Search PubMed⌕ Search

Biomedical subjects

J T Triffitt

Publications and source records attributed to J T Triffitt.

At least 55 records · Page 3Linked to original sources
PubMed1996

Arthroplasty implant biomaterial particle associated macrophages differentiate into lacunar bone resorbing cells.

OBJECTIVE: To study the pathogenesis of aseptic loosening: in particular, to determine whether macrophages responding to particles of biomaterials commonly used in arthroplasty surgery for arthritis are capable of differentiating into osteoclastic bone resorbing cells, and the cellular and hormonal conditions required for this to occur. METHODS: Biomaterial particles (polymethylmethacrylate, high density polyethylene, titanium, chromium-cobalt, stainless steel) were implanted subcutaneously into mice. Macrophages were isolated from the foreign body granulomas that resulted, cultured on bone slices and coverslips, and assessed for both cytochemical and functional evidence of osteoclast differentiation. RESULTS: Tartrate resistant acid phosphatase (TRAP) negative macrophages isolated from granulomas containing particles of all types of biomaterial composition were capable of differentiating into TRAP positive cells capable of extensive lacunar bone resorption (assessed by scanning electron microscopy). The presence of both UMR106 rat osteoblast-like cells and 1,25-dihydroxy vitamin D3 was necessary for this to occur. CONCLUSION: All implant materials produce wear particles that are the focus of a heavy foreign body macrophage response in the fibrous membrane between a loose implant component and the host bone undergoing resorption. These findings underline the importance of biomaterial wear particle generation and the macrophage response to different types of biomaterial wear particles in the pathogenesis of aseptic loosening.

Animals↗
PubMed1995

Conformational studies of osteocalcin in solution.

1H-NMR and circular dichroism studies have been carried out on osteocalcin, a 49-residue, calcium-binding protein, the sequence of which contains a disulphide bridge, a proline-rich segment and three gamma-carboxyglutamic acid (Gla) residues. These latter residues have been proposed to lie on one face of an alpha helix and interact with the mineral phase, leading to incorporation of the protein into the bone matrix. Circular dichroism shows an increase in the alpha-helical structure on Ca2+ binding to bovine osteocalcin. This induced structure is lost on heating the protein, giving a spectrum close to that of the Ca(2+)-free protein. 1H-NMR studies of rabbit osteocalcin gave a set of resonance assignments and NOEs which could be interpreted in terms of distance constraints. These did not allow a single conformation to be defined for the protein in solution but reflect rather a flexible structure which may be essential for the function of the protein. The calculated structures contain a hydrophobic core (comprising Leu2, Leu32, Val36 and Tyr42, seen to be slowly flipping in the Ca(2+)-bound form) and have the gamma-carboxyglutamic acid side chains exposed on one face of the molecule.

Amino Acid Sequence↗
PubMed1995

Human bone tissue formation in diffusion chamber culture in vivo by bone-derived cells and marrow stromal fibroblastic cells.

Direct grafts of human cells into immunocompromised or cortisone-treated animals, either alone or within carrier materials, have been used with some success to assess the developmental capability of the grafted cells. However, identification of the donor or host origins of the generated tissue in such direct grafts is essential. In an alternative and extensively used experimental system, cells are cultured within the isolated environments of diffusion chambers, which are surgically implanted in appropriate hosts. This system allows the direct study of the cellular potentials for differentiation as host tissues are excluded. In the present study, human osteoprogenitor cell populations derived from trabecular bone explants or marrow suspensions of 3 patients (2 females aged 14 years and 1 male aged 27 years) were cultured in the absence or the continuous presence of dexamethasone (10 nmol/L). Cells were impregnated into porous hydroxyapatite ceramics before subcutaneous implantation, or placed within diffusion chambers before intraperitoneal implantation, in athymic mice. All subcutaneous implants of cells in ceramic showed morphological evidence for the formation of bone tissue. In the diffusion chambers it was found that both marrow- and bone-derived fibroblastic cells cultured in the absence of dexamethasone generally produced fibrous tissue only. When cultured in the continuous presence of dexamethasone (10 nmol/L), these cell populations produced similar osteogenic tissues with active osteoblasts, wide osteoid seams, and mineralized tissue, with cartilage toward the interior of the chamber. These results validate the diffusion chamber as an experimental system to study human osteogenesis using appropriately primed cell populations.

Adolescent↗
PubMed1995

In vitro effects of growth factors and dexamethasone on rat marrow stromal cells.

Bone marrow contains multipotential stromal stem cells that can differentiate into fibroblastic, osteogenic, adipocytic, and other cell lines. There is evidence for a considerable degree of plasticity in the differentiation of these different marrow stromal lines. Additional studies have been undertaken investigating the effects of basic fibroblastic growth factor (bFGF), transforming growth factor beta (TGF beta), and dexamethasone on the proliferation and differentiation of rat marrow stromal cells in in vitro cultures. Cell proliferation was stimulated by bFGF and inhibited by dexamethasone and TGF beta. Alkaline phosphatase activity was stimulated by TGF beta and dexamethasone, whereas the expression of the enzyme was inhibited by bFGF. Adipogenesis was induced in cultures containing dexamethasone, but this was depressed by the presence of TGF beta. These observations support the authors' previous hypothesis that there may be an inverse relationship between the differentiation of osteogenic and adipocytic cell lines. The results are relevant to osteoporosis and the aging skeleton, where excess marrow fat is a common feature, and have implications for the pathology of bone and marrow.

Alkaline Phosphatase↗
PubMed1994

A number of osteocalcin antisera recognize epitopes on proteins other than osteocalcin in cultured skin fibroblasts: implications for the identification of cells of the osteoblastic lineage in vitro.

Rabbit antisera to bovine osteocalcin were produced independently in two laboratories and their specificities established by western blot analysis. By immunohistochemistry each of the five polyclonal antisera produced an intense cytoplasmic staining in human bone-derived cells. Staining intensity was strongly attenuated by preabsorption of the antisera with osteocalcin. No staining was observed using nonimmune rabbit serum. However, the choice of skin cells as negative controls for osteocalcin synthesis yielded an unexpected positive staining pattern similar to that seen with the bone-derived cells over a range of antiserum dilutions. This was not caused by the uptake of exogenous osteocalcin from the culture medium because a similar pattern of staining was observed when medium was supplemented with osteocalcin-depleted fetal calf serum. Treatment with 1,25-dihydroxyvitamin D3 induced osteocalcin mRNA expression and osteocalcin secretion in cultures of bone-derived cells but not in skin fibroblasts. The results demonstrate that these polyclonal antisera also recognize epitopes shared with other proteins synthesized in culture by skin fibroblasts. Furthermore, three mouse monoclonal antibodies to distinct regions of the osteocalcin molecule show differential staining of human bone-derived cells, skin cells, and osteosarcoma cells (MG63). These observations indicate that the shared epitope residues in the central region of osteocalcin and are consistent with the specific synthesis of osteocalcin by bone cells alone. The observed nonspecificity of many osteocalcin antisera may compromise immunocytochemical studies of the osteoblast phenotype in studies in vitro when based solely on reactivity with inadequately characterized osteocalcin antisera.

Animals↗
PubMed1994

The effects of dexamethasone and 1,25-dihydroxyvitamin D3 on osteogenic differentiation of human marrow stromal cells in vitro.

Effects of dexamethasone and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] were studied in cultures of adult human marrow stromal cells. In primary culture, dexamethasone (10(-8) M) increased the number of fibroblast colonies formed but decreased their average size. The number of colonies expressing alkaline phosphatase activity was increased, consistent with the enhancement of osteogenic differentiation by this glucocorticoid. In secondary culture, osteogenic differentiation was assessed by measurement of the steady-state levels of particular mRNAs that are characteristic of cells of the osteoblast lineage. The mRNAs for alpha 1(I)-procollagen, alkaline phosphatase, osteopontin and bone sialoprotein were expressed under all culture conditions used. In contrast, osteocalcin mRNA expression was detectable only in cultures treated with 1,25(OH)2D3 (10(-8) M). Addition of 1,25(OH)2D3 to control increased the expression of the mRNAs for alkaline phosphatase and osteopontin but had no significant effect on bone sialoprotein expression. The highest levels of expression of the mRNAs for alkaline phosphatase, bone sialoprotein and osteocalcin were observed in dexamethasone-treated cultures to which 1,25(OH)2D3 had been added. These results demonstrate that, as earlier found in other species, dexamethasone and 1,25(OH)2D3 promote the osteogenic differentiation of human marrow stromal cells as measured by expression of these osteogenic markers.

Adult↗
PubMed1992

Phenotypic characterisation of mononuclear and multinucleated cells of giant cell tumour of bone.

Studies were carried out on 3 giant cell tumours of bone (GCTB) to characterise further the cells forming the distinctive mononuclear and multinucleated components. Samples of tumours were grown as explants in vitro and implanted subcutaneously in athymic mice. Cells were characterised in terms of their cell morphology and cytochemical, antigenic and functional phenotype. In culture, giant cells formed a non-proliferative, relatively homogeneous population of cells which expressed features characteristic of the osteoclast phenotype. The mononuclear cell component was heterogeneous and included macrophage-like cells, which persisted for a short time in culture, and fibroblast-like cells which proliferated. In subcutaneous implants, the fibroblast-like cells formed a tissue which included areas of bone formation associated with regions of alkaline phosphatase activity. These observations are consistent with earlier suggestions that the neoplastic component in GCTB consists of a mononuclear stromal cell which elicits a macrophage/osteoclast response.

Adult↗
PubMed1992

Polymethylmethacrylate-induced inflammatory macrophages resorb bone.

Macrophages and their fused products are commonly found at the polymethylmethacrylate cement-bone interface, but it is not known if they contribute directly to the osteolysis associated with loosening of the cemented prosthesis. We isolated mononuclear phagocytes from granulomas formed by subcutaneous implantation of polymethylmethacrylate into mice and incubated them on bone slices in which they formed resorption lacunae after co-culture for seven to 14 days with both marrow stromal cells and osteoblast-like cells (in the presence of 1 alpha,25-dihydroxyvitamin D3 and dexamethasone). Increased numbers of tartrate-resistant acid phosphatase-positive mononuclear and multinucleated cells formed in these cultures. Both in the presence and absence of stromal cells, macrophages produced extensive superficial roughening of the bone surface. Polymethylmethacrylate-induced macrophages are thus capable of low-grade surface and high-grade lacunar osteolysis, the latter requiring the presence of specific hormonal and stromal cell elements. These two forms of bone resorption could account for the pathogenesis and clinical patterns associated with loosening of the cemented prosthesis.

Animals↗
PubMed1992

Monoclonal antibody BRL 12 reacts with bone keratan sulphate proteoglycan.

Our previous studies suggest that a monoclonal antibody, BRL 12, reacts with a specific product of differentiated cells of the osteoblastic lineage. In the present study, the bone constituent recognized by this antibody has been characterized by gel filtration, ion exchange chromatography, protein blotting and immunolocalization. Our findings show that the antibody reacts with an epitope associated with the core protein of rabbit keratan sulfate proteoglycan (KSPG), a molecule which shares considerable homology with the sialoprotein present in the bone tissue of other mammalian species.

Animals↗
PubMed1991

Adipocytic cells cultured from marrow have osteogenic potential.

Stromal colonies with fibroblastic morphology grown from rabbit marrow cells in culture supplemented with foetal calf serum. In this study the same marrow cells cultured with autologous rabbit plasma and hydrocortisone form colonies of a single lineage that express the adipocytic phenotype. A comparison of the potential for differentiation of cloned cell populations grown from fibroblastic and adipocytic colonies has been made using an in vivo diffusion chamber assay. The adipocytic colonies differentiated and grew to a limited size in medium with rabbit plasma and hydrocortisone, but attempts to isolate them and expand them in this medium failed. When the serum supplement was changed to foetal calf serum at day 10 the cells in the adipocytic colonies acquired a less differentiated morphology, there was a large increase in colony growth and cells were produced in sufficient numbers for the diffusion chamber assay. Thirty one fibroblastic colonies and twenty one adipocytic colonies were isolated either by limiting dilution or ring cloning and then expanded. Of these, eleven fibroblastic and eight adipocytic colonies provided enough cells (2 x 10(5) to 2 x 10(6] for implantation and culture in the chambers. Four of the eleven fibroblastic and three of the eight adipocytic colonies formed an osteogenic tissue in the chambers. It was concluded that cells that have differentiated in an adipocytic direction are able to revert to a more proliferative stage and subsequently to differentiate along the osteogenic pathway. Adipocytic and fibroblastic cells cultured in vitro from marrow have, with osteogenic cells, a common precursor in adult marrow.

Adipose Tissue↗
PubMed1990

Interferons and bone. A comparison of the effects of interferon-alpha and interferon-gamma in cultures of human bone-derived cells and an osteosarcoma cell line.

Recombinant human interferon-alpha 2C and recombinant human interferon-gamma (5-1000 U/ml) inhibit the proliferation of normal human bone-derived cells and a human osteosarcoma cell line. In the bone-derived cells the inhibitory effect of interferon-gamma was significantly greater than that of interferon-alpha, whereas in the osteosarcoma cell line the inhibitory effects of both interferons were quantitatively similar. Interferon-alpha did not affect the alkaline phosphatase activity of either type of cells. In contrast, interferon-gamma affected the activity of the enzyme in both cell types: in the bone-derived cells the effect of interferon-gamma was stimulatory whereas in the osteosarcoma cells the effect was inhibitory. In both cell types interferon-gamma selectively inhibited the incorporation of radiolabelled proline into type I collagen. In the osteosarcoma cells, the effects of both interferons on collagen synthesis were quantitatively similar. In the bone-derived cells, however, interferon-alpha decreased proline incorporation into collagen and non-collagen proteins to a similar extent and thus did not affect collagen synthesis when expressed as a percentage of total protein synthesis. Two-dimensional polyacrylamide gel electrophoresis of the radiolabelled proteins of the cell layer synthesised by both cell types in the presence of either interferon demonstrated that this treatment enhanced or induced the synthesis of a total of 21 individual proteins (19 in bone cells, 14 in osteosarcoma), ranging in apparent molecular mass over 14-87 kDa. The set of proteins induced was different in all four combinations of cells and interferon. A tentative identification of several of the proteins was possible based upon estimation of molecular mass, preferential induction by interferon-alpha or interferon-gamma and differential induction in normal and transformed bone-derived cells. The results of this study demonstrate that interferons have complex effects upon the proliferative and biosynthetic activities of human bone-derived cells and demonstrate significant differences between the responses of normal cells and transformed bone-derived cell line. Further investigations will be required in order to determine whether or not these differences are unique to the osteosarcoma cell line or are a characteristic of the effects of interferons on bone-derived cells in general.

Adolescent↗
PubMed1990

Presence of type III collagen in disc attachments of human temporomandibular joints.

Samples of discs and disc attachments were extracted by dissociative methods and the resultant collagenous residues cleaved with cyanogen bromide. Soluble peptides thus released were characterized by their electrophoretic mobility following SDS-PAGE and by Western blot staining with specific antibodies against type I and type III collagens. Type III collagen was identified in samples taken from the posterior discal attachments. This may explain why this disc is prone to detachment and internal derangement and the high incidence of patients with temporomandibular joint dysfunction.

Adult↗
PubMed1989

The adenylate cyclase response to parathyroid hormone in cultured rabbit marrow fibroblastic cells.

The ability of fibroblastic cells to respond to parathyroid hormone (PTH) by an increase in adenylate cyclase activity is accepted as a characteristic of the osteogenic phenotype. Whether marrow fibroblastic cells, which have osteogenic potential when assayed in vivo, demonstrate this hormonal response when cultured in vitro has been investigated. Our study has shown a level of stimulation of adenylate cyclase activity by PTH in cultured rabbit marrow fibroblasts comparable with other osteogenic cells in vitro. The effect is seen in fibroblasts grown either from multiple colonies or from single colonies. Only a proportion of colonies had osteogenic potential in vivo assay and our results show a similar finding for the PTH response in vitro. To what degree the two parameters are expressed by the same colony has not yet been established.

Adenylyl Cyclases↗
PubMed1989

Interferon effects on osteoinduction.

Xenografts of human osteosarcoma growing in athymic mice are inhibited in growth rate by human interferon-alpha (IFN-alpha) treatment. In addition, differentiation of trabecular bone occurs external to the osteosarcomatous tissue and this is entirely dependent on IFN treatment. We have used species-specific anticollagens and antivimentin antibodies to determine the species origins of this trabecular bone. By using immunohistochemical procedures, it was found that this bone is host-derived. These results suggest that IFN provoked the production of a bone-inducing agent by the human osteosarcomas.

Animals↗
PubMed1989

Immunohistochemical studies using BRL 12, a monoclonal antibody reacting specifically with osteogenic tissues.

A monoclonal antibody of immunoglobulin class G1 has been produced which reacts with a high molecular weight antigen apparently present exclusively in osteogenic tissues. Immunohistochemical studies have shown that the antigen is present throughout the mineralized matrix and in osteoid. None of the other tissues examined namely liver, intestine, kidney, spleen, thymus, heart, lung, skin, cartilage and skeletal muscle showed evidence of specific antibody binding. Immunohistochemical staining was also demonstrated in tissues developing from rabbit marrow cultured in vitro and in diffusion chambers in vivo. Temporal studies of antigen expression in the chambers indicated that the antigen occurs at sites of bone formation after the appearance of alkaline phosphatase but before the formation of a mineralized matrix. The results of these studies suggest that the monoclonal antibody recognises a product of differentiated osteoblasts. This antibody may therefore prove useful in studies of osteogenic differentiation.

Animals↗
PubMed1988

Interferon-inhibited human osteosarcoma xenografts induce host bone in nude mice.

The growth of human osteosarcoma xenografts in nude mice can be inhibited by human interferon-alpha (IFN-alpha). Histologic examination of growth-inhibited tumors has revealed mineralization and partial replacement of the tumor by normal bone tissue. We have investigated whether the normal bone tissue was formed by differentiated tumor cells or by induction of host stroma to differentiate into bone tissue. Employing antibodies to both murine and human type I collagen, it was found that the normal bone produced in IFN-inhibited osteosarcomas was host derived. These results suggest that IFN induced the osteosarcoma cells to produce a bone-inductive agent that interacts with the host cells, and leads to the formation of mature normal bone tissue in a heterotopic site.

Animals↗