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J T Triffitt

Publications and source records attributed to J T Triffitt.

At least 37 records · Page 2Linked to original sources
PubMed1999

Expression of estrogen receptor-alpha in cells of the osteoclastic lineage.

Estrogen deficiency at the menopause is associated with an increased rate of bone loss and subsequent risk of skeletal fracture. Whilst cells of the osteoblastic lineage are known to express estrogen receptors, the presence of estrogen receptors in osteoclasts remains controversial. We have examined expression of the classic estrogen receptor, estrogen receptor-alpha (ERalpha), during osteoclast differentiation. In situ mRNA hybridisation with a digoxygenin-labelled riboprobe to ERalpha mRNA, together with immunocytochemical analysis using a human ERalpha-specific monoclonal antibody demonstrated similar findings and confirmed the expression of ERalpha in chondroblasts and osteoblasts from human fetal bone and mineralising human bone marrow cultures. ERalpha expression was detected in human bone marrow cultures treated with 1,25(OH)2D3 and macrophage colony-stimulating factor and in macrophage cultures treated with 1,25(OH)2D3. However, in an in vitro model of human osteoclast formation, no ERalpha expression was observed in the osteoclasts that developed. The human preosteoclast TCG 51 cell line showed strong expression of ERalpha in contrast to the low levels observed in the more mature bone resorptive TCG 23 cell line. No expression was detectable in osteoclasts cultured from giant cell tumour of bone (GCTB) tissue or in osteoclasts in Pagetic, GCTB, or hyperparathyroid bone tissues. In conclusion, preosteoclasts express detectable levels of ERalpha, but osteoclast maturation and bone resorption is associated with loss of ERalpha expression. This indicates that ERalpha expression and regulation may play a role in osteoclast formation.

Bone Diseases↗
PubMed1999

Interconversion potential of cloned human marrow adipocytes in vitro.

Information on the interconversion potential of adipocytes and other end cells characteristic of the stromal fibroblastic cell lineages, key in the understanding of bone turnover in metabolic diseases such as osteoporosis, is limited. The object of the present study was: i) to isolate relatively pure populations of adipocytes from human bone marrow; ii) to clone single adipocytes from these populations; and iii) to examine in vitro the interconversion potential of the progeny of these single-cloned adipocytes between the osteogenic and adipogenic phenotypes. Adipogenic colonies were isolated from the low-density floating fraction of normal bone marrow cells cultured in adipogenic media for 4 days. Single adipocytes were isolated and cloned by limiting dilution. Cloned adipocytes were found to dedifferentiate into fibroblast-like cells, and subsequently to differentiate into two morphologically distinct cell types: osteoblasts and adipocytes in appropriate culture systems. The adipocytic phenotype was confirmed by morphology, oil red O staining, and immunocytochemistry using antiserum to aP2. The osteogenic phenotype was confirmed by alkaline phosphatase, osteocalcin immunostaining using specific osteocalcin antiserum, and formation of mineralized cell aggregates. These findings demonstrate the extent of plasticity between the differentiation of adipocytic and osteogenic cells in human bone marrow stromal cell cultures. We have shown the ability of isolated clonal adipogenic cells to redifferentiate into cells of the osteogenic and adipogenic lineage and the interconversion potential of human marrow stromal cells in vitro. These results provide further evidence that the osteogenic and adipogenic cells share a common multipotential precursor.

Adipocytes↗
PubMed1999

Future potentials for using osteogenic stem cells and biomaterials in orthopedics.

Ideal skeletal reconstruction depends on regeneration of normal tissues that result from initiation of progenitor cell activity. However, knowledge of the origins and phenotypic characteristics of these progenitors and the controlling factors that govern bone formation and remodeling to give a functional skeleton adequate for physiological needs is limited. Practical methods are currently being investigated to amplify in in vitro culture the appropriate autologous cells to aid skeletal healing and reconstruction. Recent advances in the fields of biomaterials, biomimetics, and tissue engineering have focused attention on the potentials for clinical application. Current cell therapy procedures include the use of tissue-cultured skin cells for treatment of burns and ulcers, and in orthopedics, the use of cultured cartilage cells for articular defects. As mimicry of natural tissues is the goal, a fuller understanding of the development, structures, and functions of normal tissues is necessary. Practically all tissues are capable of being repaired by tissue engineering principles. Basic requirements include a scaffold conducive to cell attachment and maintenance of cell function, together with a rich source of progenitor cells. In the latter respect, bone is a special case and there is a vast potential for regeneration from cells with stem cell characteristics. The development of osteoblasts, chondroblasts, adipoblasts, myoblasts, and fibroblasts results from colonies derived from such single cells. They may thus, theoretically, be useful for regeneration of all tissues that this variety of cells comprise: bone, cartilage, fat, muscle, tendons, and ligaments. Also relevant to tissue reconstruction is the field of genetic engineering, which as a principal step in gene therapy would be the introduction of a functional specific human DNA into cells of a patient with a genetic disease that affects mainly a particular tissue or organ. Such a situation is pertinent to osteogenesis imperfecta, for example, where in more severely affected individuals any improvements in long bone quality would be beneficial to the patient. In conclusion, the potentials for using osteogenic stem cells and biomaterials in orthopedics for skeletal healing is immense, and work in this area is likely to expand significantly in the future.

Animals↗
PubMed1998

Modulation of osteogenic differentiation in human skeletal cells in Vitro by 5-azacytidine.

Cellular differentiation is controlled by a variety of factors including gene methylation, which represses particular genes as cell fate is determined. The incorporation of 5-azacytidine (5azaC) into DNA in vitro prevents methylation and thus can alter cellular differentiation pathways. Human bone marrow fibroblasts and MG63 cells treated with 5azaC were used as models of osteogenic progenitors and of a more mature osteoblast phenotype, respectively. The capacity for differentiation of these cells following treatment with glucocorticoids was investigated. 5azaC treatment led to significant expression of the osteoblastic marker alkaline phosphatase in MG63 osteosarcoma cells, which was further augmented by glucocorticoids; however, in human marrow fibroblasts alkaline phosphatase activity was only observed in glucocorticoid-treated cultures. MG63 cells represent a phenotype late in the osteogenic lineage in which demethylation is sufficient to induce alkaline phosphatase activity. Marrow fibroblasts are at an earlier stage of differentiation and require stimulation with glucocorticoids. In contrast, the expression of osteocalcin, an osteoblastic marker, was unaffected by 5azaC treatment, suggesting that regulation of expression of the osteocalcin gene does not involve methylation. These models provide novel approaches to the study of the control of differentiation in the marrow fibroblastic system.

Alkaline Phosphatase↗
PubMed1998

Growth and differentiation of human bone marrow osteoprogenitors on novel calcium phosphate cements.

Materials that augment bone cell proliferation and osteogenic activity have important therapeutic implications for bone regeneration and for use in skeletal reconstruction and joint replacement. We have studied the growth and interactions of human bone marrow cells on a variety of new cement composites in vitro. These cement materials are composed of calcium-deficient hydroxyapatites, carbonated apatite and amorphous calcium phosphate. Cell proliferation was significantly reduced and cell differentiation increased in the presence of these cements compared with cells cultured on tissue culture plastic. Alkaline phosphatase, one of the markers of the osteoblast phenotype, was dramatically stimulated by 3 of the 4 cements examined between day 4 and day 10, above levels observed following culture of human osteoblasts on plastic alone. Photomicroscopic examination demonstrated growth and close integration of bone marrow cells and 3 of the composites. Longer term marrow cultures (15 day) on the cements confirmed the stimulation of cell differentiation over proliferation. From these studies, enhanced osteoblastic differentiation was observed on a 70% carbonated apatite, which has a composition similar to bone mineral, whereas, cell toxicity was observed on cells grown on amorphous calcium phosphate. This in vitro culture system demonstrates the use of human bone marrow cells for the potential evaluation of new biomaterials and the development of a novel carbonated apatite that may be of potential use in orthopaedic implants.

Adult↗
PubMed1998

Skeletal progenitor cells and ageing human populations.

1. Stem and progenitor cells present within bone marrow give rise to colony forming units-fibroblastic (CFU-F) which can differentiate into fibroblastic, osteogenic, myogenic, adipogenic and reticular cells. The decrease in skeletal bone formation and rate of fracture repair observed with ageing and in osteoporosis has been suggested to be due to a decrease in numbers of these progenitors, but human studies are limited. 2. We have tested the potential to form CFU-F in a total of 99 patients undergoing corrective surgery (16 controls, 14-48 years of age) or hip arthroplasty for osteoarthritis (57 patients, 28-87 years of age) or osteoporosis (26 patients, 69-97 years of age). Total colony number, alkaline phosphatase-positive colony number and colony size were determined. 3. No decrease in colony forming efficiency under the culture conditions used was observed in all populations examined irrespective of age, disease or gender, as determined by the lack of correlation between colony formation and age. 3. Examination of colony sizes showed a significant reduction in colony size with age in osteoarthritis and in control populations indicating a change in cellular proliferative potential with age. 4. Examination of number and percentage of alkaline phosphatase-positive CFU-F showed a significant decrease in osteoporotic patients compared with controls and osteoarthritis patients, indicating altered differentiation potential. 5. These results suggest that the reduction in bone mass with ageing may be due to reduction of the proliferative capacity of progenitor cells or their responsiveness to biological factors leading to alteration in subsequent differentiation. The maintenance of CFU-F number and alkaline phosphatase activity in these osteoarthritis patients may, in part, explain the inverse relationship observed for the preservation of bone mass between generalized osteoarthritis and primary osteoporosis.

Adolescent↗
PubMed1998

Patients with primary osteoarthritis show no change with ageing in the number of osteogenic precursors.

The variation in marrow colony forming unit-fibroblastic (CFU-F) number in 59 patients (14-87 years of age) undergoing corrective surgery (14 controls; 14-48 years of age) or hip arthroplasty for primary osteoarthritis (45 OA; 46-87 years of age) was examined to determine whether marrow CFU-F, derived from marrow stromal fibroblastic stem cells, are maintained with the development of primary osteoarthritis (OA). Total colony number, colony size as well as alkaline phosphatase-positive colonies were determined. The mean fibroblast colony forming efficiency from the whole patient group was 2.4 x 10(-5) +/- 1.4 x 10(-5). Ageing had no effect on the colony forming efficiency or on the alkaline-phosphatase-positive colony forming efficiency, irrespective of gender. Thus precursor cells with the potential for osteogenic differentiation are maintained in OA with ageing. However, colony size showed a significant reduction with age, implying altered proliferation potential of osteogenic progenitors with ageing. This ageing effect may not be as significant in OA as in the rest of the population as bone mineral density is often preserved in osteoarthritis. As there is no apparent deficit in primitive progenitor cells, this preservation may be the result of altered regulation of osteoprogenitor activity in OA.

Adolescent↗
PubMed1998

Effects of novel calcium phosphate cements on human bone marrow fibroblastic cells.

The identification and characterization of biocompatible materials that augment bone cell proliferation and osteogenic activity have important therapeutic implications in skeletal reconstruction and joint replacement. In the present study, we have examined the effects of three biocements, biocement H, calcium-deficient apatite; biocement F, apatite + CaHPO(4); biocement D, carbonated apatite + CaHPO(4) + CaCO(3) and an amorphous calcium phosphate (ACP) proposed as implant fixing materials, on the growth, differentiation, and cell surface interaction of human bone marrow fibroblastic cells. These cells are known to be progenitors of osteoblasts, chondroblasts, adipocytes, myoblasts, and reticulocytes. Alkaline phosphatase enzyme activity, a marker of the osteoblast phenotype, was increased by a factor of two- to sixfold on carbonated apatite, one- to sixfold on apatite and three- to 10-fold on calcium-deficient apatite, over levels observed on plastic. Cell proliferation was significantly reduced. Photomicroscopic examination indicated high biocompatibility with close adhesion of the bone marrow fibroblastic cells to composites D, F, and H. Longer term marrow cultures (15 days) confirmed the stimulation of cell differentiation, as assessed by collagen production, over cell proliferation, of cells grown on carbonated apatite. Enhanced osteoblastic differentiation was observed on a 70% carbonated apatite, which has a composition similar to bone mineral, whereas cell toxicity was observed on cells grown on amorphous calcium phosphate. This in vitro human bone marrow fibroblast culture system provides a simple and effective method for the evaluation of new biomaterials. The development of these novel cements may be of potential use in orthopedic implants.

Alkaline Phosphatase↗
PubMed1998

Localization of estrogen receptor-alpha in human and rabbit skeletal tissues.

Estrogen is essential for the development and maintenance of optimal bone mass in women and men, and acts through activation of estrogen receptors (ER). We have examined the pathways of estrogen action on the skeleton by seeking to localize the "classical" estrogen receptor, ER alpha, to particular cells to test the hypotheses that 1) estrogen directly influences growth plate chondrocytes; and 2) estrogen has a principal action on bone tissue via osteoblasts. ER alpha messenger ribonucleic acid (mRNA) was localized by in situ hybridization in human specimens from five males (11-15 yr old), two females (9 and 11 yr old), and three growing rabbits. In all of the human material examined, ER alpha mRNA was consistently identified in chondrocytes. In all of the rabbit tissue studied, ER alpha mRNA was localized in chondrocytes of the growth plate and the subarticular epiphyseal growth center. ER alpha mRNA signals were readily observed in both active osteoblasts and lining cells on trabecular surfaces of all samples. No clear evidence of positive staining was detectable in osteoclasts or osteocytes in either species. The distribution of ER alpha mRNA coincided with immunolocalization of the ER protein in the human specimens. These data suggest a direct action of estrogen on growth plate chondrocytes that may affect longitudinal growth and subsequent fusion of the growth plate and also on osteoblasts to affect bone formation at trabecular sites.

Adolescent↗
PubMed1998

Expression of BMP-4 mRNA during distraction osteogenesis in rabbits.

We characterized the presence and localization of bone morphogenetic protein 4 (BMP-4) mRNA in the regenerating tissues produced by distraction osteogenesis following tibial osteotomy in the rabbit. The findings are consistent with previous reports on the localization of BMP-4 mRNA expression during fracture repair. The BMP-4 gene is expressed by less differentiated osteoprogenitor cells (fibroblastic mesenchymal cells and preosteoblasts), and not by fully differentiated osteoblasts. BMP-4 gene expression is localized in callus-forming tissue (muscle, periosteum) during callus formation. Our observations suggest that the BMP-4 gene product is one of the local contributing factors in regulating bone and cartilage formation in distraction osteogenesis.

Animals↗
PubMed1998

Modulation of bone morphogenetic protein-2 and bone morphogenetic protein-4 gene expression in osteoblastic cell lines.

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-beta (TGF-beta) superfamily and are crucial factors in the process of bone formation. Despite knowledge on their wide distribution and expression, however, there is very little information on the biological factors that affect gene transcription of these osteoinductive agents. To investigate this aspect of BMP gene regulation we have studied the effect of a number of factors known to affect osteogenic cells. Northern analysis showed modulation of the expression of BMP-2 and BMP-4 mRNAs in two human osteosarcoma cell lines, MG63 and Saos-2, by prostaglandin E2 (PGE2), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), interferon-alpha (IFN-alpha), retinoic acid and 1,25(OH)2 vitamin D3. mRNA expressions of the normally used "housekeeping genes", glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin, were found to be susceptible to influence by some of the factors used. Hence, an oligo(dT)15-18 probe was used to reliably estimate the relative quantities of mRNA present for normalization of data. In general, all factors down-regulated mRNA expressions of BMP-2 and BMP-4 in MG63 cells. IL-6 completely abolished detectable expression of BMP-2 mRNA, which was also greatly reduced by IL-1beta, retinoic acid and 1,25(OH)2 vitamin D3. PGE2 had similar influences on BMP-2 and BMP-4 expressions, showing reductions to approximately 60% of normal. In Saos-2 cells only 1,25(OH)2 vitamin D3 had any great effect on BMP-2 expression, which was down-regulated to approximately 60% of control values. BMP-4 was down-regulated by IFN-alpha (approximately 60%) and IL-1beta (approximately 20%). We conclude that BMPs are subject to regulation by a variety of factors and that this is dependent on the stage of the cell in the osteogenic lineage. Furthermore, the use of GAPDH and beta-actin genes as "housekeeping genes" in expression-modulation studies must be treated with care.

Actins↗
PubMed1997

Assessment of cell proliferation in regenerating bone during distraction osteogenesis at different distraction rates.

An experimental model of lengthening of the lower limb was used to study the morphology and cellular proliferation of regenerating bone tissue after 20% lengthening at four rates of distraction. Groups of rabbits were killed at different times 1-8 weeks after surgery. The regenerated area was divided into three zones: fibrous, primary mineralization front, and new bone. As the rate of distraction increased, the size of the fibrous zone increased and that of the new bone zone decreased. Necrosis, formation of cysts, and cartilage were found in the regenerated area at the higher distraction rates. Cell proliferation was assessed by in vivo labelling with bromodeoxyuridine, and the positive staining index for anti-bromodeoxyuridine antibody was calculated in the zones of the regenerated tissue. The index values for the fibrous zones and the new bone zones did not differ significantly in any of the groups. The value increased significantly (p < 0.05) in the primary mineralization front as the rate of distraction increased from 0.3 to 0.7 mm/day, but there was no further significant increase at higher distraction rates. In conclusion, cell proliferation was increased at all of the higher rates (more than 0.3 mm/day) of distraction studied. Higher rates of distraction caused tissue damage. A distraction rate of 0.7 mm/day appeared optimal for cell proliferation and histological characteristics.

Animals↗
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

Identification and enrichment of human osteoprogenitor cells by using differentiation stage-specific monoclonal antibodies.

A major problem in developmental bone biology is the inability to clearly identify early progenitor cells of the osteogenic and related lineages. Identification of these cells is important for the study of their normal development and for determination of potential changes in skeletal diseases. The objective of the present study was to obtain specific markers for early progenitor cells. Monoclonal antibodies were raised against human marrow stromal fibroblastic cell cultures, known to be rich in progenitors for the stromal lineages. Antibodies were selected initially by their reactivity with these marrow cultures and their immunohistochemical localization in human fetal tissues, in progenitor cell regions adjacent to osteoblastic cells. Antibody HOP-26 was strongly reactive with cells in marrow stromal colonies at early stages of differentiation, before the induction of alkaline phosphatase activity, and decreased dramatically after the cells reached confluence. In sections of human fetal limb, binding of HOP-26 was restricted to cells in close proximity to the developing bone, in periosteum, and between the developing bone trabeculae. In adult trabecular bone tissue, HOP-26 was reactive with occasional cells present within the marrow spaces with osteoblasts, adipocytes, and fibrous tissue unreactive. No antibody binding was detected in sections of skin, muscle, appendix, brain, tonsil, or liposarcoma, or cultured SaOS II, MG63, or skin cells. In primary cell suspensions, HOP-26 was unreactive with blood cells but strongly reactive with 0.59 +/- 0.27% of nucleated marrow cells. The antigen associated with these cells was detectable both intracellularly and on the cell surface, and by using immunopanning, HOP-26 selected the marrow stromal fibroblastic colony-forming units (CFU-F). HOP-26 provides the means to identify osteogenic progenitor cells directly and with high specificity. The present studies demonstrate the value of this antibody in providing enriched populations of progenitor cells for experimental studies of osteogenic differentiation and in histopathology.

Alkaline Phosphatase↗
PubMed1997

Interactions of human osteoprogenitors with porous ceramic following diffusion chamber implantation in a xenogeneic host.

Porous calcium phosphate ceramics are useful bone graft substitutes on account of their osteoconductive properties and lack of toxicity, but they lack osteogenicity and are brittle in nature. Osteogenic properties, and increased biomechanical properties, could be induced by combining them with human bone-forming cell populations. Progress has been hampered both by the lack of a suitable experimental assay of in vivo human bone formation and a suitable in vivo test system with which to study such cells in association with biomaterials. Here, trabecular bone-derived cells and marrow stromal fibroblastic cells from four human donors aged between 14 and 27 y have been cultured in vitro then combined with a porous ceramic within diffusion chambers and implanted into athymic mice. Bone and cartilage formation was found within the chambers primed with cells cultured in the continuous presence of dexamethasone and ascorbate. These tissues were found in close apposition to the ceramic, confirming that the material is biocompatible and bioactive. These findings demonstrate both that appropriately primed human-cell populations can express the fully differentiated osteoblastic phenotype in the diffusion-chamber model, and also that this is a useful system in which to test the interactions of such cell populations with putative biomaterials.

Journal Article↗
PubMed1997

The localization of androgen receptors in human bone.

Androgens have important effects on the human skeleton, and deficiency has been associated with bone loss in both males and females. The skeletal actions of androgens may be mediated directly via the androgen receptor (AR) or indirectly via the estrogen receptor after aromatization to estrogens. The presence of androgen receptors has been demonstrated in bone cells and chondrocytes in vitro, but their presence in human bone in situ has not been reported. In order to provide further evidence for a direct action of androgens on bone via androgen receptors, we have used specific monoclonal antibodies to investigate the expression of human AR in normal developing and osteophytic bone of both sexes. In the growth plates from the developing bone, androgen receptors were predominantly expressed in hypertrophic chondrocytes and in osteoblasts at sites of bone formation. They were also observed in osteocytes in the bone, and in mononuclear cells and endothelial cells of blood vessels within the bone marrow. In the osteophytes, androgen receptors were widely distributed at sites of endochondral ossification in proliferating, mature, and hypertrophic chondrocytes and at sites of bone remodeling in osteoblasts. They were also expressed in osteocytes and mononuclear cells within the bone marrow. The pattern and number of cells expressing the receptor was similar in both sexes. Our results show for the first time the presence and distribution of androgen receptors in normal developing human and osteophytic bone in situ and further provide evidence for a direct action of androgens on bone and cartilage cells.

Adenocarcinoma↗
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↗