuclear cell and fibroblast CM did not alter collagen synthesis. Initial characterization studies demonstrate that the inhibitor is a heat-labile factor with a molecular weight greater than 3500. In summary, authentic osteoclasts, tumor osteoclast-like cells, and chicken and human multinucleated giant cells produce a soluble factor that alters osteoblast collagen synthesis, suggesting that osteoclasts play a role in the modulation of osteoblast activity.

Osteoporosis in rheumatoid arthritis: a molecular biological aspect of connective tissue gene activation.

Osteoporosis, especially the juxtaarticular osteoporosis of involved joints, is a characteristic manifestation of rheumatoid arthritis (RA). Histomorphometric studies suggest the existence of increased bone turnover in RA: impaired bone formation and hightened osteoclasic bone resorption. Recent studies show that important mediators in the pathogenesis of RA such as prostaglandin E, interleukin 1 (IL1) or tumor necrosis factor (TNF) alpha also play important roles in bone remodelling. Prostaglandin E2 promotes maturation of osteoclasts from hematopoietic precursor cells. IL1 inhibits collagen synthesis in osteoblasts. IL1 enhances collagenase and stromelysin gene expression and stimulates osteoclastic bone resorption. TNF alpha inhibits bone collagen synthesis and causes osteoclastic bone resorption. TNF alpha, and possibly IL1, enhances collagenase and stromelysin gene expression by stimulating the AP-1 promoter sites of the genes. Constitutive expression of c-fos induces joint destruction without lymphocyte infiltration in antigen-induced arthritis in mice, and supports cell growth of human rheumatoid synovial cells, possibly acting on the AP-1 sites. Furthermore, constitutive c-fos expression decreases collagen synthesis in osteoblasts and increases the mediator secretion from osteoblasts thereby stimulating osteoclastic bone resorption. These findings suggest that signal transduction through AP-1 transcriptional regulation sites may play an important role in the pathogenesis of joint destruction and osteoporosis in RA.

Proliferation and collagen biosynthesis of osteoblasts and chondrocytes in short rib syndrome type beemer

We report on a case of lethal short-limbed skeletal dysplasia with extremely short ribs, median cleft upper lip and palate, malrotation of intestine, lung hypoplasia with bilateral segmentation defect, atrial septum defect, union of distal urethra and vagina, and complex brain malformations. Based on radiological criteria and the pattern of associated abnormalities a short rib syndrome without polydactyly (Type Beemer) was diagnosed. Morphologically, the growth plate showed a reduced proliferation zone and an enlarged zone of hypertrophic cartilage. In addition, islands of persistent hypertrophic cartilage were present even in the metaphysis. In monolayer cell cultures supplemented with 10% fetal calf serum proliferation was normal in articular chondrocytes, reduced in costal chondrocytes, and elevated in osteoblasts from the patient. Clonal growth of costal and articular chondrocytes in methylcellulose could be stimulated normally by insulin-like growth factor-I (IGF-I), IGF-II, and human growth hormone (hGH). However, the response to transforming growth factor beta 1 (TGF-beta 1) was markedly elevated in articular chondrocytes of the patient compared to those of 3 fetal controls. Quantitative collagen synthesis in both osteoblasts and chondrocytes from the patient did not differ significantly from that of controls. Osteoblasts synthesized predominantly collagen I and minor amounts of collagen III, chondrocytes synthesized primarily collagen II. All collagen chains including CNBr-peptides of collagen II showed normal migration in PAA gel electrophoresis.

Biological activities of surface-associated material from Porphyromonas gingivalis

Surface-associated material (SAM) from Porphyromonas gingivalis was tested for in vitro biological activities that may be relevant to the pathogenesis of chronic periodontitis. SAM was found to stimulate bone resorption at a concentration of 1.0 µg/ml and this was inhibited by indomethacin, interleukin-1 receptor antagonist protein and anti-tumour necrosis factor antibody. At a concentration of 10 ng/ml, the SAM inhibited DNA and collagen synthesis in osteoblasts and murine calvaria and DNA synthesis in fibroblasts, monocytes and epidermal cells. Therefore, easily solubilised surface components from P. gingivalis could play a role in the pathogenesis of chronic periodontitis if these activities operate in vivo.