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At least 523 records · Page 29Linked to original sources

PubMed2003

[Expression of proto-oncogenes c-fos and c-jun in osteoblasts activated by excessive fluoride].

OBJECTIVE: To study expression of proto-oncogenes c-fos and its accompanying gene c-jun in osteoblasts activated by action of excessive fluoride in vivo and in vitro. METHODS: Experimental Wistar rats were exposed to sodium fluoride (NaF) added to their drinking water, and NaF was also added in cell culture supernatant for osteoblast-like cells in vitro. Expression of both mRNA and protein of c-fos and c-jun in bone-tissue of rats with chronic fluorosis and cultured osteoblast-like cells were determined by hybridization in situ, Western blot and immunohistochemistry at varied time periods after exposure. RESULTS: Sodium fluoride could stimulate the proliferation of osteoblast in rats with chronic fluorosis and induce expression of both c-fos and c-jun in all envelops of the spine bone, as compared with its control group. Value of optical absorption in mRNA expression of c-fos and c-jun was 139.63 and 126.37, respectively, in rats with NaF plus high-calcium, significantly lower than that in control group with high-calcium only (107.74 and 117.48, respectively) (P < 0.001). Immunohistochemical analysis showed that protein level of c-fos and c-jun was significantly higher in rats with NaF plus high-calcium than that in control rats with high-calcium only, with values of optical absorption of 139.16, 131.15, 149.98 and 149.19 (P < 0.05), respectively, and protein level of c-fos and c-jun was significantly higher in rats with NaF plus low-calcium than that in control rats with low-calcium only, with values of optical absorption of 117.24, 111.46, 132.46 and 129.79 (P < 0.05), respectively. Western blotting showed that level of protein expression of c-fos and c-jun in periosteal osteoblasts was significantly higher in all rat groups with NaF than that in all control groups, with values of optical absorption of 123.32, 116.60, 115.97 and 108.30, respectively. mRNA expression of c-fos and c-jun in osteoblast-like cells treated with NaF for 12 h increased obviously, and remained at high level 48 h after exposure, with values of optical absorption of 114.80, 161.14, 118.20, and 150.41, respectively, as compared with that in control group (P < 0.001 and P < 0.05). CONCLUSIONS: Exposure to excessive fluoride could stimulate activation and proliferation of both osteoblasts in rats and cultured osteoblast-like cells in vitro, and cause enhanced expression of mRNA and protein of both c-fos and c-jun. Over-expression of c-fos could play an important role in development and proliferation of skeletal lesions in rats with chronic fluorosis.

Animals↗

PubMed2003

[Effects of soybean isoflavone and calcium on proliferation, differentiation and mineralization of cultured osteoblasts in vitro].

To explore the effects of soybean isoflavone and calcium on proliferation, differentiation and mineralization of cultured osteoblastic cells. Osteoblastic cells from newborn rat calvarial was cultured. Osteoblastic cells proliferation was measured by MTT, activity of APL was observed with Golden's method and ARS was used to measure the mineral nodes of osteoblasts. The results showed that the calcium group had not different from the control group on stimulating the proliferation, differentiation and mineralization of rat calvarial osteoblastic cells. The soybean isoflavone group had significant effects on stimulating the proliferation, differentiation of osteoblasts and increase the forming of the mineral nodes of osteoblasts in rat. Calcium and soybean isoflavone group had more benefit for forming mineral nodes. The soybean isoflavone stimulates the proliferation, differentiation and mineralization of rat calvarial osteoblastic cells, and increase calcium in the same time is more benefit for mineralization.

Alkaline Phosphatase↗

PubMed2004

[Effects of recombinant bovine basic fibroblast growth factor on osteoblast's growth and c-fos expression].

Rat's osteoblasts cultured in vitro were stimulated by recombinant bovine basic fibroblast growth factor(rb-bFGF). After 1-2 days, the osteoblast grew long protuberance, the numbers of osteoblasts were greater than the control groups', the vitality of osteoblasts was better than that of control groups. After 1 hour, the expression of c-fos in osteoblast increased when compared with that in the control group. After the osteoblasts having been stimulated for 1-2 days, the expression of c-fos increased more conspicuously. These results show that bFGF can boost the osteoblast to grow and proliferate and can enhance the expression of c-fos gene. The increase of c-fos gene's expression may be an important step in the signal transformation process of all kinds of stimulation boosting osteoblast to proliferate.

Animals↗

PubMed2004

[Study on biological behavior of osteoblast and vascular endothelial cell culture].

OBJECTIVE: To study the biological behavior of osteoblast and vascular endothelial cell culture. METHODS: The osteoblasts and vascular endothelial cells were obtained from calvarial bone and renal cortex of 2-week rabbits respectively. The experiment were divided into group A (osteoblasts), group B (vascular endothelial cells) and group C (co-cultured osteoblasts and vascular endothelial cells). The cells were identified with cytoimmunochemical staining. The cellular biological behavior and compatibility were observed under inverted phase contrast microscope and with histological staining. The cells viability and alkaline phosphatase (ALP) activity were measured. RESULTS: The cytoimmunochemical staining showed that the cultured cells were osteoblasts and vascular endothelial cells. The cellular compatibility of osteoblasts and vascular endothelial cells was good. The ALP activity was higher in group C than in group A and group B (P<0.01), and it was higher in group A than in group B (P<0.05). In group C, the cell proliferation were increased slowly early, but fast later. CONCLUSION: The cellular compatibility of osteoblasts and vascular endothelial cells were good. The vascular endothelial cells can significantly increased the osteoblast viability and ALP activity, and the combined cultured cells have greater proliferation ability.

Alkaline Phosphatase↗

PubMed2005

[Influence of different-sized titanium particles loading on osteoblastic differentiation and mineralization].

Studies have recently suggested that the coupling mechanism of bone formation and bone resorption are affected by particulate wear debris inducing aseptic loosening around the bone-prosthesis microenviroment. There may be direct impacts on osteoblasts, resulting in net decrease in bone formation. In addition, the influences of particulate wear debris in different size on the osteogenesis should be various. In order to investigate the hypothesis that particulate wear debris derived from prosthetic biomaterials affects the osteogenesis of osteblasts, we studied the influence of different-sized titanium particles loading on the osteoblastic differentiation by assaying the secretion of alkaline phosphatase (ALP), osteocalcin (OCN), N-terminal type I procollagen (PINP), and on the osteoblastic mineralization with the use of calcified node number, calcified node area and Alizarin Red S (ARS) concentration. Upon in vitro culture in the absence of titanium particles, we observed that cultures of osteoblasts isolated from newborn Japanese rabbits' cranium were excellently capable of differentiation and mineralization. Phi6.9 microm titanium particles did not evidently alter osteoblastic differentiation and mineralization. In comparison, phi2.7 microm and phi0.9 microm titanium particles, especially phi0.9 microm (submicron), significantly suppressed ALP expression, reduced PINP production, decreased OCN secretion and inhibited matrix mineralization. Results of transmission electron microscopy (TEM) of titanium particles-loaded osteoblastic cultures revealed that osteoblasts phagocytized titanium particles and exhibited ultrastructional changes consistent with cellular dysfuction. Combined with our previous studies in vitro findings, these results suggest that particles size play a key role in the process of aseptic loosening, which submicron particles are closely associated with inhibition of bone formation while bigger particles with enhancement of bone resorption. Further understanding the nature of osteoblastic bioreactivity to different-size wear particles should provide additional insights into mechanisms underlying aseptic loosening.

Animals↗

PubMed2005

Fibronectin preadsorbed on hydroxyapatite together with rough surface structure increases osteoblasts' adhesion "in vitro": the theoretical usefulness of fibronectin preadsorption on hydroxyapatite to increase permanent stability and longevity in spine implants.

OBJECTIVE: The aim of this study was to investigate the contribution of fibronectin (FN) preadsorption to enhance osteoblast adhesion and strength on hydroxyapatite (HA) used either as osteoconductive bone substitute or precoating of pedicle screws and cages in spine surgery. METHODS: HA substrata with two different surface roughness values (rough HA180 and smooth HA1200) were produced, and human osteoblasts were seeded after culturing on them. Prior to osteoblast seeding, the HA substrata were immersed in FN solution. Osteoblast attachment on each of the two HA substrata was evaluated by recording the number of cells, whereas osteoblast adhesion strength was determined by measuring the shear stress required to detach the cells from the HA substrates. RESULTS: FN preadsorption increased the number of attached osteoblasts on smooth and rough HA substratum at 40% and 62%, respectively, whereas it increased osteoblast attachment strength on the smooth and rough substratum at 165% and 73%, respectively. CONCLUSIONS: This study showed that FN preadsorption and rough HA surface texture synergistically increased "in vitro" both the number and the adhesion strength of human osteoblasts. Further studies in primates and humans should be carried out to disclose the clinical relevance (increase implant's stability and longevity) of the above-mentioned observations.

Adsorption↗

PubMed2005

Albumin regulates Runx2 and alpha1 (I) collagen mRNA expression in osteoblastic cells: comparison with insulin-like growth factor-I.

Albumin has been shown to be expressed in osteoblastic cells. The role of albumin in osteoblastic cells was investigated. Osteoblastic MC3T3-E1 cells with subconfluent monolayers were cultured for 24 or 48 h in a medium without fetal bovine serum (FBS) in the presence or absence of albumin (0.5 or 1.0 mg/ml of medium) or insulin-like growth factor-I (IGF-I; 10(-9) or 10(-8) M). The number of osteoblastic cells was significantly increased by culture for 48 h in the presence of albumin (0.5 or 1.0 mg/ml) or IGF-I (10(-9) or 10(-8) M). The effect of albumin (0.5 or 1.0 mg/ml) in increasing the cell number was not significantly modulated in the presence of IGF-I (10(-9) or 10(-8) M). Protein content in osteoblastic cells was significantly increased by culture with albumin (1.0 mg/ml). This effect was not significantly altered in the presence of IGF-I (10(-9) or 10(-8) M). Alkaline phosphatase activity was significantly decreased by culture with albumin (0.5 or 1.0 mg/ ml), while it was significantly increased in the presence of IGF-I (10(-9) or 10(-8) M). The effect of IGF-I in increasing the enzyme activity was not significantly altered in the presence of albumin (0.5 or 1.0 mg/ ml). The effect of albumin (0.5 mg/ ml) plus IGF-I (10(-9) M) in increasing the cell number was completely prevented in the presence of PD98059, suggesting that the effect of albumin or IGF-I is partly mediated through mitogen-activated protein (MAP) kinase cascade. The expression of Runx2 (type 1) mRNA using transcription-polymerase chain reaction (RT-PCR) analysis in osteoblastic cells was significantly decreased by culture for 24 or 48 h with albumin (0.5 or 1.0 mg/ml) or IGF-I (10(-9) M). alpha1 (I) collagen mRNA expression in osteoblastic cells was significantly increased by culture for 24 h with albumin (0.5 or 1.0 mg/ml) or IGF-I (10(-9) M). Albumin (0.5, 1.0, or 2.0 mg/ml) did not have a significant effect on osteoclast-like cell formation induced by culture with parathyroid hormone (PTH; 10(-7) M) in mouse marrow culture. This study demonstrates that albumin has a role in the regulation of Runx2 or alpha1 (I) collagen mRNA expression, which may be mediated through intracellular signaling pathway, in osteoblastic cells.

3T3 Cells↗

PubMed2006

[A model for studying on mechanical responses of osteoblast seeded in three dimensional scaffold].

OBJECTIVE: To establish a model for studying on mechanical responses of osteoblasts seeded in 3 dimensional(3D) scaffold. METHODS: Fifty pieces of bio-derived cancellous bones, whose holes were 500 to 800 microm and density was 0.36 to 0.45 g/cm3, were obtained as the scaffolds. They were cultured with the third passage suspension of Wistar rat. Twenty-four of the 50 scaffolds were constructed under apparent strain sine waveform with amplitude of 1 000 microepsilon, frequency of 3 Hz, and duration of 3 min/d, as experimental group. The other scaffolds were control group. After 3-day coculture, osteoblasts were observed with scanning electron microscope. The proliferation of the osteoblasts was checked by MTT on scheduled date. RESULTS: Scanning electron microscopic observation showed that osteoblasts attached and spread on the trabeculae, which presented the validity of the model under proper mechanical condition. Experiment showed that mechanical environment promoted the proliferation of osteoblasts. The observation of proliferation of osteoblasts showed that the quantity of osteoblasts in the experimental group was higher than that in the control group 1, 4, 8, 12, 16, 20, 24, and 28 days after culturing. There was significant difference between the two groups 12, 16, 20, 24, and 28 days after culturing (P < 0.05). CONCLUSION: The establishment of the model can facilitate the study of mechanical responses of osteoblasts under different conditions.

Animals↗

PubMed2006

Inhibitory effects of osteoblasts and increased bone formation on myeloma in novel culture systems and a myelomatous mouse model.

BACKGROUND AND OBJECTIVES: Multiple myeloma (MM) growth in the bone marrow is associated with increased osteoclast activity and a reduced number of osteoblasts. Experimental studies suggest that bone disease drives the progression of MM. Whereas those studies focused on the critical role of myeloma-induced osteoclastogenesis in disease progression, little is known about the impact of osteoblasts and increased bone formation on MM. DESIGN AND METHODS: We investigated the effect of isolated osteoblasts and osteoclasts on survival and proliferation of primary MM plasma cells (PC) in co-cultures and triple-cultures, and tested the effect of mesenchymal stem cells (MSC) on bone mineral density and MM growth in myelomatous human bones of SCID-hu mice. RESULTS: Whereas osteoclasts promoted survival and proliferation of MM PC, osteoblasts supported or inhibited MM PC, depending on the source of the MM cells. In triple-cultures osteoblasts attenuated the effect of osteoclasts on MM PC in 18 of 24 experiments. The anti-MM response to osteoblasts correlated with advanced clinical stage. Injection of MSC into myelomatous bones resulted in marked inhibition of tumor growth in three of nine experiments and stabilization of disease in two additional experiments. The anti-MM response of MSC was associated with increased human bone mineral density. Immunohistochemical analysis indicated that the MSC were well engrafted and, in responding mice, differentiated into osteogenic cells. INTERPRETATION AND CONCLUSIONS: MM PC from the majority of patients are susceptible to growth inhibition by osteoblasts; however, growth of MM PC from certain patients is accelerated by osteoblasts. In vivo, increased bone formation is associated with reduced myeloma burden.

Animals↗

PubMed2006

Implication of prostaglandin receptors in the accumulation of osteoprotegerin in human osteoblast cultures.

OBJECTIVE: Prostaglandins are important mediators in bone metabolism and in pathologies such as rheumatoid arthritis and osteoarthritis. We investigated the roles of cyclooxygenases (COX) and prostaglandin receptors in the accumulation of osteoprotegerin (OPG) in the supernatants of human osteoblasts in culture. METHODS: Three different cellular models were used, the human osteosarcoma cell lines MG-63 and Saos-2, and primary cultures of human osteoblasts. OPG concentrations were determined by ELISA. RESULTS: RT-PCR analysis showed that, like primary human osteoblasts, MG-63 cells express DP, EP4, FP, IP, and TP receptors, whereas the Saos-2 cells lack IP. Concentration of OPG was highest in MG-63 cell supernatants (36 +/- 12.5 ng/ml), followed by human osteoblasts (12.77 +/- 2.2 ng/ml) and Saos-2 (3.6 +/- 0.76 ng/ml). COX inhibitors did not alter these values. Prostaglandin E2 and BW 245C (a synthetic DP receptor agonist) decreased OPG in the supernatants of human osteoblasts but not in immortalized cell lines. These effects were concentration-dependent and were inhibited by EP4 and DP receptor antagonists. Fluprostenol, an FP receptor agonist, increased the accumulation of OPG in MG-63 but not in primary human osteoblasts or Saos-2. CONCLUSION: Our results show that activation of EP4 or DP receptors decreased the accumulation of OPG in supernatants of osteoblasts in culture, and suggest that these receptors could be interesting pharmacological targets in bone diseases. They also demonstrate important differences between primary osteoblasts and immortalized cell lines, both in the distribution and in the effects mediated by prostaglandin receptors.

Cell Line, Transformed↗

PubMed2007

Genistein and zinc synergistically enhance gene expression and mineralization in osteoblastic MC3T3-E1 cells.

Genistein, a phytoestrogen, and zinc, an essential trace element, have an anabolic effect on bone components. We investigated whether the combination of genistein and zinc has additive and synergistic effects in osteoblastic cells in vitro. Osteoblastic MC3T3-E1 cells with subconfluency were cultured for 48 h in medium containing either vehicle, genistein (10(-6) or 10(-5) M), zinc sulfate (10(-5) or 10(-4) M) or genistein (10(-6) or 10(-5) M) plus zinc sulfate (10(-5) or 10(-4) M). The protein content in the osteoblastic cells was significantly increased in the presence of genistein (10(-5) M) or zinc (10(-4) M). This increase was significantly enhanced with a culture of genistein (10(-5) M) plus zinc (10(-4) M). The expression of mRNAs of alpha1(I) collagen and osteocalcin, which are bone matrix protein, in osteoblastic cells was demonstrated using reverse transcription-polymerase chain reaction (RT-PCR) analysis with a specific primer. This expression was not significantly increased with genistein (10(-6) or 10(-5) M) or zinc (10(-5) or 10(-4) M). The combination of genistein (10(-6) or 10(-5) M) and zinc (10(-5) or 10(-4) M) caused a significant increase in alpha1(I) collagen and osteocalcin mRNA expression in osteoblastic cells. Alkaline phosphatase mRNA expression in osteoblastic cells was significantly enhanced with a culture of genistein (10(-5) M) plus zinc (10(-4) M). The effect of genistein (10(-5) M) plus zinc (10(-4) M) in enhancing the expression of alpha1(I) collagen, alkaline phosphatase, or osteocalcin mRNAs in osteoblastic cells was completely prevented with a culture of cycloheximide (10(-7) M), an inhibitor of protein synthesis, or 5,6-dichloro-1-beta-D-ribofuranosylbenimidazole (DRB) (10(-6) M), an inhibitor of transcriptional activity. Moreover, a culture with genistein (10(-5) M) or zinc (10(-4) M) for 14 or 21 days caused a significant increase in mineralization. This increase was markedly enhanced with a culture of the combination of genistein (10(-5) M) and zinc (10(-4) M). This study demonstrates that the combination of genistein and zinc can synergistically enhance gene expression and mineralization in osteoblastic cells.

Alkaline Phosphatase↗

PubMed1989

Difference in shape of bone formed by isolated calvarial and scapular osteoblasts transplanted under various conditions.

To study the influence of transplantation conditions on early stages of osteogenesis, isolated calvarial or scapular osteoblasts were injected into the leg or dorsal muscles (free transplants) or implanted after seeding on fragments of devitalized parietal bones (supported transplants) into dorsal muscles. The cross-sections of bone islands formed by calvarial osteoblasts in the different types of transplants were then compared according to their maximal breadth and length. Moreover, the same dimensions of pieces of bone formed by scapular osteoblasts in supported transplants were compared with those of bones formed in free transplants into leg muscles. Finally, comparison of the dimensions of cross-sections of supported transplants of calvarial and scapular osteoblasts was done. Calvarial osteoblasts in dorsal muscles produced a slightly higher percentage of wider and longer islands than those in leg muscles. In supported transplants of calvarial osteoblasts the percentage of narrow bone islands (breadth less than 100 microns) was considerably higher than in free transplants. Similarly, the percentage of narrow cross-sections in bones formed by scapular osteoblasts was higher in supported than in free transplants. In supported transplants of calvarial osteoblasts the percentage of narrow islands was higher than in similar transplants of scapular bone cells. It is suggested that the differences in shape of pieces of bone formed in supported and free transplants reflect the difference in mechanical conditions to which the bone cells were subjected. Furthermore, in supported transplants devitalized parietal bones could form a barrier for diffusion of nutrients.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1996

Human osteoblasts support human hematopoietic progenitor cells in vitro bone marrow cultures.

Hematopoietic stem cell differentiation occurs in direct proximity to osteoblasts within the bone marrow cavity. Despite this striking affiliation, surprisingly little is known about the precise cellular and molecular impact of osteoblasts on the bone marrow microenvironment. Recently, we showed that human osteoblasts produce a variety of cytokine mRNAs including granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, and interleukin-6. We examined here the ability of osteoblasts to support the development of hematopoietic colonies from progenitors as well the ability to maintain long-term culture-initiating cells (LTC-IC) in vitro. Examination of the hematopoietic cells recovered after 2 weeks of culture showed that osteoblasts support the maintenance of immature hematopoietic phenotypes. In methylcellulose assays, osteoblasts stimulate the development of hematopoietic colonies to a level at least 10-fold over controls from progenitor cells. Using limiting dilutional bone marrow cultures, we observed an activity produced by osteoblasts resulting in an threefold to fourfold expansion of human LTC-IC and progenitor cells in vitro. Thus, the presence of hematopoietic stem cells in close proximity to endosteal surfaces in vivo may be due in part to a requirement for osteoblast-derived products.

Adult↗

PubMed1995

Effects of the tripeptide glycyl-L-histidyl-L-lysine copper complex on osteoblastic cell spreading, attachment and phenotype.

We have studied the effects of the complex Glycyl-L-Histidyl-L-Lysine:Cu (GHK:Cu), the GHK sequence present in the alpha 2 (I) chain of human collagen (Coll I), and bone matrix glycoproteins containing either RGD (fibronectin, FN), or RGD and GHK (Coll I), on the spreading, attachment and markers of the osteoblast phenotype in rat calvaria cells (RC), human trabecular osteoblastic cells (HT) and human marrow stromal cells (HM). Coll I (20 micrograms/ml) and FN (20 micrograms/ml) coating enhanced osteoblastic cell spreading, whereas free GHK:Cu and GHK coating (10(-10)-10(-8) M) had no effect. FN and Coll I, as well as GHK:Cu and GHK, increased the attachment of RC and HT cells. The attachment of both total number of cells and alkaline phosphatase (ALP)-positive osteoblastic cells was increased, showing no preferential effect on cells expressing this early marker of the osteoblast phenotype. In addition, immunocytochemical analysis showed that FN, Coll I and GHK:Cu coating increased both the total number of HM cells and the number of HM cells expressing Coll I or osteocalcin, indicating that GHK:Cu and RGD-containing proteins acted similarly on cells expressing different maturational stages. In contrast to its effect on cell attachment, GHK:Cu coating slightly inhibited the basal and 1,25(OH)2D-induced stimulation of ALP activity or osteocalcin production in rat and human osteoblastic cells. The finding that GHK promotes cell attachment and decreases the phenotype of normal rat and human osteoblastic cells suggests that osteoblasts may interact with free GHK or GHK-containing proteins in the bone matrix.

Alkaline Phosphatase↗

PubMed1997

Age-related changes in bone formation, osteoblastic cell proliferation, and differentiation during postnatal osteogenesis in human calvaria.

We have determined the age-related changes in the growth characteristics and expression of the osteoblast phenotype in human calvaria osteoblastic cells in relation with histologic indices of bone formation during postnatal calvaria osteogenesis. Histomorphometric analysis of normal calvaria samples obtained from 36 children, aged 3 to 18 months, showed an age-related decrease in the extent of bone surface covered with osteoblasts and newly synthesized collagen, demonstrating a progressive decline in bone formation during postnatal calvaria osteogenesis. Immunohistochemical analysis showed expression of type I collagen, bone sialoprotein, and osteonectin in the matrix and osteoblasts, with no apparent age-related change during postnatal calvaria osteogenesis. Cells isolated from human calvaria displayed characteristics of the osteoblast phenotype including alkaline phosphatase (ALP) activity, osteocalcin (OC) production, expression of bone matrix proteins, and responsiveness to calciotropic hormones. The growth of human calvaria osteoblastic cells was high at 3 months of age and decreased with age, as assessed by (3H)-thymidine incorporation into DNA. Thus, the age-related decrease in bone formation is associated with a decline in osteoblastic cell proliferation during human calvaria osteogenesis. In contrast, ALP activity and OC production increased with age in basal conditions and in response to 1,25(OH)2 vitamin D3, suggesting a reciprocal relationship between cell growth and expression of phenotypic markers during human postnatal osteogenesis. Finally, we found that human calvaria osteoblastic cells isolated from young individuals with high bone formation activity in vivo and high growth potential in vitro had the ability to form calcified nodular bone-like structures in vitro in the presence of ascorbic acid and beta-glycerophosphate, providing a new model to study human osteogenesis in vitro.

Age Factors↗

PubMed1997

Increased proliferation of osteoblastic cells expressing the activating Gs alpha mutation in monostotic and polyostotic fibrous dysplasia.

We studied the osteoblastic abnormalities resulting from activating mutation of the Gs alpha gene in two patients with McCune-Albright syndrome and one patient with monostotic fibrous dysplasia. Histomorphometric analysis of dysplastic lesions showed a low number of differentiated osteoblasts along the bone surface and numerous immature alkaline phosphatase-positive mesenchymal cells actively depositing a woven bone matrix. Osteoblastic cells isolated from dysplastic bone lesions expressed a missense mutation in the Gs alpha gene in position 201 and showed increased intracellular basal cyclic adenosine 3',5'-monophosphate levels compared with normal cells isolated from a noninvolved area in the same patient. Cell proliferation evaluated by DNA synthesis was two-fold to threefold greater in osteoblastic cells expressing the mutation compared with normal cells from the same patient and was greater in cells isolated from more severe than less severe fibrotic lesions. In contrast, the synthesis of osteocalcin, a marker of mature osteoblasts, was lower in osteoblastic cells expressing the Gs alpha mutation compared with normal cells from the same patient and was lower in cells isolated from severe compared with less severe fibrotic lesions, indicating that the increased growth in mutated osteoblastic cells was associated with reduced cell differentiation. The results show that activating mutation of Gs alpha in osteoblastic cells leads to constitutive activation of adenylate cyclase, increased cell proliferation, and inappropriate cell differentiation, resulting in overproduction of a disorganized fibrotic bone matrix in polyostotic and monostotic fibrous dysplasia.

Adolescent↗

PubMed1998

Multiple levels of steroid hormone-dependent control of osteocalcin during osteoblast differentiation: glucocorticoid regulation of basal and vitamin D stimulated gene expression.

We have examined the contribution of transcriptional mechanisms to the pleiotropic effects of glucocorticoids on basal and vitamin D stimulated expression of the developmentally regulated bone-specific osteocalcin (OC) gene. OC expression was systematically investigated at the level of protein, mRNA, and newly synthesized transcripts during maturation of the bone cell phenotype in cultures of fetal rat calvarial-derived osteoblasts. Our results indicate that transcriptional control of basal and hormone-regulated OC expression predominates in immature osteoblasts prior to matrix mineralization. However, in mature osteoblasts OC expression is controlled primarily by posttranscriptional mechanisms reflected by elevated mRNA levels with a decline in transcription. Vitamin D, alone or in combination with Dex, is a significant factor contributing to mRNA stabilization in mature osteoblasts with a mineralized extracellular matrix. Transcriptional modifications in response to Dex are reflected by quantitative differences between proliferating and mature osteoblasts in the formation of glucocorticoid receptor binding complexes at the proximal OC glucocorticoid response element. Vitamin D and glucocorticoid receptor mRNA levels are significantly higher in mature osteoblasts than in early stage bone cells. However, receptor complexes do not appear to be rate limiting in proliferating osteoblasts when the OC gene is not transcribed. Our results indicate (1) developmental stage-specific effects of steroid hormone on transcriptional regulation of bone expressed genes, and (2) inverse relationships between levels of transcription and cellular representation of mRNA with OC message stabilized in mature osteoblasts.

Animals↗

PubMed2000

Growth and adhesion of osteoblast-like cells derived from neonatal rat calvaria on calcium phosphate ceramics.

The effects of biocompatible ceramics on the growth and adhesion of osteoblast-rich rat calvarial cell cultures were investigated. Osteoblast-like cells and mouse fibroblast-like L-929 cells were cultured on composite sinters of hydroxyapatite (HAP) and beta-tricalcium phosphate (TCP) culture carriers, whose Ca/P molar ratios were adjusted to values of 1.50, 1.55, 1.60, 1.64 and 1.67. The growth rates of both cell types were accelerated on the TCP-HAP ceramics as compared to those on polystyrene plastic (LUX) or bioinert zirconia ceramics. The population of osteoblast-like cells reached a density of 2.28 x 10(5) cells/cm2 on 100% HAP (Ca/P ratio 1.67) at 9 d of culture, while the corresponding cell density was 1.66 x 10(5) cells/cm2 on LUX and 1.26 x 10(5) cells/cm2 on zirconia. Adhesion of the osteoblast-like cells on TCP-HAP ceramics was similarly increased as compared with that on LUX or zirconia ceramics. The adhesion of L-929 cells on TCP-HAP ceramics was found to be weaker than that on cultures on LUX or zirconia ceramics. The time-dependent variations in the alkaline phosphatase activity of the osteoblast-like cells showed that the osteoblastic phenotype was potentiated by culturing the cells in calcium-rich media. The surface analyses of the Ca/P ratio and the microstructure by XRD and FTIR suggest that the Ca-rich surface was newly formed by recombination on the surface layer in the culture medium containing fetal bovine serum. These results suggest that the surface of TCP-HAP ceramics, especially that of 100% HAP ceramics, are effective for accelerating growth and differentiation of osteoblast-like cells. This is most probably due to the chemical and physical instability and composition of 100% HAP, which promote the formation of a Ca-rich layer at the cell-material interface and provision of Ca ions to the osteoblast-like cells.

Journal Article↗