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R O Oreffo

Publications and source records attributed to R O Oreffo.

34 records · Page 2Linked to original sources
PubMed1997

Effects of beta mercaptoethanol on the proliferation and differentiation of human osteoprogenitor cells.

Antioxidants are known to influence metabolism and promote cell survival in a number of cell culture systems. However, their effects on the modulation of bone cell differentiation in vitro are not clearly defined. In the present studies we have investigated the effects of beta-mercaptoethanol (beta ME) and ascorbate alone and in combination on human osteoprogenitors derived from bone marrow fibroblasts. In primary marrow cultures, beta ME stimulated colony formation (2-fold), alkaline phosphatase activity (3.5-fold) and, increased DNA synthesis (8-fold) after 21 days. Cell proliferation was increased significantly by beta ME during the first 4 days of a 10-day culture period, indicating stimulation of marrow osteoprogenitor proliferation. Ascorbate did not significantly augment the effects of beta ME in primary cultures or long-term cultures of passaged bone marrow fibroblasts. These findings indicate a potential beneficial role for beta ME addition for the optimal maintenance of colony formation, cell proliferation and differentiation of marrow osteoprogenitor cells in primary human bone marrow fibroblast cultures.

Alkaline Phosphatase↗
PubMed1997

Effects of retinol on activation of latent transforming growth factor-beta by isolated osteoclasts.

The multifunctional cytokine, transforming growth factor-beta (TGF beta), is found in many tissues in a latent or inactive form. The nature and composition of the latent complex can vary depending on tissue type. The release of active TGF beta from its latent complex is a potentially important mechanism for regulation of TGF beta activity. We have shown previously that osteoclasts activate latent TGF beta produced by bone and that bone cells produce a 100-kDa latent complex that lacks the latent TGF beta-binding protein. Here we investigated the effects of retinol on osteoclast activation of various forms of latent TGF beta. Two sources of osteoclasts were used that provide either mature avian osteoclasts or avian osteoclast precursors. Whereas both cell populations activate latent TGF beta, only mature osteoclasts respond to retinol with an increase in activation of latent TGF beta over basal levels. Activation could not be ascribed to pH changes in conditioned medium. Nonacid-dissociable 100-kDa latent complex, which is also produced by bone cells, was added to mature osteoclasts and to osteoclast precursors, but no activation was observed. Platelet latent TGF beta, which contains the 130-kDa latent TGF beta-binding protein, was activated by both osteoclast populations. Conditioned medium from the precursor population activated latent complex, whereas conditioned medium from mature cells did not. Activation of latent TGF beta by retinol-treated mature cells was not blocked by inhibitors of plasmin, nor was activation by conditioned medium from precursor cells. These data suggest that retinol-induced activation of latent TGF beta by osteoclasts is dependent on the stage of differentiation of these cells and the presence of other cell types, and that unlike other cell systems, the plasmin-plasminogen activator mechanism is not involved.

Animals↗
PubMed1997

Modulation of osteogenesis and adipogenesis by human serum in human bone marrow cultures.

Knowledge of the controlling mechanisms of human osteoprogenitor cell differentiation has important implications for understanding bone turnover. The in vitro differentiation of human bone marrow fibroblasts into adipogenic and osteogenic cells and the interaction of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) and dexamethasone in this process has been investigated together with the effects of human serum. Marrow fibroblasts cultured in human serum and dexamethasone for 28 days, generated lipid containing cells as confirmed by morphology, Oil red O staining and immunocytochemistry using antiserum to the adipocyte-specific protein, adipocyte P2 (aP2). In cultures containing 1,25(OH)2D3 and dexamethasone, adipogenesis was stimulated within 21 days. Osteocalcin expression, as assessed by in situ hybridization, was dependent on the presence of 1,25(OH)2D3 and was decreased in cultures treated with dexamethasone. Northern analysis confirmed the decrease in osteocalcin expression and increase in lipoprotein lipase expression with the appearance of the adipogenic phenotype in these cultures. Marrow cultures maintained for 14 days in human serum and osteotropic agents before switching to fetal calf serum indicated the continuous requirement of human serum in these cultures for adipogenesis. These results demonstrate that human serum contains factors that exert dramatic effects on human bone marrow cell differentiation to augment the osteogenic and adipogenic activity of 1,25(OH)2D3 and dexamethasone.

Adipocytes↗
PubMed1994

Cells cultured from the growing tip of red deer antler express alkaline phosphatase and proliferate in response to insulin-like growth factor-I.

Deer antler growth provides a unique natural model of rapid and complete bone regeneration. In this study, the distal antler tips of male red deer (Cervus elaphus) were collected post-mortem during the annual growth period (April-August), and an in vitro system established for the culture of cells from three regions; the inner layer of the perichondrium, the reserve mesenchyme and the cartilage zone. Alkaline phosphatase (ALP) expression by cultured cells, as demonstrated by enzyme histochemistry and biochemical assay, reflected the stage of cellular differentiation. ALP activity was highest in cells cultured from the hypertrophic cartilage region (3.6 +/- 0.2 mumol/micrograms cell protein/minute), and lowest in undifferentiated mesenchymal cells (0.3 +/- 0.01 mumol/microgram cell protein/minute). ALP expression was lost with passage in culture. Levels of ALP activity in cultured cells correlated with the pattern and extent of enzyme expression in tissue sections as demonstrated by histochemical staining. Insulin-like growth factor (IGF)-I (10(-9)M-10(-7)M) was found to be mitogenic for cultured cells from all three zones as shown by increased incorporation of [3H]thymidine into DNA. These results demonstrate that cells from three different regions of the antler tip can be maintained in culture, and that antler cells share certain phenotypic characteristics of growth plate chondrocytes. These data provide further evidence of a role for IGF-1 in the regulation of antler growth. Antler regrowth is a potentially useful model for the study of the factors that regulate bone formation.

Alkaline Phosphatase↗
PubMed1993

Characterization of a cell line derived from a human giant cell tumor that stimulates osteoclastic bone resorption.

Giant cell tumors of bone are common but unusual tumors that are comprised of multiple cell types. Most attention has been focused on the giant cells, which resemble osteoclasts morphologically and functionally. This study examines the properties of a cell line derived from mononuclear cells from one of these tumors, since it appears likely that these cells may be able to influence the activities of cells with the osteoclast phenotype. This cell line, C433, has the following characteristics: (1) it represents undifferentiated cells, not recognized by any known antigenic markers for leukocytes; (2) it contains tartrate-resistant acid phosphatase; (3) it responds to the osteotropic factors 1,25 dihydroxyvitamin D3, insulin-like growth factor I and II, but not to parathyroid hormone; (4) it forms sarcomas in nude mice; and (5) it produces an activity that stimulates isolated avian and rat osteoclasts to resorb bone. This cell line may be useful in examining interactions between osteoclasts and accessory cells involved in bone resorption.

Alkaline Phosphatase↗
PubMed1991

Inhibition of bone resorption by inorganic phosphate is mediated by both reduced osteoclast formation and decreased activity of mature osteoclasts.

High concentrations of inorganic phosphate (Pi) are known to inhibit bone resorption, although the mechanism(s) underlying this effect is unclear. To investigate whether Pi can inhibit the formation of osteoclasts we studied the effects of changes in Pi concentration between 1 and 4 mM on osteoclast-like cell formation in 1 week cultures of mouse bone marrow. Osteoclast-like cells were identified by multinuclearity, positive staining for tartrate-resistant acid phosphatase (TRAP), and contraction in response to calcitonin. Increasing concentrations of Pi inhibited formation of these cells in a dose-dependent manner. To study effects of Pi on the bone-resorbing activity of mature osteoclasts we isolated osteoclasts from calcium-deficient egg-laying hens or rat pups and incubated them on sperm whale dentine slices. High Pi concentrations markedly reduced both the number of resorption pits formed per dentine slice and the mean area of each pit in both avian and mammalian systems. These data indicate that high concentrations of Pi act on bone directly, both to inhibit generation of new osteoclasts from their precursor cells and to inhibit bone resorption by mature osteoclasts. These effects of extracellular Pi concentration may play an important modulatory role on bone turnover in vivo and have potential importance in several disease states in which Pi metabolism is perturbed.

Animals↗
PubMed1991

Latent forms of transforming growth factor-beta (TGF beta) derived from bone cultures: identification of a naturally occurring 100-kDa complex with similarity to recombinant latent TGF beta.

Transforming growth factor-beta (TGF beta) is produced by most tissues, including bone, as a complex that is biologically inert. Release of TGF beta homodimer from this latent complex is necessary for TGF beta to exert effects on target cells. Thus, the nature of the latent complex and the mechanisms responsible for TGF beta release are the key to understanding TGF beta actions. We have found that murine calvarial bone cultures secrete multiple latent forms of TGF beta. Using analytical chromatography and Western blot analysis, we have compared bone latent TGF beta with the previously characterized latent complex present in platelets and with simian TGF beta precursor, which is stably expressed in a latent form by Chinese hamster ovarian (CHO) cells. A major component of the bone material appears to be a latent complex of 100 kDa, consisting of mature TGF beta (25-kDa homodimer). Like the recombinant TGF beta precursor, it elutes from a Mono-Q fast pressure liquid chromatography anion exchange column at 0.2 M NaCl and shows a very similar banding pattern on Western blots. Thus, this bone complex closely resembles recombinant TGF beta precursor expressed in a latent form by CHO cells and differs from the naturally occurring platelet complex, which has an additional 135-kDa binding protein that is bound through disulfide bonds to the precursor proregion. Western blot analysis also indicates that, like CHO cells, which express recombinant TGF beta precursor, but unlike other cell types, the bone cultures secrete detectable amounts of uncleaved TGF beta precursor. The bone calvarial culture is the first example of a naturally occurring system that expresses the 100-kDa latent TGF beta complex.

Animals↗
PubMed1990

Oxygen-derived free radicals stimulate osteoclastic bone resorption in rodent bone in vitro and in vivo.

The mechanisms by which bone resorbing osteoclasts form and are activated by hormones are poorly understood. We show here that the generation of oxygen-derived free radicals in cultured bone is associated with the formation of new osteoclasts and enhanced bone resorption, identical to the effects seen when bones are treated with hormones such as parathyroid hormone (PTH) and interleukin 1 (IL-1). When free oxygen radicals were generated adjacent to bone surfaces in vivo, osteoclasts were also formed. PTH and IL-1-stimulated bone resorption was inhibited by both natural and recombinant superoxide dismutase, an enzyme that depletes tissues of superoxide anions. We used the marker nitroblue tetrazolium (NBT) to identify the cells that were responsible for free radical production in resorbing bones. NBT staining was detected only in osteoclasts in cultures of resorbing bones. NBT staining in osteoclasts was decreased in bones coincubated with calcitonin, an inhibitor of bone resorption. We also found that isolated avian osteoclasts stained positively for NBT. NBT staining in isolated osteoclasts was increased when the cells were incubated with bone particles, to which they attach. We confirmed the formation of superoxide anion in isolated avian osteoclasts using ferricytochrome c reduction as a method of detection. The reduction of ferricytochrome c in isolated osteoclasts was inhibited by superoxide dismutase. Our results suggest that oxygen-derived free radicals, and particularly the superoxide anion, are intermediaries in the formation and activation of osteoclasts.

Animals↗
PubMed1990

Inhibitory effects of the bone-derived growth factors osteoinductive factor and transforming growth factor-beta on isolated osteoclasts.

Demineralized bone matrix contains a number of growth factors for osteoblast-like cells. Two of these, the novel glycoprotein osteoinductive factor (OIF) and transforming growth factor-beta (TGF beta), act together to cause ectopic bone formation in vivo. Since OIF, like TGF beta, is likely released from bone when the matrix is resorbed, we examined the effects of homogeneous OIF and TGF beta on osteoclast function. Osteoclast function was tested in isolated avian osteoclasts and was measured in terms of tartrate-resistant acid phosphatase (TRAP) activity, oxygen-derived free radical production, and formation of characteristic resorption lacunae on slices of sperm whale dentine. OIF (50-100 ng/ml) inhibited the capacity of these osteoclasts to form lacunae whether assessed by the number of excavations per slice or by the total area resorbed. OIF (10-100 ng/ml) or TGF beta (10-20 ng/ml) caused a decrease in TRAP activity as well as a reduction in oxygen-derived free radical generation detected by nitroblue tetrazolium staining. TGF beta had no effect on the resorption capacity of isolated osteoclasts in concentrations that inhibited TRAP activity and nitroblue tetrazolium staining. These results suggest that growth regulatory factors, such as OIF and TGF beta, released during the resorption of bone may be endogenous inhibitors of continued osteoclastic activity. This cessation of osteoclast activity may be an essential preliminary step to the new bone formation that occurs at resorption sites during bone remodeling.

Acid Phosphatase↗
PubMed1989

Activation of the bone-derived latent TGF beta complex by isolated osteoclasts.

Although TGF beta is unquestionably an important growth regulatory polypeptide with effects on many cell types, the cellular mechanisms which release it from the binding proteins which mask its biological activity are not well understood. Here we show that when isolated osteoclasts are activated, they release active TGF beta from the latent TGF beta complex produced by bone organ cultures. Since active TGF beta has powerful inhibitory effects on osteoclast formation and bone resorption and stimulates osteoblast activity, is present in abundant amounts in the bone matrix and is released during hormone-stimulated osteoclastic bone resorption, the activation of TGF beta by stimulated osteoclasts may be an important regulatory step in normal bone remodeling.

Animals↗
PubMed1988

Effect of vitamin A on bone resorption: evidence for direct stimulation of isolated chicken osteoclasts by retinol and retinoic acid.

The effects of retinol (vitamin A) and retinoic acid on primary cultures of isolated chicken osteoclasts have been studied. The experiments were performed to establish the direct actions of these two agents on the organization of cytoskeletal structures, on the acid phosphatase contents, and on the bone resorption activities of these cells. The results showed that by treating the cultures with retinol or retinoic acid, from 10(-8) to 10(-5) M, there were dose-related responses of the osteoclasts. Adhesion to the substratum was stimulated by increasing the number of cells exhibiting the specialized dot-like adhesion structures, or podosomes, which represent the active part of the sealing zone. The treatments also induced rearrangement of the microtubular patterns with reversible depolymerization of microtubules. Acid phosphatase activity was significantly higher both in vitamin A-treated osteoclasts and in their media. When [3H]proline-labeled bone particles were added to the retinoid-treated osteoclasts, the release of [3H]proline was increased significantly compared to controls. These results suggest that the two vitamin A metabolites cause several modifications of the metabolic status of isolated osteoclasts that result in augmented rates of bone resorption.

Acid Phosphatase↗
PubMedDate not supplied

In vitro and in vivo methods to determine the interactions of osteogenic cells with biomaterials.

To assess new biomaterials for possible use as bone graft substitutes, a number of techniques allow interactions with osteoblastic cells to be studied, with respect to effects on proliferation and differentiation of osteoprogenitors. In vitro models include the use of bone explant cultures, fetal rat calvarial-derived osteoblast cells, primary stromal populations, transformed and non-transformed cell lines and immortalized osteoblast cell lines. However, these assessments are limited by the extent of osteogenic differentiation and bone formation that can be observed in vitro, species differences and phenotypic drift of cells cultured in vitro. The use of in vivo experimental systems such as the segmental/calvarial bone defect model, the subcutaneous implant model and the diffusion chamber implantation model circumvent some of these issues and, in the appropriate model, provide data on efficacy, biocompatibility and osteointegration of a biomaterial. The combination of in vitro and in vivo approaches together with the development of new cell labeling techniques, in particular the ability to genetically mark and select specific human bone cell populations provides new avenues for their potential evaluation in combination with appropriate biomaterials for clinical use. These in vitro and in vivo techniques are reviewed and those recently developed for assessment of human osteogenic cells should be applicable to many other cell systems where knowledge of specific human tissue or cell interactions with biomaterials is required.

Journal Article↗