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PubMed1998

[Response of osteoblast cultures to titanium, steel and hydroxyapatite implants].

The biocompatibility of bone implants and substitutes is usually tested on cell-lines only, despite a different and more relevant behaviour on primary osteoblasts as final targets can be expected. "Osteoblast-like" cell line (MC3T3-E1) and fresh human osteoblasts (HOB), cultured from cancellous bone grafts from the iliac crest were used for the study. Three different clinically used biomaterials were compared regarding biocompatibility: titanium, steel and hydroxyapatite (Bio-Oss). "Osteoblast-like" cell line and fresh human osteoblasts (5x104) were seeded on the three bone implants. Cell proliferation and osteocalcin synthesis were determined 1, 3, 7, and 10 days after the cells were plated on the biomaterials. All experiments were performed in five times (pro culture double measurements). HOB proliferation on hydroxyapatite was decreasing after 3 days, whereas cells from "Osteoblast-like" cell line showed comparable proliferation to the control group. The most interesting observation was the significant decrease of the osteocalcin levels (in conditioned medium) of "osteoblast like" cells and HOB on HA (Bio-Oss). We conclude that HA disturbs the proliferation and osteocalcin synthesis of HOB.

Bone Substitutes↗

PubMed1998

Growth factor stimulation of bone healing. Effects on osteoblasts, osteomies, and implants fixation.

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.

Animals↗

PubMed2006

Chemokine gene activation in human bone marrow-derived osteoblasts following exposure to particulate wear debris.

Particulate wear debris induces the expression of pro-inflammatory cytokine and chemokine genes in various cell types of the periprosthetic region. We have previously reported that titanium particles stimulate the selective induction of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) chemokines in human osteoblast-like osteosarcoma cells. In this study, we characterize the human bone marrow-derived osteoblast chemokine response to titanium particles. We demonstrate that titanium particles result in enhanced IL-8 and MCP-1 protein secretion as well as differential chemokine gene activation. Osteoblast chemokine expression was regulated at the level of gene transcription, with a time-dependent induction of NF-kappaB activation. Inhibition studies with N-acetyl-L-cysteine (Nac) and MG-132 suggest that titanium particle activation of NF-kappaB activity and IL-8 chemokine expression involves oxidant signaling and IkappaBalpha-proteasomal degradation. Activation of the NF-kappaB transcription factor, as well as the IL-8 gene, are redox-regulated. We also demonstrate that while cytochalasin D, a potent inhibitor of phagocytosis, suppressed the titanium particle effect on IL-8 protein release in human bone marrow-derived osteoblasts, the inhibitor had no effect on IL-8 expression in MG-63 osteoblast-like cells. Collectively, these results provide insight into the potential mechanisms responsible for the particulate activation of osteoblast chemokine expression and suggest an important role for the osteoblast in the pathogenesis of periprosthetic osteolysis.

Adult↗

PubMed2007

Modeled microgravity stimulates osteoclastogenesis and bone resorption by increasing osteoblast RANKL/OPG ratio.

Mechanical unloading causes detrimental effects on the skeleton, but the underlying mechanisms are still unclear. We investigated the effect of microgravity on osteoblast ability to regulate osteoclastogenesis. Mouse osteoblast primary cultures were grown for 24 h at unit gravity or under simulated microgravity, using the NASA-developed Rotating Wall Vessel bioreactor. Conditioned media (CM) from osteoblasts subjected to microgravity increased osteoclastogenesis and bone resorption in mouse bone marrow cultures. In these osteoblasts, the RANKL/OPG ratio was higher relative to 1g. Consistently, treatment with high concentrations of OPG-inhibited osteoclastogenesis and bone resorption in the presence of CM arising from osteoblasts cultured under microgravity. Microgravity failed to affect osteoblast differentiation and function in the time frame of the experiment, as we found no effect on alkaline phosphatase mRNA and activity, nor on Runx2, osteocalcin, osteopontin, and collagen1A2 mRNA expression. In contrast, microgravity induced a time dependent increase of ERK-1/2 phosphorylation, while phospho-p38 and phospho-JNK remained unchanged. Apoptosis, revealed by bis-benzimide staining, was similar among the various gravity conditions, while it was increased under microgravity after treatment with the MEK-1/2 inhibitor, PD98059, suggesting a protection role by ERK-1/2 against cell death. In conclusion, microgravity is capable to indirectly stimulate osteoclast formation and activity by regulating osteoblast secretion of crucial regulatory factors such as RANKL and OPG. We hypothesize that this mechanism could contribute to bone loss in individuals subjected to weightlessness and other unloading conditions.

Animals↗

PubMed2001

Effect of water soluble extract of nacre (Pinctada maxima) on alkaline phosphatase activity and Bcl-2 expression in primary cultured osteoblasts from neonatal rat calvaria.

The nacre (mother of pearl) layer of the oyster Pinctada maxima shell can initiate bone formation by human osteoblasts in vivo and in vitro and is a new biomaterial that induces osteogenesis. This activity of nacre could be due to its water-soluble matrix. We examined the action of a water-soluble extract of nacre on the osteoblast phenotype of cells isolated from rat neonatal calvaria by measuring alkaline phosphatase (ALP) activity and by localization of the anti-apoptotic protein Bcl-2 by immunocytochemistry. ALP activity was increased 7% (p<0.001) by 100 microg proteins/ml extract and 20% (p<0.001) by 50 microg proteins/ml extract, but a low concentration of extract decreased the ALP activity by 8%. Cells treated with a high aspartic acid content fraction of the extract had increased ALP activity (23%, p<0.0001). Nacre extract and the fraction have no effect on the proliferation of mature osteoblasts. Immunoreactive Bcl-2 was overproduced in the cytoplasm and nuclei of osteoblasts at all stages of culture. Bcl-2 was found over the whole chromatin in quiescent and mitotic cells at the end of mitosis in the two nuclei in one cell, before cytodieresis. Bcl-2 was also found over chromosomes. Thus, nacre extract stimulates Bcl-2 production in osteoblasts, that is correlated with the cell cycle. Bcl-2 was also abundant in the nucleoli of extract-treated cells. Thus, the concentration and subcellular distribution of Bcl-2 in osteoblasts in primary cultures is influenced by nacre extract, and related to the cell cycle and the regulation of gene expression. Hence, knowledge of how water-soluble extracts of Pinctada maxima nacre act on osteoblasts in vitro may reveal the mechanisms involved in its action in vivo on bone cells and bone regeneration.

Journal Article↗

PubMed1998

Human cementum tumor cells have different features from human osteoblastic cells in vitro.

Cells obtained from human cementoblastoma and alveolar bone were isolated and cultured. Initial and late stages of mineralization were assessed by using atomic force microscopy, scanning electron microscopy and X-ray microanalysis. In cultures of cementoblastoma-derived cells the initial stages of mineralization showed well-defined spherical-shaped structures, while the osteoblastic cells showed plaque-like deposits. These morphological patterns of mineral deposition could serve as nucleation centers for hydroxyapatite crystals. Late stages of mineralization at 28 and 35 d maintained those morphological differences established in initial cultures. The material deposited by cementoblastoma and osteoblastic cells, analyzed by EDX spectra, revealed similar Ca/P ratios for both cell types. These values were similar to those reported for hydroxyapatite in enamel and bone. Alkaline phosphatase specific activity (AlP), of osteoblastic cells at 3, 7 and 11 d, showed an increase of 27.9, 50.9 and 37.0% (p < 0.001), respectively. However, at 15 and 19 d there was an increase of AlP activity of cementoblastoma cells by 39.4 and 34.5% over osteoblastic cells (p < 0.001). Immunostaining of cementoblastoma and osteoblastic cells using a specific mAb against a cementum-derived attachment protein revealed strong immunostaining of cementoblastoma cells which was localized to the cell membrane and fibril-like structures (96.2 +/- 1.3). A few osteoblastic cells also stained weakly with the anti-CAP mAb (6.4 +/- 0.6). Sections of decalcified paraffin embedded cementoblastoma specimens, when immunostained with anti-CAP mAb, showed strong immunostaining of the cells surrounding the regular and irregularly-shaped calcified masses of the tumor. Putative cementocytes also stained positively. Immunostaining with a polyclonal antibody against osteopontin strongly stained the osteoblastic cells (89.0 +/- 3.6). Cementoblastoma cells showed weaker staining (54.2 +/- 2.4). The results suggest that cementoblastoma cells could be a major source of specific cementum proteins. These cells could provide the opportunity to elucidate the regulation of the cementogenesis process.

Actin Cytoskeleton↗

PubMed2000

[In vitro studies about the effects of implant materials on calmodulin, introcellular calcium of rat osteoblasts].

OBJECTIVE: 1. To evaluate the effects of the four implant materials, hydroxyl poly calcium sodium phosphate (HPA), hydroxyapatite (HA), bioglass ceramics (BGC) and titanium (Ti) on the calmodulin (CaM) content of osteoblasts, the concentration of intracellular free calcium and the ATPases activity of cell membrane of rat osteoblasts cultured in vitro; 2. To reveal the biological compatibility of the four implant materials and the index to estimate the biological compatibility condition. METHODS: The powders of HPA, HA, BGC and Ti were sterilized and made into extracted solutions with 199 culture medium. The isolated osteoblasts, which were taken from three-day-old sparague Dawley rats' calvaria, were incubated in 199 culture medium also. Then the osteoblasts were mixed and incubated into the four kinds of extracted solutions of the implant materials. After three days of co-incubation, the cells were taken out, and the cellular calmodulin content were measured with radiobiological analysis. The concentration of intracellular free calcium were under fluorescent analysis, and the ATPases activities of cell membrane were estimated with colorimetric analysis. RESULTS: 1. Calmodulin content of the osteoblasts co-incubated with all the implant materials decreased, and moreover, only that of the Ti group decreased more obviously than that of the Ti and HPA group (P < 0.05). 2. The concentration of intracellular free calcium of all the co-incubated osteoblasts were as normal as that of the control group. 3. Ca(2+)-ATPases activities of osteoblasts co-incubated with HPA, HA, BGC increased (P < 0.05), and Na+/K(+)-ATPases activities of the cells co-incubated with HPA, HA were strenghtened (P < 0.05). CONCLUSION: The indexes of cellular calmodulin content, concentration of intracellular free calcium and ATPases activity of cell membrane can reflect the biological compatibility of implant materials, and they may be used as indexes of estimating biological compatibility condition.

Animals↗

PubMed2000

[A study of fluoride and dexamethasone affecting the proliferation in osteoblast].

OBJECTIVE: Investigate the effects of low dose of fluoride and dexamethasone on the proliferation of MC3T3-E1 osteoblast cell line in vitro, which was for determining the possible effects of fluoride and dexamthasone affecting the development and differentiation of osteoblast. METHODS: Observe the growing shape of MC3T3-E1 osteoblast cultured in vitro, and investigate the effects of low dose of fluoride and dexamethasone on the proliferation of osteoblast by cell count. RESULTS: MC3T3-E1 osteoblast grew stably. The cells in D group increased obviously in the early stage of the cell culture, however the cells in F group had no tendency of increasing during the whole culture. CONCLUSION: In vitro low doses of dexamethasone may promote the proloferation of osteoblast precursors,however,low dose of fluoride has no effect on promoting proliferation of MC3T3-E1 osteoblast.

English Abstract↗

PubMed2004

[Effects of 1,25-dihydroxyvitamin D3 on the expressions of osteoprotegerin and receptor activator of NF-kappaB ligand in mouse osteoblasts].

OBJECTIVE: To study the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the expression of osteoprotegerin (OPG) and receptor activator of NF-kappaB ligand (RANKL) mRNA in mouse osteoblasts. METHODS: Calvariae derived from CD-1 neonatal mouse (after born 24 h). Bone samples were processed by the collagenase/trypsin digestion method. Mouse osteoblasts were cultured in vitro. After 48 hours of addition of 1,25(OH)2D3 (0, 10(-8), 10(-9), 10(-11) mol/L) to the culture medium of mouse osteoblasts, the content of the OPG protein in culture medium was estimated with enzyme linked immunosorbent assay. Total RNA was prepared from mouse osteoblasts. mRNA expression of OPG and RANKL were detected by reverse transcription-polymerase chain reaction. RESULTS: The mRNA expression of OPG in osteoblasts added with 1,25(OH)2D3 significantly decreased compared with the controls, which was markedly dose-dependent. OPG protein production in the medium decreased after treatment with 1,25(OH)2D3. In contrast, RANKL mRNA expression levels in osteoblasts significantly increased after 48 h of culture with 1,25(OH)2D3. CONCLUSION: 1,25 (OH)2D3 can stimulate RANKL mRNA expression, but decrease OPG mRNA levels in vitro in mouse osteoblasts.

Animals↗

PubMed1997

Mechanisms of the development of osteoblastic metastases.

Although several neoplasms may produce osteoblastic metastases, carcinoma of the prostate is by far the most common. Biochemical and histologic studies indicate that osteolysis also is a manifestation of prostate carcinoma. Furthermore, factors such as parathyroid hormone-related peptide, which mediate osteolysis in other cancers, also appear to be operative in the bone breakdown induced by prostate carcinoma. However, the most unique skeletal effect of this tumor is its consistent capacity to stimulate osteoblasts to deposit new bone. Several bone growth factors have been detected in prostatic tissue and may contribute to this process. These include transforming growth factor-beta, fibroblast growth factor, and bone morphogenetic proteins. The author isolated an amino-terminal fragment (ATF) of the protease urokinase (uPA) from the conditioned medium of the prostate carcinoma cell line PC-3 and demonstrated that this fragment has mitogenic activity for osteoblastic cells. The activity appears to reside in an epidermal growth factor-like growth factor domain (GFD) within the ATF. Subsequently, the author cloned the rat uPA receptor (uPAR). uPAR is known to bind the ATF and can permit the uPA molecule to exhibit focal proteolysis. It was shown that the ATF also can induce c-myc, c-jun, and c-fos in osteoblastic cells. This effect of ATF can be mimicked by the GFD and suggests that this signalling pathway in osteoblasts is via the uPAR. Consequently, the uPA molecule may contribute to growth factor effects in osteoblasts via the NH2-terminal fragment and to tumor invasiveness via its COOH-terminal proteolytic domain. This scenario is supported by results from studies with uPA-overexpressing prostate carcinoma cells in rats. Additional studies will be required to further define the mechanisms of interaction of prostate carcinoma and other cancers with bone but each site of molecular interaction may provide a therapeutic window for curtailing the effects of these tumors on the skeleton.

Animals↗

PubMed1996

Analysis of osteoblast activity at biomaterial-bone interfaces by in situ hybridization.

To investigate the effects of bioactive materials on bone formation in vivo, a new experimental model using in situ hybridization has been developed. A hole was drilled bilaterally in the distal epiphysis of rabbit femurs with subsequent implantations of beta-tricalcium phosphate (beta-TCP) cylinders in a press-fit manner. Specimens were collected at 3, 7, 14, and 28 days after operation. Femurs with empty drilling holes, and normal distal femurs without operation were used as controls. All specimens were decalcified and hybridized with a procollagen alpha 1(I) complementary RNA probe labeled with digoxygenin. In normal-bone sections, procollagen alpha 1(I) RNA was clearly demonstrated in periosteal osteoblasts, in osteoblasts in the mineralizing zone adjacent to growth plates, and in osteoblasts lining remodeling canals. As for beta-TCP, labeled osteoblasts around the material were not found at day 3, whereas they were most intensively observed at day 7 and a little less at day 14, in accordance with new-bone formation around the material. Weaker signals were also detected in fibroblasts at day 7. At day 28, osteoblasts lining the surface of newly formed bone were mainly negative, whereas those adjacent to the resorption sites of the beta-TCP showed positive signals, demonstrating an active remodeling at the material surface. The temporal expression of procollagen alpha 1(I) RNA in the beta-TCP specimens was fundamentally the same as that in the empty-hole specimens, suggesting no remarkable acceleration or suppression of bone-forming activity of osteoblasts by beta-TCP, which is consistent with osteoconductive bone formation. This in situ hybridization method was suggested to be a powerful tool in analyzing the biological effects of bioactive materials.

Animals↗

PubMed1999

In vitro reactions of human osteoblasts in culture with zirconia and alumina ceramics.

The biocompatibility of two implantable materials, zirconia and alumina ceramics, was investigated in vitro using human osteoblast cell cultures. The viability of osteoblast cells with the materials was evaluated by the methylthiazole sulfate test that revealed an absence of any cytostatic or cytotoxic effect. Cell proliferation kinetic and total protein synthesis in osteoblasts with zirconia or alumina were similar to that observed in control cells cultured on glass coverslips. Light and scanning electron microscopic examinations showed an intimate contact between osteoblasts and the substrates; well-spread cells were observed on the surfaces of both materials. Adhesion ability and morphological characteristics were preserved in osteoblast cultures with these substrates. Moreover, immunohistochemical staining in osteoblasts with zirconia and alumina showed the capacity of these cells to elaborate the extracellular matrix composed of types I and V collagen, osteocalcin, osteonectin, bone sialoprotein, and cellular fibronectin. Finally, DNA image cytometry and interphase silver-nucleolar organizer regions quantification were applied as complementary biocompatibility tests to detect any changes in DNA synthesis and cell proliferation, respectively. The results showed that neither material altered cell ploidy or cell growth rate in accordance with the absence of any inducing effect on DNA synthesis or proliferation.

Aluminum Oxide↗

PubMed1998

TGF-beta1 modifications in nuclear matrix proteins of osteoblasts during differentiation.

Nuclear matrix protein (NMP) composition of osteoblasts shows distinct two-dimensional gel electrophoretic profiles of labeled proteins as a function of stages of cellular differentiation. Because NMPs are involved in the control of gene expression, we examined modifications in the representation of NMPs induced by TGF-beta1 treatment of osteoblasts to gain insight into the effects of TGF-beta on development of the osteoblast phenotype. Exposure of proliferating fetal rat calvarial derived primary cells in culture to TGF-beta1 for 48 h (day 4-6) modifies osteoblast cell morphology and proliferation and blocks subsequent formation of mineralized nodules. Nuclear matrix protein profiles were very similar between control and TGF-beta-treated cultures until day 14, but subsequently differences in nuclear matrix proteins were apparent in TGF-beta-treated cultures. These findings support the concept that TGF-beta1 modifies the final stage of osteoblast mineralization and alters the composition of the osteoblast nuclear matrix as reflected by selective and TGF-beta-dependent modifications in the levels of specific nuclear matrix proteins. The specific changes induced by TGF-beta in nuclear matrix associated proteins may reflect specialized mechanisms by which TGF-beta signalling mediates the alterations in cell organization and nodule formation and/or the consequential block in extracellular mineralization.

Animals↗

PubMed1999

Identification of DERMO-1 as a member of helix-loop-helix type transcription factors expressed in osteoblastic cells.

Several members of the basic helix-loop-helix (bHLH) type of transcription factors have now been reported, and almost every member of this class has been implicated in transcriptional regulation in cell type determination and differentiation. Previously, we reported that dominant negative HLH proteins are involved in osteoblastic phenotype expression, such as osteocalcin, and hence differentiation (Tamura and Noda [1994] J. Cell Biol. 126:773-782). In this work, we used degenerate PCR cloning in order to identify cDNA clones encoding bHLH proteins expressed in osteoblastic osteosarcoma ROS17/2.8 cells. Sequence analyses of the 47 clones revealed that 11 clones encoded products with a characteristic motif of the bHLH transcription factor family. Of these clones, sequences in the amplified region of seven clones were homologous to the mouse twist, and three clones were homologous to the mouse twist-related HLH protein, Dermo-1. To confirm Dermo-1 mRNA expression in osteoblastic cells, we performed reverse transcription polymerase chain reaction (RT-PCR) analysis using mRNA from ROS17/2.8 cells and MC3T3-E1 cells by Dermo-1 specific primers and Northern blot analysis. These analyses demonstrated that Dermo-1 mRNA was expressed in these osteoblast-like cell lines. Nucleotide sequence analysis of the partial rat Dermo-1 cDNA cloned from ROS17/2.8 library revealed that it has the highest degree of homology with the mouse Dermo-1 cDNA, and the partial amino acid sequence deduced from the obtained rat Dermo-1 was identical with the corresponding region of the mouse Dermo-1 amino acid sequence. To further examine the role of Dermo-1 in the regulation of osteoblastic differentiation, we examined mRNA levels of Dermo-1 and twist in C3H10T1/2 cells treated with recombinant human bone morphogenetic protein (rhBMP)-2. Using the RT-PCR method, the mRNA levels of Dermo-1 and twist were found to be decreased by the treatment with rhBMP-2 in C3H10T1/2 cells. We also observed that the mRNA level of Dermo-1 was decreased about fourfold by the treatment with rhBMP-2 in C3H10T1/2 cells by Northern blot analysis. Moreover, Dermo-1 mRNA was detected at lower levels in 21-day-old differentiated MC3T3-E1 cells compared with 3-day-old undifferentiated MC3T3-E1 cells. These results suggested that Dermo-1 could be involved in the osteoblastic differentiation in a negative manner.

Amino Acid Sequence↗

PubMed1999

Inhibitory effects of activin-A on osteoblast differentiation during cultures of fetal rat calvarial cells.

Activin-A is a member of the transforming growth factor-beta (TGF-beta) superfamily and is expressed by osteoblasts. However, the role of activin-A on osteoblasts is not clearly understood. We examined the effects of activin-A on osteoblast proliferation or differentiation, and mineralization by the osteoblasts in the first subcultures of fetal rat osteoblasts obtained from calvarial bones. Exogenous activin-A led to impaired formation of bone nodules in a dose-dependent manner, although it did not influence cell proliferation using an MTT assay. This inhibitory effect depended upon the time at which activin-A was added to the culture media, and the effect was most significant when addition took place at the early phase of the culture. In addition, exogenous activin-A inhibited gene expression of type I procollagen, alkaline phosphatase, osteonectin, and osteopontin in the cultured cells using Northern blot analysis. The peak of osteocalcin mRNA was delayed. Gene expression for TGF-beta was not influenced by exogenous activin-A. The betaA subunit (activin-A) mRNA was detected during the early phase of this culture. These results indicate that activin-A inhibited early differentiation of the fetal rat calvarial cells, or osteoblasts.

Activins↗

PubMed1996

Expression of parathyroid hormone-related protein in cells of osteoblast lineage.

The expression of parathyroid hormone-related protein (PTHrP) was studied in a range of cell cultures representative of the osteoblast lineage and in rat calvarial sections. Primary newborn rat calvarial cells, a rat preosteoblastic cell line (UMR 201), a mouse stromal cell line (ST 2), a mouse calvaria-derived osteoblastic cell line (KS 4), and rat osteosarcoma cell lines (UMR 106-01 and -06), all expressed PTHrP when examined by reverse transcription polymerase chain reaction (RT-PCR). Using a radioimmunoassay we also demonstrated PTHrP in the conditioned medium of the cultured cells, with the exception of UMR 106-01 and -06 cells. Treatment of UMR 201 cells with all-trans-retinoic acid which induces them to acquire a more differentiated phenotype, also led to a time-dependent decrease in PTHrP mRNA levels as determined by RT-PCR, Northern blot analysis, and in situ hybridization. Decreased PTHrP levels in the conditioned medium of the treated cells was also observed. These results suggested that PTHrP production might be greater in less mature osteoblasts. Examination of the populations obtained from newborn rat calvariae by sequential collagenase digestion revealed that the early digests exhibited low ALP activity, low expression of PTH/PTHrP receptor mRNA, and no adenylate cyclase response to PTHrP(1-34). These populations showed the highest level of mRNA and production of PTHrP. Cells from later digests, the "osteoblast-rich" populations, had reduced PTHrP expression. Immunohistochemistry and in situ hybridization in sections of newborn rat calvariae showed PTHrP expression in cuboidal osteoblasts located adjacent to bone and in spindle-shaped cells in the periosteal region. It is concluded that PTHrP is produced by cells of the osteoblast lineage, supporting the hypothesis that PTHrP may function physiologically as a paracrine factor in bone.

Animals↗

PubMed1998

Three novel type I collagen mutations in osteogenesis imperfecta type IV probands are associated with discrepancies between electrophoretic migration of osteoblast and fibroblast collagen.

In three cases of type IV osteogenesis imperfecta (OI), we identified unique point mutations in type I collagen alpha1(I) cDNA. In two cases, the appearance of dimers indicated the presence of cysteine substitutions in the alpha1(I) protein chain. Cyanogen bromide digestion localized these cross-links to CB8 and 3, respectively. In the third case, the overmodification pattern of the CNBr peptides was compatible with a substitution in the aa 123-402 region of either type I collagen chain. We identified a unique point mutation in each proband, which resulted in substitutions for glycine residues in a 300-aa region of the alpha1(I) helix, specifically, Gly to Ala at codon 220 (GGT-->GCT), Gly to Cys at codon 349 (GGT-->TGT) and Gly to Cys at codon 523 (GGT-->TGT). We compared each proband's fibroblast and osteoblast collagen directly, as well as with fibroblast and osteoblast controls. For all cases, the OI osteoblast collagen was more electrophoretically delayed than OI fibroblast collagen. In the patient with G349C, OI fibroblast and osteoblast collagen synthesized in the presence of alpha,alpha'-dipyridyl co-migrated on gels, demonstrating that the electrophoretic discrepancy resulted from differences in post-translational modification. Melting temperature curves for stability of the collagen helix yielded an identical Tm for control fibroblast and osteoblast collagen (41.2 degrees C). By contrast, for collagen with the gly349-->cys substitution, the Tm of the fibroblast collagen was 1 degree C lower than the Tm of the osteoblast collagen. These data indicate that the metabolism of mutant collagen might be cell-specific and has significant implications for understanding the phenotype/genotype correlations and the pathophysiology of OI.

Adolescent↗

PubMed1999

p53 transactivity during in vitro osteoblast differentiation in a rat osteosarcoma cell line.

We previously demonstrated a correlation between wild-type p53 expression and appearance of osteoblastic-specific differentiation characteristics, as evidenced by basal osteocalcin gene expression in a mouse osteosarcoma tumor. The study reported here further explored the possibility of p53's having a distinct transcription-activating role in bone differentiation, in addition to its proposed role in G1 arrest and apoptosis. ROS17/2.3 osteoblastic osteosarcoma cells were stably transfected with a plasmid containing wild-type p53 binding sequences fused to the chloramphenicol acetyltransferase reporter gene. These cells were used to determine the transactivating role of p53 in regulation of osteocalcin gene expression. We chose two conditions under which osteocalcin expression is known to be upregulated: exposure of osteoblastic cells to differentiation-promoting medium and to vitamin D3. Exposure of the transfected cells to differentiation-promoting medium produced an increase in p53 transactivating activity correlating with the appearance of osteocalcin expression after about 1 wk. Vitamin D3 treatment resulted in upregulation of osteocalcin activity without a corresponding change in p53 transactivation activity or expression. In separate experiments, we tested whether changes in osteocalcin expression accompanied changes in p53 activity under conditions of downregulation of cell proliferation mediated by inhibition of DNA synthesis. Hydroxyurea treatment was used to inhibit DNA synthesis and produce growth arrest in osteoblastic cells. Inhibition of osteoblast cell proliferation was associated with a fourfold increase in p53 transactivating activity and a transient increase in osteocalcin steady-state expression. These results demonstrated a close relationship between p53 and osteocalcin and suggested a regulatory role for wild-type p53 in the control of basal osteocalcin gene expression in osteoblasts.

Animals↗