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PubMedDate not supplied

Interleukin-1 alpha: regulation of cellular proliferation and collagen synthesis in cultured human osteoblast-like cells.

In this work the authors studied the effects of interleukin-1 alpha on metabolic activities of human osteoblast-like cells in vitro. The bone nature of the cells was established by assaying for specific bone protein, the osteonectin, and the parathormone receptor, an osteoblast marker. Administration of interleukin-1 alpha to cultured osteoblasts produce an increase in cellular proliferation as suggested by 3H-thymidine incorporation and cell growth studies. Interleukin-1 alpha also affected collagen synthesis confirming its potential role on bone-formation and resorption processes.

Adenylyl Cyclases↗

PubMed2004

Decreased levels of osteopontin and bone sialoprotein II are correlated with reduced proliferation, colony formation, and migration of GFP-MDA-MB-231 cells.

MDA-MB-231 human breast cancer cells transfected with GFP were used as model to determine the reduction in proliferation, colony formation, and migration in response to agents with anti-metastatic properties. These agents consisted of antisense oligonucleotides (ASOs) directed against osteopontin (OPN), bone sialoprotein II (BSP II), and osteonectin (ON), as well as an antibody directed against BSP II. A bisphosphonate derivative (ibandronate) and an alkylphosphocholine (erucylphospho-NNN-trimethylpropanolamine; ErPC3) were used as positive controls. The ASOs directed against OPN, BSP II and ON suppressed the expression of their respective target proteins by 81%, 74% and 69%, respectively. They were barely but significantly active in inhibiting the proliferation, but intermediately to highly active in inhibiting the colony formation and migration of GFP-MDA-MB-231 breast cancer cells. The antibody against human BSP II was significantly more active than all ASOs used and was equally active or even surpassed the activity of ibandronate and ErPC3 in all three assays. The results obtained suggest a specific anti-metastatic activity of this antibody as well as of the ASOs found effective in decreasing OPN and BSP II expression.

Adult↗

PubMed2003

Measurement of gene expression following cryogenic mu-CT scanning of human iliac crest biopsies.

An important consideration in interpreting indices of gene expression in human bone is relating mRNA levels to functional endpoints such as bone architecture. In the present study, a method was developed for quantitative measurement of gene expression and bone morphology in the same specimen. Three-dimensional images of iliac crest bone biopsies from healthy premenopausal women were obtained using a novel high resolution cryogenic mu-CT scanner. RNA was isolated from the biopsies and mRNA levels were measured for genes related to bone metabolism. The gene expression profile and variability of expression within iliac crest biopsies of women was similar to human osteoblastic cell lines and rat long bones. mRNA for alkaline phosphatase, bone matrix proteins, and selected cytokines and cytokine receptors were consistently detected in biopsies. As previously shown in rat bone, there was a tight correlation between mRNA levels for type 1 collagen and osteonectin, a weaker correlation between type 1 collagen and osteocalcin and no correlation between bone matrix proteins and alkaline phosphatase. The relative abundance of the mRNA for the three most prevalent transforming growth factor-beta (TGF-beta) isoforms in bone (TGF-beta(1)>> TGF-beta(3)> TGF-beta(2)) was the same as the known abundance of the corresponding TGF-beta peptides in bone matrix. The results demonstrate the feasibility of analyzing the three-dimensional architecture of a bone biopsy using cryogenic mu-CT imaging and then measuring expression of genes related to bone cell function within the same specimen following RNA extraction and analysis.

Journal Article↗

PubMed2005

Gene expression patterns in bone after 4 days of hind-limb unloading in two inbred strains of mice.

INTRODUCTION: An improved understanding of the interdependence of transcriptional and genomic control of bone loss is critical for the design of effective and safe countermeasures for osteoporosis in space and on Earth. In an effort to test whether molecular pathways modulating the loss of functional weight bearing are dependent on genetic makeup, we quantified the differential expression of genes critical to the early stages of bone remodeling in two different strains of mice. METHODS: Adult (4-mo-old) female BALB/cByJ (BALB) and C3H/HeJ (C3H) mice, strains with different sensitivities to unloading, were subjected to hind-limb unloading (HLU) or normal cage activities. RNA was extracted from the tibia following 4 d of HLU and expression levels were determined. RESULTS: In the BALB mice, HLU significantly altered transcriptional levels of osterix (-36%), alkaline phosphatase (-36%), osteonectin (-44%), collagen type 1 (-55%), MMP2 (-36%), osteocalcin (-68%), and osteopontin (+28%). This expression pattern was highly correlated (R2 = 0.75) with altered expression levels in the C3H mice, but the magnitude of altered mRNA levels was less than half of those in the BALB mice. These strain-specific changes in gene expression were consistent with the differential changes in bone formation, as determined in a second group of BALB and C3H mice. DISCUSSION AND CONCLUSIONS: These data indicate that genetics may influence the absolute changes in gene expression of genes during spaceflight, but that the molecular pathways targeted by countermeasures of bone loss may not need to be specific to an individual's genetic makeup.

Animals↗

PubMed2005

[Development and congenital malformations of the skeleton].

The article presents current knowledge on skeleton development, which is initiated by mesenchymal cells condensation. The mesenchymal to epithelial transformation, vascular regression, cartilage formation and ossification are involved in normal skeletogenesis. All the processes are influenced by various genes and regulatory molecules, e.g., N-cadherin, vascular endothelial growth factor, transforming growth factor-beta, syndecan-3 and others. The chondrogenesis depends on the Sox genes expression - Sox5, Sox6 and especially Sox9, which continued action produce persistent cartilage. The essential role in osteogenesis is played by the transcription factor Runx2 that directly activates expression of genes coding typical bone proteins like collagen type I, osteocalcin, osteopontin and osteonectin. Development of fibrous, cartilaginous and synovial joints, bone maturation and remodeling, and mechanisms leading to skeleton dysmorphogenesis are also discussed.

Antigens, CD↗

PubMed2005

Tissue engineering of cardiac valves: re-seeding of acellular porcine aortic valve matrices with human mesenchymal progenitor cells.

BACKGROUND AND AIM OF THE STUDY: Tissue-engineered heart valves have the potential to overcome the limitations of present heart valve replacements. This study investigated the potential for re-seeding an acellular porcine heart valve matrix using human mesenchymal progenitor cells (MPC). METHODS: MPC were isolated from the bone marrow of patients undergoing hip replacement operations. Putative MPC were then cultured in several differentiation media in order to determine the multipotential differentiation capacity of the cells. The MPC were also characterized by FACS analysis. Cells at passage 8 were then seeded at between 1 x 10(4) and 1 x 10(5) cells/cm2 onto a decellularized porcine aortic valve matrix, and recellularization of the matrix was assessed. The phenotype of the re-seeded cells and re-seeded cell density was then determined by histology and immunohistochemistry. RESULTS: Putative MPC were successfully isolated and differentiated into cells of the adipogenic, neurogenic, and myogenic lineages. FACS analysis showed the cells to have a similar phenotype to those isolated by others (CD45-, CD13+, D7FIB+, CD105+, CD10+/-, LNGFR+/-, CD55+, BMP- and AP+/-). Cells seeded onto an acellular valve matrix penetrated the center of the tissue after four weeks to 2% of homograft cell density. Phenotypic analysis of the cells in the re-seeded matrix revealed the cells to have a similar phenotype to native valve interstitial cells (vimentin+, alpha-smooth muscle actin+, heavy chain myosin slow-, desmin-). However, re-seeded cells also expressed osteogenic markers (alkaline phosphatase, osteonectin, and osteopontin). CONCLUSION: This study has shown, for the first time, that human MPC have the capacity to infiltrate an acellular porcine valve matrix under static conditions in vitro. Future studies will comprise culture under pulsatile flow in a physiological heart valve bioreactor to maintain the desired cell phenotype and increase cell density.

Animals↗

PubMed1992

[Vitamin D: biosynthesis, metabolism and mechanism of action at the cellular level].

The term vitamin D includes various chemical species. Vitamin D3 a true endogenous or alimentary prohormone is converted into its main metabolite, calcitriol, by successive hydroxylations in the liver in position 25 and in the kidney in position 1, the production of which is controlled by several factors including parathyroid hormone, blood calcium and phosphorus or insulin as well as by the metabolites of the hormone itself. It controls the synthesis of numerous peptides by acting on gene expression. Indeed, several structural proteins are involved including procollagen alpha 1l, core protein of proteoglycans, diverse regulatory peptides such as protooncogene c-myc and growth factors, "Tumor Necrosis Factor or TNF" and "Nerve Growth Factor or NGF" or hormones such as parathyroid hormone, and finally constitutive proteins of the mineralized tissues such as osteonectin, osteocalcin, osteopontin and calbindins. Therefore, it modulates very different cellular processes. It acts via a nuclear receptor the structure and function of which have been investigated by genetic engineering (cloning of genes encoding for the receptor and hormono-dependent peptides, transfection assays, directed mutagenesis). Actual studies investigate its role in the formation of the complex for transcription initiation near ADN sites, the "Vitamin D Responsive Element or VDRE", located upstream vitamin D-responsive genes and approximately RNA polymerase II. The receptor, which is present in many cell types at various concentrations, would determine spatial and temporal patterns of calcitriol action during development in conjunction with chromatin factors.

Cell Physiological Phenomena↗

PubMed2006

The effect of osteogenic medium on the adhesion of rat bone marrow stromal cell to the hydroxyapatite.

OBJECTIVE: To investigate the adhesive properties of bone marrow stromal cell (BMSC) on the hydroxyapatite (HA) particles and analyze their behavior. METHODS: The study took place in the Department of the Histology and Embryology, Celal Bayar University, Manisa and in the Department of Bioengineering, Ege University, Izmir, Turkey between 2004 and 2005. We cultured BMSC from the mature rat tibia and differentiated to the osteoblasts by osteogenic medium. The BMSCs were subcultured and were taken to the HA substrate. We measured their proliferation capacity and viability with MTT assay using the spectrophotometric method. Furthermore, we identified the osteoblast-like cells by immunohistochemical staining of osteonectin and osteocalcin and we analyzed the behavior of the cells on different sized HA particles by SEM at the end of 3 days incubation. RESULTS: Osteogenic medium caused the proliferation capacity of BMSC to speed up and the effects appeared earlier. We confirmed the osteoblastic differentiation by staining of most cells with osteoblastic markers. Subcultured cells were similarly adhesive to the HA particles and the osteogenic medium did not alter this behavior. They spread on the substrate similarly. Most of the cells demonstrated the cytoplasmic protrusion. Morphology of the cells did not change much with or without osteogenic medium. Different sizes of HA particles did not affect the adhesive properties of these cells except HA gel. The spreading and attachment ratios of the cells on HA gel were more than the others CONCLUSION: We found that there was heterogeneity in BMSC on differentiation capacity to the osteoblast, which was a sign of a subpopulation. Adhesive cells showed similar morphology and behavior under the effect of osteogenic medium. The only difference was the spreading capacity on the HA gel where cell used this substrate more effectively for adhesion.

Animals↗

PubMed2005

Cardiovascular disease and osteoporosis.

Cardiovascular disease (CVD) and osteoporosis (OP) are public health problems with numerous epidemiological links and important economic consequences. Recent studies have demonstrated that CVD and cardiovascular mortality are associated with reduced bone mineral density (BMD) and bone fractures. These two conditions may be sustained by similar or common pathophysiological mechanisms and risk factors. There are several matrix proteins, such as type 1 collagen, proteoglycan, osteopontin, and osteonectin, which are found in bone and vascular matrix components. Matrix proteins play an important role both in bone formation and in the development of atherosclerosis. Estrogens also play a role in both CVD and OP through their effects on cytokines, such as IL-1, IL-6 and TNF-alpha and osteoprotegerin (OPG). The lack of estrogens induces an increase in these cytokines and a decrease in OPG, both implicated in the mechanisms of bone loss and atherogenesis. An additional link between CVD and OP seems to be related to the action of some drugs, such as bisphosphonates, statins and raloxifene. Several studies suggest that the mechanism of action of these drugs at cellular level may not be mutually exclusive, acting either in bone or in atherosclerotic plaque. However, further studies are necessary to define the relationship between CVD and OP more specifically and to understand the complex interaction of similar or common risk factors and genetic or molecular determinants.

Atherosclerosis↗

PubMed2006

[In vitro study on proliferation and multi-lineage differentiation potential of adipose-derived cells].

Adipose were obtained from patients underwent liposuction treatment (total 19 female donors, 31.5 +/- 5.8 years old). Liposuction tissues were digested with type I collagenase, cells were isolated and cultured up to passage 10. To evaluate the proliferation potential of ADCs, growth curve and cumulative population doubling were achieved by cell counting. CD29,CD105, CD106, CD166, CD49d, CD34, CD31, 3G5 were analyzed by flow cytometry and immunocytochemistry to characterize the cell population. The multi-lineage potential of ADCs was testified by differentiating cells with osteogenic,chondrogenic and adipogenic inducer. A total of 5x10(7) nucleared cells could be obtained from 300ml liposuction tissues. After in vitro cultivation,cumulative population doubling number reached 15.53 at passage 10 (average 1.59 +/- 0.224 /passage). Flow cytometry and immunocytochemistry showed that ADCs expressed high level (>60%) of stem cell-related antigen (CD29, CD105, CD106, CD166), while cells expressed hematopoiesis-related antigen CD34 and CD31 around 7.3% and 29.2% respectively. Collagen II (both in mRNA and protein level) was detected in chondrogenic differentiation. The calcified nodules were observed by von Kossa and Alizarn Red staining and the expressions of AKP and Osteonectin were detected by RT-PCR in osteogenic differentiation. PPARr2, GLU-4, and Leptin genes were detected in adipogenic differentiation and intracellular lipid droplets could be observed by Oil Red staining. ADCs can be abundantly harvested and have high proliferative and multi-lineage differentiation potential.

Adipocytes↗

PubMed1991

Gene expression in osteoblastic cells.

Osteoblasts, the bone-forming cells, synthesize the macromolecules of the bone matrix including: type I collagen; osteocalcin; osteonectin; osteopontin; proteoglycan I and II; bone sialoprotein; matrix gla-protein; bone glycoprotein 75; several other proteins, which have not been extensively characterized; growth factors, including transforming growth factor beta and fibroblast growth factor. Osteoblasts also have high levels of the membrane-bound enzyme, alkaline phosphatase, which plays a role in matrix mineralization, and receptors for tissue-specific hormones, such as parathyroid hormone, as well as many other hormones, cytokines and growth factors, which regulate bone growth, differentiation and metabolism. The expression of these various proteins, most of which are not unique to bone but which together characterize the bone phenotype, is induced during osteoblastic differentiation in a stepwise fashion, suggestive of multiple regulatory factors. The detailed sequence of the expression of osteoblastic genes in situ has not been fully characterized. It appears that type I collagen and alkaline phosphatase are expressed early during the commitment to the osteoblastic phenotype, whereas osteopontin and osteocalcin appear late during osteoblastic differentiation. Diversity among "osteoblastic" cells is also apparent, probably not all osteoblastic cells express all the features. A large number of osteoblastic models are currently available to study the expression of osteoblast-related genes in vitro. These include primary cultures from calvaria or trabecular bone from several species, including humans, osteosarcoma-derived cell lines, and experimentally immortalized cells. Some of these in vitro models, especially the calvaria-derived cultures, undergo changes which mimic osteoblastic differentiation in vivo. The study of these and other cell models started providing insights into the regulation of gene expression in osteoblastic cells. In addition to a vast body of information on the conditions required for the expression of various proteins in culture and their regulation by hormones and growth factors, more detailed information on specific genes has recently been obtained. For example, regulation of type I collagen gene expression has been studied in osteosarcoma cell lines where 1,25(OH)2 vitamin D3 was shown to act via specific DNA segment(s) in the 5' flanking region of the gene, while parathyroid hormone affected gene expression by altering the stability of the transcripts. TGF beta 1, which stimulates osteogenesis, was shown to promote the transcription of osteopontin and type I collagen, the latter effect requiring the binding site for the transactivating protein, nuclear factor I.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Nutritional requirements for boron, silicon, vanadium, nickel, and arsenic: current knowledge and speculation.

Definition of specific biochemical functions in higher animals (including humans) for the ultratrace elements boron, silicon, vanadium, nickel, and arsenic still has not been achieved although all of these elements have been described as being essential nutrients. Recently, many new findings from studies using molecular biology techniques, sophisticated equipment, unusual organisms, and newly defined enzymes have revealed possible sites of essential action for these five elements. Based on these findings and the response of animals and/or humans to low intakes of these elements, the following speculations have been presented: 1) Boron has a role that affects cell membrane characteristics and transmembrane signaling. 2) Silicon is necessary for the association between cells and one or more macromolecules such as osteonectin, which affects cartilage composition and ultimately cartilage calcification. 3) Vanadium reacts with hydrogen peroxide to form a pervanadate that is required to catalyze the oxidation of halide ions and/or stimulate the phosphorylation of receptor proteins. 4) Nickel is needed for the CO2-fixation to propionyl-CoA to form D-methylmalonyl-CoA. 5) Arsenic has an important role in the conversion of methionine to its metabolites taurine, labile methyl, and the polyamines. If any of these speculations are found to be true, the element involved will be firmly established as having a nutritional requirement because the body obviously cannot synthesize it. Based on animal findings, the dietary requirement is likely to be small; that is, expressed in micrograms per day.

Amino Acids↗

PubMedDate not supplied

Biochemical markers of bone metabolism.

Bone metabolic homeostasis is regulated by a number of hormones and local modulators, and the study of these factors has been of major help in our understanding of bone disease. However, these parameters do not, in a strict sense reflect the metabolic and biochemical changes in the diseased bone tissue. Thus, there is a great interest in the study of biochemical specific "markers" of bone metabolic processes, namely of bone formation and bone resorption. Alkaline phosphatase, osteocalcin, osteonectin, and procollagen type I propeptides are the currently known markers of bone formation, whereas urinary hydroxyproline and hydroxylysine glycosides, plasma tartrate resistant acid phosphatase, and urinary hydroxy-pyridinium crosslinks of collagen are considered markers of bone resorption. In this paper, we review the background work on each of these markers, and subsequently give an overview of the currently available data on their usefulness in metabolic bone diseases, namely in Paget's disease of bone, primary hyperparathyroidism, osteoporosis, and renal osteodystrophy.

Biomarkers↗

PubMed1990

[The usefulness of biochemical markers of bone remodelling in the diagnosis and follow-up of Paget's disease of bone, primary hyperparathyroidism, tumoral hypercalcemia and postmenopausal osteoporosis. I. The markers of bone formation].

Rapid detection of the exact changes in bone remodelling is exceptionally important. In this paper, the latest bone remodelling biochemical markers are reviewed. Some of them have already been used for a long time, and their utility has been widely demonstrated. The newest ones, in experimental stage, can be used as a complement to the others. The bone remodelling markers reviewed are: 1) Alkaline phosphatase; 2) osteocalcin; 3) Other noncollagen of bone matrix such as osteonectin, GLA-protein of the matrix, osteopontine and alpha 2-HS-glycoprotein; 4) Procollagenous and other collagenous peptides of the matrix (C terminal of type-I procollagen and urinary elimination of nondialysis hydroxyproline. Amongst the bone resorption markers studied are: 1) Calcium/creatinine urinary quotient; 2) Tartrate resistant acid phosphatase; 3) Urinary hydroxyproline; 4) Other substances derived from collagen disruption such as hydroxilysin glycoside, piridinolinic intermolecular bridges and the enzymatic activity of proline iminopeptidase. We endeavoured to collect all the most important references on the matter, especially those relating to Paget's disease of the bone, primary hyperparathyroidism, tumoral hypercalcemia and postmenopausal osteoporosis.

Biomarkers↗

PubMed1990

[Usefulness of bone remodelling biochemical markers in the diagnosis and follow-up of Paget's bone disease, primary hyperparathyroidism, tumor hypercalcemia, and postmenopausal osteoporosis. II. Bone resorption markers].

Rapid detection of the exact changes in bone remodelling is exceptionally important. In this paper, the latest bone remodelling biochemical markers are reviewed. Some of them have already been used for a long time, and their utility has been widely demonstrated. The newest ones, in experimental stage, can be used as a complement to the others. The bone remodelling markers reviewed are: 1) Alkaline phosphatase; 2) osteocalcin; 3) other noncollagen of bone matrix such as osteonectin, GLA-protein of the matrix, osteopontine and alpha 2-HS-glycoprotein; 4) Procollagenous and other collagenous peptides of the matrix (C terminal of type I procollagen and urinary elimination of non-dialysis hydroxyproline. Amongst the bone resorption markers studied are: 1) Calcium/creatinine urinary quotient; 2) Tartrate resistant acid phosphatase; 3) Urinary hydroxyproline; 4) Other substance derived from collagen disruption such as hydroxylysine glycoside, piridinolinic intermolecular bridges and the enzymatic activity of proline iminopeptidase. We endeavored to collect all the most important references on the matter, especially those relating to Paget's disease of the bone, primary hyperparathyroidism, tumoral hypercalcemia and postmenopausal osteoporosis.

Acid Phosphatase↗

PubMed1989

Mechanism of mineral formation in bone.

The mechanism of mineral formation in bone is seen best where active new bone formation is occurring, e.g., in newly forming subperiosteal bone of the embryo, in the growing bone of young animals, and in healing rickets where the calcification process in osteoid is reactivated. A large body of ultrastructural evidence, using conventional and anhydrous methods for tissue preparation, has shown convincingly that extracellular matrix vesicles are present at or near the mineralization front in all of the above, and that these vesicles are the initial site of apatite mineral deposition. Thus bone resembles growth plate cartilage, predentin, and turkey tendon in having calcification initiated by matrix vesicles. Once the calcification cascade is begun, matrix vesicles are no longer needed to support mineralization and are consumed by the advancing mineralization front in which performed crystals serve as nuclei for the formation of new crystals. The rate of crystal proliferation is promoted by the availability of Ca2+, PO4(3-), and the presence of collagen, and retarded by naturally occurring inhibitors of mineralization such as proteoglycans and several noncollagenous calcium-binding proteins of bone including bone-Gla protein (osteocalcin), phosphoproteins, osteonectin, and alpha-2HS-glycoproteins. New electron microscopic immunocytochemical findings in our laboratory suggest that the origin of alkaline phosphatase-positive bone matrix vesicles is polarized to the mineral-facing side of osteoblasts and may be concentrated near the intercellular junctions of human embryonic osteoblasts.

Alkaline Phosphatase↗

PubMed1989

Immunohistochemistry of extracellular matrix proteins during various stages of dentinogenesis.

During dentinogenesis the expression of extracellular matrix (ECM) proteins by (pre)odontoblasts changes concomitantly with the stage of differentiation. Of these ECM proteins some are present throughout all stages of dentinogenesis, while others can only be demonstrated at particular stages of differentiation. Utilizing immunohistochemical techniques, positive detection of ECM proteins within the (pre)odontoblast or in their extracellular matrices has been demonstrated for (pro)collagen type I, III, IV, V and VI, fibronectin, tenascin, laminin, basement membrane heparan sulfate, nidogen, dentinophosphophoryns (DPP), osteocalcin (OC), osteonectin, osteopontin and 95 kDal glycoprotein. Early predentin before onset of dentin mineralization also reacts with antibodies to enamel matrix proteins. Of these ECM proteins, only DPP are exclusively synthesized by odontoblasts; DPP thus can be regarded as specific biochemical markers for odontoblast activity. A second marker for odontoblasts (but also synthesized by osteoblasts and osteocytes) is OC. In some species however OC levels in dentin seem very low. The initiation of dentin mineralization may be a matrix-mediated process in which preameloblasts also seem to be involved. Current data suggest that the DPP-collagen complex is associated with the mineralization process in dentin.

Animals↗

PubMedDate not supplied

[Osteocalcin and endocrine pathology].

The field of metabolic bone diseases has recently received useful information from the examination of the role of new collagenous and non-collagenous bone proteins, such as osteocalcin (also called bone Gla-protein) and osteonectin. It appears that at present, serum osteocalcin is the one bone protein that has the most promise for assisting in the diagnosis and management of high turnover metabolic bone disease states. Since endocrine diseases are frequently associated with altered bone turnover due to a disequilibrium between formation and bone resorption, we examine the role of osteocalcin, a specific marker of osteoblastic activity, in these clinical conditions.

Bone Diseases, Metabolic↗