Osteoblast Differentiation Process Research Articles

Clodronate stimulates bone formation as well as inhibits bone resorption and increases bone mineral density in rats fed a low-calcium diet.

The pharmacological actions of bisphosphonates are due to the inhibitory effects on bone resorption, but little is known about the bisphosphonate action on bone formation. The purpose of this study is to elucidate the actions of bisphosphonates, clodronate, on bone formation in the experimental in vivo and in vitro rat models. The bone mineral density (BMD) was decreased in the rats fed a low-calcium diet (0.05% Ca) for 6 days compared with the rats fed a normal-calcium diet (0.5% Ca). The decrease in BMD was suppressed in the 2 mgP/day and the 4 mgP/day clodronate administrations. Bone formation rate (BFR) in rats fed a low-calcium diet was significantly increased compared with the rats fed a normal-calcium diet, and the 2 mgP clodronate administration further increased the BFR. In the cultured rat bone marrow cells, the area of mineralized nodules was significantly increased at 10(-7) and 10(-6) M clodronate, but high concentration of clodronate decreased the area. From these results, it is concluded that clodronate stimulates bone formation when the drug was given to a rat with a relatively lower dose that is sufficient to prevent bone resorption and that this effect may be due to the stimulatory effect on the differentiation process of osteoblasts.

Microarray analysis of bone morphogenetic protein, transforming growth factor beta, and activin early response genes during osteoblastic cell differentiation.

Bone morphogenetic protein (BMP) 2, a member of the transforming growth factor (TGF) beta family, is a potent regulator of osteoblast differentiation. In addition, both TGF-beta and activin A can either induce bone formation or inhibit bone formation depending on cell type and differentiation status. Although much is known about the receptors and intracellular second messengers involved in the action of TGF-beta family members, little is known about how selectivity in the biological response of individual family members is controlled. In this study, we have investigated selective gene induction by BMP-2, TGF-beta1 and activin A in relation to their ability to control differentiation of mouse mesenchymal precursor cells C2C12 into osteoblastic cells. TGF-beta1 can inhibit BMP-2-induced differentiation of these cells, whereas activin A was found to be without morphogenetic effect. Using a gene expression microarray approach covering 8636 sequences, we have identified a total of 57 established genes and expressed sequence tags (ESTs) that were either up-regulated or down-regulated 2 h after treatment with at least one of these three stimuli. With respect to the established genes, 15 new target genes for TGF-beta family members thus were identified. Furthermore, a set of transcripts was identified, which was oppositely regulated by TGF-beta1 and BMP-2. Based on the inverse biological effects of TGF-beta1 and BMP-2 on C2C12 cells, these genes are important candidates for controlling the process of growth factor-induced osteoblast differentiation.

Behavior of osteoblast, adipocyte, and myoblast markers in genome-wide expression analysis of mouse calvaria primary osteoblasts in vitro

Several genes, such as alkaline phosphatase, osteocalcin, and Cbfa1/Osf2, are known to be regulated during osteoblastic differentiation and are commonly used as “osteoblast markers” for in vitro or in vivo studies. The number of these genes is very limited, however, and it is of major interest to identify new genes that are activated or repressed during the process of osteoblast differentiation and bone formation as well as to extend the available information on gene families relevant to this particular differentiation pathway. To identify such genes, we have implemented a genome-wide analysis by determining changes in expression levels of 27,000 genes during in vitro differentiation of primary osteoblasts isolated from mouse calvaria. This study focuses on the description of the analytical and filtering process applied; on the transcriptional analysis of well-established “bone,” “adipocyte,” and “muscle” pathway markers; and on a description of the regulation profiles for genes recently described in the Skeletal Gene Database. We also demonstrate that new array technologies constitute reliable and powerful tools to monitor the transcription of genes involved in osteoblastic differentiation, allowing a more integrated vision of the biological pathways regulated during osteoblast commitment, differentiation, and function.

Progressive Development of the Osteoblast Phenotype during Differentiation of Osteoprogenitor Cells Derived from Fetal Rat Calvaria: Model for in Vitro Bone Formation.

Osteoblasts are the primary cells responsible for bone formation and are thought to originate from mesenchymal osteoprogenitor cells within skeletal tissues. To elucidate the osteoblastic differentiation process, fetal rat calvariae (FRC) were enzymatically digested and fractionated to provide an osteoprogenitor-enriched cell population. The third fraction of cells from the five sequential digestions tested showed a significant osteogenic response to dexamethasone (Dex), a well-known differentiation hormone, which was demonstrated by high alkaline phosphatase activity early in culture and enhanced calcium deposition and bone nodule formation in late stage cultures. These data indicate that fraction three contains a large number of osteoprogenitor cells. During the osteoblastic differentiation of the third fraction of FRC cells, the formation of collagen cross-links (pyridinoline and deoxypyridinoline) was time-dependently accelerated with the accumulation of collagens, which coincided with an onset of mineralization of the cultures, i.e., calcium deposition and bone nodule formation. Moreover, noncollagenous matrix proteins, bone sialoprotein and osteocalcin, were also increased at both mRNA and protein level in Dex-treated cultures with advancing culture periods. Further examination for mRNA expression of bone morphogenetic proteins (BMPs) and TGF-beta1 revealed a notable elevation in BMP-6 mRNA expression on days 3 and 10, and no significant change in TGF-beta1 expression. These observations suggested that the progressive formation of collagen cross-links, production of noncollagenous proteins, and up regulation of BMP-6 mRNA play an important role in the osteoblastic differentiation process of osteoprogenitor cells isolated from FRC. This culture system provides us a suitable model for in vitro bone formation.

Overexpression of the Granulocyte Colony-Stimulating Factor Gene Impairs Bone Morphogenetic Protein Responsiveness in Mice

Granulocyte colony-stimulating factor (G-CSF) is the major hematopoietic growth factor regulating the production and differentiation of neutrophils. We previously demonstrated that permanent overexpression of G-CSF in transgenic mice produces a dramatic enlargement of the bone cavity and reduction of bone mass. This phenotype was shown to be associated with an increase of osteoclast-mediated bone resorption. As a way of determining the role of G-CSF in bone formation in vivo, an ectopic bone was induced subcutaneously into G-CSF transgenic mice by bone morphogenetic protein (BMP)-2, a potent initiator of bone and cartilage from undifferentiated mesenchymal cells. A BMP-2/atelocollagen pellet containing recombinant human BMP-2 was implanted into a dorsal subfascial pocket. At one week after implantation, proliferation of mesenchymal cells around the implant was significantly decreased in transgenic mice compared with control mice. At three weeks, an ectopic bone containing bone marrow was formed both in transgenic and control mice. However, the ectopic bones of the transgenic mice were smaller and less consistent than those of control mice, and the calcium contents were reduced to 56.2% of those of controls. The ectopic bone in the G-CSF mice showed poor development of both lamellar and trabecular bone. Semiquantitative reverse transcription-polymerase chain reaction analysis of the ectopic bone at 3 weeks disclosed no significant differences in the mRNA levels of type I collagen, osteopontin, and osteocalcin between G-CSF mice and control mice. Immunohistochemical study in G-CSF mice showed reduced staining of osteocalcin in the bone matrix surrounding the reduced number of osteoblasts. The half-life of BMP in the implants was prolonged to 7 to 9 days in the G-CSF mice, whereas it was 5 days in the control mice. Collectively, the permanent expression of G-CSF may retard the differentiation process of osteoblasts by impairing the initial induction of mesenchymal cells, resulting in reduction of bone mass, suggesting that G-CSF regulates the bone metabolism by modulating both osteoclast and osteoblast function. Furthermore, it is suggested that G-CSF is a potent modulator of the BMP-2 signal pathway in vivo.

Molecular characterization of gene expression changes in ROS 17/2.8 cells cultured in diffusion chambers in vivo.

Transplantation of diffusion chambers (DC) containing osteoblast-like cells to extraskeletal sites has been highly studied and proven to be a useful technique to investigate the process of osteoblast differentiation and bone formation. To investigate the molecular basis of osteogenesis in DC, we examined the temporal pattern of gene expression of the proliferation marker histone H4, immediate early response genes (IEGs), c-fos, c-jun, c-myc, osteoblast phenotype-associated genes, osteocalcin (OC), osteopontin (OP), type I collagen (COL1A1), alkaline phosphatase (ALP), parathyroid hormone receptor (PTHR) and matrix modifying enzyme, matrix metalloproteinase-9 (MMP-9). DC containing ROS 17/2.8 were implanted intraperitoneally into rat hosts and cultured in vivo for various times up to 56 days. Histological analysis of von Kossa stained sections of the DC contents showed a well-organized connective tissue and the production of mineralized matrices/nodules. In contrast, histological examination of DC containing Rat-2 fibroblast cells revealed the lack of an organized mineralized matrix. Molecular analysis of DC containing ROS 17/2.8 cells at 0, 3, 10, 28, and 56 days demonstrated a time-dependent decrease in DNA content associated with cell death. In the surviving cells, an increase in histone H4 mRNA (consistent with an increase in cell proliferation) was evident by 3-10 days and thereafter expression returned to control levels. In vitro, ROS 17/2.8 cells expressed detectable levels of c-fos, c-jun, c-myc, OC, OP, ALP, COL1A1, and PTHR but not MMP-9. In vivo, the expression of c-fos increased 2-fold in 3-28 days and by 56 days was 4-5 fold above control levels. In 3-10 days, c-jun expression increased 1.6-1.8-fold above control levels. In contrast, by day 28, c-jun expression decreased to control levels, but increased to 2.1-fold above control by 56 days. c-myc mRNA expression increased 3-fold within 3 days and then dropped to below control values by 10-56 days. After transplantation in vivo, the expression of OC and PTHR decreased to undetectable levels. Similarly, ALP mRNA decreased to </=28% of preimplantation values. In contrast, OPN mRNA levels increased up to 7-fold by day 10 and thereafter, returned to 1.7-fold above control values. COL1A1 mRNA decreased 2-fold at day 3 and increased to 3.5-, 1.6-, and 2.8-fold above control at days 10, 28, and 56, respectively. MMP-9 levels increased 5- to 10-fold by days 3-10, but fell to undetectable levels by 28-56 days. These results indicate that the formation of mineralized matrix (bone nodules) seen in the 56-day DC of ROS 17/2.8 cells was preceded by coordinate temporal expression of IEGs, matrix proteins, and matrix-modifying enzymes. Additionally, these results substantiate that measurement of molecular parameters in tissues formed by cells incubated in DC in vivo may be a useful predictor of the osteogenic process.

Changes in Osteoblast Phenotype During Differentiation of Enzymatically Isolated Rat Calvaria Cells

Osteoblasts enzymatically isolated from newborn rat calvariae show various phenotypes including formation of mineralized bone nodules in culture. We investigated the temporal changes in osteoblast phenotype in these cells up to day 20 in culture. These cells formed unmineralized nodules by day 5. Mineralization was observed at the center of nodules by day 10, and nodules became larger on day 15. The nodules were surrounded by numerous alkaline phosphatase (ALP)-positive cells. ALP activity gradually increased by day 20. Parathyroid hormone (PTH) responsiveness increased with time in culture. Osteoblasts produced no osteocalcin by day 10, but its synthesis was detected from day 15. These cells expressed substantial levels of ALP and PTH/PTHrP receptor mRNAs as early as day 5 in culture, but very weak expression of osteocalcin mRNA on day 5. The levels of expression of these transcripts increased with time in culture. In situ hybridization demonstrated that PTH/PTHrP receptor and osteocalcin mRNAs were strongly expressed in nodules, but the former appeared much earlier than the latter. BMP-2 and BMP-4 mRNAs also appeared in the cells forming nodules. Immunohistochemical analysis demonstrated that cells expressing either BMP-2/4 or their receptors (BMPR-IA, BMPR-IB, and BMPR-II) preferentially appeared in nodules. These observations suggested that BMPs play an important role in the formation of mineralized bone nodules in an autocrine and/or paracrine fashion in these cells. The present study confirmed that osteoblasts enzymatically isolated from newborn rat calvariae are a useful tool for studying the differentiation process of osteoblasts.

Prostate-derived soluble factors block osteoblast differentiation in culture.

Bone metastasis is a common event and a major cause of morbidity in prostate cancer patients. After colonization of bone, prostate cells induce an osteoblastic reaction which is not associated with marrow fibrosis (i.e., osteoblast but not fibroblast proliferation). In the present study we test the hypothesis that the tumoral prostatic cell line (PC-3) secretes factors that block the osteoblast differentiation process, resulting in an increase of the relative size of the proliferative cell pool. Our results, using fetal rat calvaria cells in culture, show that conditioned medium from PC-3 cells (PC-3 CM) stimulates osteoblast proliferation and inhibits both alkaline phosphatase (AP) activity (an early differentiation marker) and the mineralization process, measured as calcium accumulation (late differentiation marker). The inhibition of the expression of AP and mineralization depends on the presence of PC-3 CM during the proliferative phase of culture and suggests that both processes occur in a nonsimultaneous fashion. The inhibitory effect of PC-3 CM was not reverted by dexamethasone, which would indicate that prostatic-derived factors and the glucocorticoid do not share a common site of action. Measurement of the proliferative capacity of subcultures from control and treated cells demonstrates that PC-3 CM treatment induces the maintenance of the proliferative potential that characterizes undifferentiated precursor cells.

Load strain gauge test of 150-ton floating crane for the bureau of yards and docks, U. S. navy department

Strontium Ranelate has been utilized as a preventative treatment option for osteoporosis with the release of Sr ions having a direct effect on preventing osteoclast activation and promoting osteoblast differentiation. Previously our group has prepared and characterized a porous Sr-mesoporous bioactive glass (Sr-MBG) scaffold demonstrating its ability to enhance new bone formation when compared to MBG alone. The goal of the present study was to elucidate the bone-inducing properties of Sr by utilizing RNA-seq on in vivo tissue samples to investigate potential target genes responsible for Sr-induced new bone formation. The results demonstrated an increased expression and affiliation of Setd2 in the Sr-MBG group when compared to MBG group alone. Immunofluorescent staining further demonstrated a localization of Setd2 and H3K36me3 in Runx2-positive cells in defects treated with Sr-MBG scaffolds. It was detected that specifically MAPK pathway was activated in MG63 stimulated by Sr. To verify the role of Setd2 in bone formation in the presence of SrCl2, Setd2 was knocked-down and overexpressed in MG63 with/without SrCl2 stimulation. The result showed that Setd2 plays a positive role in osteoblast differentiation which was enhanced by SrCl2. Furthermore, it was found that Setd2 regulated the activation of ERK, which set up a positive feedback in the osteoblast differentiation process. Based on these findings, it was shown that Setd2 has an active role in osteoblast differentiation. As a histone methylase, Setd2 may also turn to be an epigenetic target for new treatment options of osteoporosis.Our research group recently demonstrated that the combination of MBG scaffolds with Sr, efficiently promoted bone regeneration in rat femoral defects even in severely compromised osteoporotic animals, however, the epigenetic mechanism by which Sr ions function to promote bone generation remains unclear. This study showed an increased expression and affiliation of Setd2 and H3K36me3. In vitro, the increased expression of Setd2 promoted osteoblastic differentiation of MG63 stimulated by SrCl2 in MAPK-dependent way, which activated ERK in turn leading to a positive feedback. Based on these findings, it was shown that Setd2 has an active role in osteoblast differentiation and may also turn to be an epigenetic target for new treatment options of osteoporosis and the development of novel bone regeneration scaffold.