Search PubMed⌕ Search

Biomedical subjects

C W Archer

Publications and source records attributed to C W Archer.

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

Organisation of the chondrocyte cytoskeleton and its response to changing mechanical conditions in organ culture.

Articular cartilage undergoes cycles of compressive loading during joint movement, leading to its cyclical deformation and recovery. This loading is essential for chondrocytes to perform their normal function of maintenance of the extracellular matrix. Various lines of evidence suggest the involvement of the cytoskeleton in load sensing and response. The purpose of the present study is to describe the 3-dimensional (3D) architecture of the cytoskeleton of chondrocytes within their extracellular matrix, and to examine cytoskeletal responses to experimentally varied mechanical conditions. Uniformly sized explants of articular cartilage were dissected from adult rat femoral heads. Some were immediately frozen, cryosectioned and labelled for filamentous actin using phalloidin, and for the focal contact component vinculin or for vimentin by indirect immunofluorescence. Sections were examined by confocal microscopy and 3D modelling. Actin occurred in all chondrocytes, appearing as bright foci at the cell surface linked to an irregular network beneath the surface. Cell surface foci colocalised with vinculin, suggesting the presence of focal contacts between the chondrocyte and its pericellular matrix. Vimentin label occurred mainly in cells of the deep zone. It had a complex intracellular distribution, with linked networks of fibres surrounding the nucleus and beneath the plasma membrane. When cartilage explants were placed into organ culture, where in the absence of further treatments cartilage imbibes fluid from the culture medium and swells, cytoskeletal changes were observed. After 1 h in culture the vimentin cytoskeleton was disassembled, leading to diffuse labelling of cells. After a further hour in culture filamentous vimentin label reappeared in deep zone chondrocytes, and then over the next 48 h became more widespread in cells of the explants. Actin distribution was unaffected by culture. Further experiments were performed to test the effects of load on the cytoskeleton. Explants were placed in culture and immediately subjected to static uniaxial radially unconfined compressive loads of 0.5, 1, 2 or 4 MPa for 1 h using a pneumatic loading device. Loads greater than 0.5 MPa maintained the vimentin organisation over the culture period. At 0.5 MPa, the chondrocytes within the explant behaved as in free-swelling culture. The rapid change in vimentin organisation probably relates to rapid swelling of the explants--under free-swelling conditions, these reached their maximum swollen size in just 15 min of culture. The chondrocytes' response to change in tissue dimensions, and thus to their relationship to their immediate environment, was to disassemble their vimentin networks. Loading probably counteracts the swelling pressure of the tissue. Overall, this work suggests that chondrocytes maintain their actin cytoskeleton and modify their vimentin cytoskeleton in response to changing mechanical conditions.

Actins↗
PubMed1999

Mechanisms of GDF-5 action during skeletal development.

Mutations in GDF-5, a member of the TGF-beta superfamily, result in the autosomal recessive syndromes brachypod (bp) in mice and Hunter-Thompson and Grebe-type chondrodysplasias in humans. These syndromes are all characterised by the shortening of the appendicular skeleton and loss or abnormal development of some joints. To investigate how GDF-5 controls skeletogenesis, we overexpressed GDF-5 during chick limb development using the retrovirus, RCASBP. This resulted in up to a 37.5% increase in length of the skeletal elements, which was predominantly due to an increase in the number of chondrocytes. By injecting virus at different stages of development, we show that GDF-5 can increase both the size of the early cartilage condensation and the later developing skeletal element. Using in vitro micromass cultures as a model system to study the early steps of chondrogenesis, we show that GDF-5 increases chondrogenesis in a dose-dependent manner. We did not detect changes in proliferation. However, cell suspension cultures showed that GDF-5 might act at these stages by increasing cell adhesion, a critical determinant of early chondrogenesis. In contrast, pulse labelling experiments of GDF-5-infected limbs showed that at later stages of skeletal development GDF-5 can increase proliferation of chondrocytes. Thus, here we show two mechanisms of how GDF-5 may control different stages of skeletogenesis. Finally, our data show that levels of GDF-5 expression/activity are important in controlling the size of skeletal elements and provides a possible explanation for the variation in the severity of skeletal defects resulting from mutations in GDF-5.

Amino Acid Sequence↗
PubMed1998

P-glycoprotein and metallothionein expression and resistance to chemotherapy in osteosarcoma.

The expression of the drug resistance (DR) mediators P-glycoprotein (P-gp) and the metallothioneins (MT) was assessed immunohistochemically in biopsy material from patients with high-grade malignant osteosarcoma (OS). No significant difference was found in survival rate between expressors of both P-gp and MT and non-expressors. Thus, it was concluded that lack of expression of these two drug resistance-related proteins does not appear to confer any advantage in terms of patient survival in osteosarcoma.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Overexpression of BMP-2 and BMP-4 alters the size and shape of developing skeletal elements in the chick limb.

Bone morphogenetic proteins are members of the transforming growth factor beta (TGF beta) superfamily which are involved in a range of developmental processes including modelling of the skeleton. We show here that Bmp-2 is expressed in mesenchyme surrounding early cartilage condensations in the developing chick limb, and that Bmp-4 is expressed in the perichondrium of developing cartilage elements. To investigate their roles during cartilage development, BMP-2 and BMP-4 were expressed ectopically in developing chick limbs using retroviral vectors. Over-expression of BMP-2 or BMP-4 led to a dramatic increase in the volume of cartilage elements, altered their shapes and led to joint fusions. This increase in volume appeared to result from an increase in the amount of matrix and in the number of chondrocytes. The latter did not appear to be due to increased proliferation of chondrocytes, suggesting that it may result from increased recruitment of precursors. BMP-2 and BMP-4 also delayed hypertrophy of chondrocytes and formation of the osteogenic periosteum. These data provide insights into how BMP-2 and BMP-4 may model and control the growth of skeletal elements during normal embryonic development, suggesting roles for both molecules in recruiting non-chondrogenic precursors to chondrogenic fate.

Animals↗
PubMed1996

High resolution magnetic resonance imaging of the proximal interphalangeal joints. Correlation with histology and production of a three-dimensional data set.

The magnetic resonance imaging appearance of the proximal interphalangeal joints of cadavers was correlated with histology of the same specimen allowing many small-scale features to be identified that might otherwise have been misinterpreted. It enabled the magnetic resonance signal to be understood at a tissue and cellular level, allowing identification of synovial folds extending from the extensor tendon and volar plate, the entheses of the proper collateral ligament, the epitenon of the flexor tendons and the presence of osteophytes and sites of cartilage erosion. The main difficulties in matching two-dimensional magnetic resonance images with histology were the differing section thicknesses of the two methods and shrinkage of histological specimens. There are many advantages in producing high resolution three-dimensional datasets-the magnetic resonance section thickness is reduced and the individual components of the joint can be viewed simultaneously in two or more planes. A unique magnetic resonance atlas of three dimensional joint structure is presented.

Aged↗
PubMed1996

The development of articular cartilage: I. The spatial and temporal patterns of collagen types.

Articular cartilage is both morphologically and biochemically heterogeneous. Its susceptibility to degenerative diseases such as arthritis and its limited repair capacity have made cartilage the focus of intense study; surprisingly, little is known of its development. Using a panel of specific antibodies, we have documented the temporal and spatial patterns of collagen types I, II, III, VI and X in the developing knee cartilage of the marsupial Monodelphis domestica from parturition to adulthood. Type I collagen was initially detected in the presumptive articular cartilage of the epiphyses in addition to the perichondrium. By 14 d postparturition, type I collagen was not detectable in the epiphyseal cartilage apart from insertion sites of ligaments and tendons of the joint. Similarly, type III collagen was detected at insertion sites of the major ligaments and tendons and within the perichondrium/periosteum but was never detected in the cartilage per se. Type II collagen was predictably distributed throughout the cartilage matrix and was also detected in the perichondrium. Type VI collagen was widely distributed throughout the cartilage matrix at parturition, but during development became restricted to a pericellular location particularly towards the presumptive articular cartilage, i.e. the epiphysis. Interestingly, generalised matrix immunopositivity was only retained in the hypertrophic cartilage of the secondary centre of ossification. After the formation of the secondary centre, type VI collagen became localised pericellularly in the deeper regions of the articular cartilage but was absent in the cartilage of the growth plate. Type X collagen showed a novel distribution pattern. In addition to being synthesised by hypertrophic chondrocytes, this collagen type was also expressed transiently by some cells at the presumptive articular surface. Furthermore, these surface chondrocytes also stained histochemically for alkaline phosphatase, suggesting that they were terminally differentiated. The fate of these terminally differentiated cells is unknown.

Alkaline Phosphatase↗
PubMed1996

The development of articular cartilage: II. The spatial and temporal patterns of glycosaminoglycans and small leucine-rich proteoglycans.

Articular cartilage is both morphologically and biochemically heterogeneous. Its susceptibility to degenerative diseases such as arthritis and its limited repair capacity has made cartilage the focus of intense study; surprisingly, little is known of its development. Using a panel of specific antibodies, we have documented the temporal and spatial patterns of the small leucine-rich proteoglycans fibomodulin, decorin and biglycan in the developing knee cartilage of the marsupial South American opposum (Monodelphis domestica) from parturition to adulthood. The major proteoglycan of cartilage, aggrecan, can be substituted with a variety of isomers of chondroitin sulphate (CS) and keratan sulphate (KS) glycosaminoglycans. Consequently, we have used monoclonal antibodies to determine the distribution of the chondroitinase generated epitopes of CS isomers (delta di-6S and delta di-4S oligosaccharide 'stubs'). Other monoclonal antibodies (3B3[-], 7D4) were used to investigate temporal changes in the expression of specific sulphation patterns within native chondroitin sulphate chains in addition to keratan sulphate chains (5D4). We found the distributions of the small proteoglycans (PGs) to be highly dynamic during development. Both fibromodulin and biglycan appeared to specifically label early articular cartilage as opposed to epiphyseal or growth plate cartilage. All 3 small PGs become preferentially distributed to the upper half of the adult articular cartilage depth. Similarly, delta di-6S, delta di-4S oligosaccharide 'stubs', KS and epitope 7D4 were variably distributed during development but all were again preferentially located to the upper depth of the mature tissue. The epitope recognised by antibody 3B3[-] was extensively distributed in the neonate, but became more restricted to hypertrophic chondrocytes by day 19. It was not detected in the adult tissue. These data suggest that in Monodelphis, proteoglycans are preferentially synthesised and elaborated in the upper half of the tissue depth and contrasts with the patterns observed in eutherian mammals. The data also pose questions as to the functional significance of these molecules within the tissues and to the idea that global patterns of matrix components exist in mammalian articular cartilages.

Animals↗
PubMed1995

Attachment and proliferation of osteoblasts and fibroblasts on biomaterials for orthopaedic use.

Using a variety of cell types, cell attachment and growth was studied on prospective (polyethersulphone (PES) and polyetheretherketone) and currently used (titanium 318 alloy, cobalt chrome molybdenum alloy and ultra-high molecular weight polyethylene (UHMWPE)) orthopaedic biomaterials. Proliferation of fibroblasts and osteoblasts was measured using incorporation of tritiated thymidine into total DNA. Attachment of cells was assessed by indirect immunofluorescent labelling of vinculin, a component of the cell's focal adhesion plaque. The degree of cell attachment was quantified on the materials by determining the mean number of adhesion plaques and using an image analysis system to determine the mean total area of plaques per cell. Fibroblasts and osteoblasts responded differently to the materials tested. When grown on PES surfaces, rat tail fibroblasts synthesized significantly greater amounts of DNA than cells on all other surfaces, whilst fibroblasts on UHMWPE synthesized significantly less DNA than cells on all other materials. Interestingly, there was no significant difference between the amounts of DNA synthesized by osteoblasts grown on the various materials. Determination of the number of vinculin adhesion plaques per cell and the mean total area of the plaques per cell showed that the attachment of fibroblasts to UHMWPE was significantly reduced compared with other materials. In contrast there was no significant difference in the adhesion of osteoblasts to different materials. Scanning electron microscope (SEM) observations of cells on the materials correlated with the morphometric data. Cells with the greatest number and area of adhesion plaques were well spread and flattened whilst those with the least number of adhesion plaques were more rounded and less spread.

Actins↗
PubMed1995

Alterations in hyaluronan synthesis during developing joint cavitation.

The mechanisms essential for generating diarthrodial joint cavities between skeletal elements in developing limbs remain enigmatic. Histochemical localization of hyaluronan (HA) at joint interzones concomitant with cavitation led to the postulation that HA may be pivotal in this process. HA synthesis involves the transfer of UDP-glucuronate and UDP-N-acetyl glucosamine to nascent HA by HA synthase. Uridine diphosphoglucose dehydrogenase (UDPGD) activity is responsible for the prior conversion of UDP-glucose to UDP-glucuronate. We have assessed the relationship between the appearance of HA and enzyme activities (quantitatively where possible) involved in HA synthesis during metatarsophalangeal joint development in embryonic chicks. Microspectrophotometric assessment of UDPGD activity using an in situ biochemical assay indicated that cells immediately adjacent to forming cavities contained increased UDPGD activity, which was subsequently maintained after cavitation. Immunocytochemistry showed that high levels of expression of HA synthase were localized to these same cells. In addition, radiolabeled sulfate autoradiography showed that cells bordering developing cavities incorporated relatively little sulfate, suggesting that UDP-glucuronate is utilized in the synthesis of undersulfated or non-sulfated glycosaminoglycans. These results indicate that the differentiation of cells bordering presumptive spaces may involve alterations associated specifically with differential synthesis of HA, which appears to be a primary event in joint cavity formation.

Animals↗
PubMed1994

Cytoskeleton of cartilage cells.

The cytoskeleton of chondrocytes consists of microfilaments made of actin, microtubules made of tubulin, and intermediate filaments made of a variety of subunits. Actin filaments are not prominent in vivo but may form in vitro. In culture, changes in filament polymerisation are important in determining cell shape, initiating chondrogenesis, and maintaining the chondrogenic phenotype. Microtubules, besides their role in cell division, organise the distribution of organelles and are involved in secretory transport mechanisms in collagen and proteoglycan synthesis. A variety of intermediate filaments may be present, frequently forming large whorled aggregates. The filaments include vimentin, cytokeratins, and glial fibrillary acidic protein. These may occur at different depths in articular cartilage. Vimentin accumulates during development of some fibrocartilages with increased mechanical loading. Together with other elements of the cytoskeleton, intermediate filaments could form part of a mechanotransduction system by which cells respond to external forces and sense changes in their external environment.

Actin Cytoskeleton↗
PubMed1994

Proteoglycan depletion alone is not sufficient to stimulate proteoglycan synthesis in cultured bovine cartilage explants.

Articular cartilage comprises a small number of cells embedded within a matrix primarily composed of collagen and proteoglycan (PG). The functional integrity of the tissue is highly dependent on the maintenance of matrix structure, which, in turn, is controlled by the chondrocytes. In normal tissue there is a slow but steady turnover of matrix components such that their levels remain constant. In certain diseased states the equilibrium is upset, resulting in a net loss of matrix components. The object of the present study was to artificially upset the synthetic/loss equilibrium by enzymic depletion and assess the ability of chondrocytes to respond by increasing PG synthesis. Cultured bovine articular cartilage explants were depleted using enzymes as follows; 10 U/ml Streptomyces hyaluronidase (induced a 30% loss of PG), 2000 U/ml testicular hyaluronidase (70% loss of PG) and 100 U/ml collagenase (35% loss of PG) and control (6% loss of PG). Collagenase also induced a 50% loss of collagen. Collagenase treatment induced a 50% stimulation of PG synthesis above control levels. Elevated synthesis levels were maintained for 9 days. Testicular hyaluronidase induced a brief elevation in PG synthesis on day 3 of culture. Streptomyces hyaluronidase treatment did not cause an alteration in the rate of PG synthesis above control levels. Histological examination indicated that collagenase-treated explants formed outgrowths consisting of rounded chondrocytes within a fine fibrous matrix which stained intensely with safranin-O, indicating a high concentration of PG. The production of repair-like outgrowths may explain the elevated PG synthesis rates measured. It appears, therefore, that collagen and matrix organization is more important than PG levels in the control of PG synthesis in articular cartilage explant cultures.

Animals↗
PubMed1994

Cellular aspects of the development of diarthrodial joints and articular cartilage.

There have been many reports on the histological development of mammalian diarthrodial or synovial joints. While these are useful for comparative purposes, they tell us little of the cellular basis of joint morphogenesis which must underlie a number of morphogenetic defects. The process of joint morphogenesis is complex and can be subdivided into a number of facets and this report will focus on 2 of them. First, the process of joint cavitation in the chick metatarsophalangeal joint, where we propose that the selective secretion of hyaluronan into the presumptive cavity plays a central role. Secondly, the development of articular cartilage where we have used the South American opossum Monodelphis domestica as a model for mammalian development. Like most marsupials, the young are born at a much earlier developmental stage than eutherian mammals. Using antibodies which detect proliferating chondrocytes and those synthesising insulin-like growth factors 1 and 2 and insulin-like growth factor 1 binding protein, we report that the majority of growth (as assessed by these indicators) appears appositional.

Animals↗
PubMed1993

Molecular heterogeneity of chondroitin sulphate in the early developing chick wing bud.

Proteoglycans are ubiquitous extracellular matrix molecules whose role in development remains poorly understood. In the developing chick limb, the nature and possible roles of a number of extracellular matrix proteins is well documented. Much less is known of the biochemical nature, and more importantly, the roles of proteoglycans. Using a panel of monoclonal antibodies (Mabs) which recognise specific epitopes on the constituent chondroitin/dermatan sulphate chains, we show that distinct sub-populations of proteoglycans are dynamically expressed within the limb ectoderm, the ectodermal basement membrane and the limb mesenchyme. In particular, prior to chondrogenesis, chondroitin-6-sulphate-rich proteoglycans containing over-sulphated domains residue predominantly within the mesenchymal extracellular matrix ECM, whilst chondroitin-4-sulphate (C-4-S) is associated with the ectodermal basement membrane and subjacent mesenchymal ECM. At stage 24, C-4-S is also localized in the prechondrogenic condensation. Concomitantly with overt chondrogenesis, the epitopes recognized by the Mabs become restricted to the chondrifying skeletal elements and the undifferentiated distal mesenchyme. The significance of these findings has yet to be elucidated.

Animals↗
PubMed1993

The control of cell division in articular chondrocytes.

Our objective was to investigate the factors that control chondrocyte division, namely, (1) steric hindrance caused by the surrounding matrix, (2) growth factors and (3) cell shape. Bovine articular cartilage explants and isolated chondrocytes were cultured in medium containing [3H]-TdR to measure DNA synthesis. The effect of enzyme treatment, growth factors and cell shape was investigated. Proteoglycan (PG) depletion did not stimulate DNA synthesis whilst disruption of the collagen architecture promoted incorporation of [3H]-TdR into DNA. DNA synthesis was also stimulated by IGF-1 in flattened chondrocytes but not in rounded cells. It was found that the control of chondrocyte division depends on several variably related factors that may act singly or together to induce cell division and that PG depletion alone is insufficient to induce DNA synthesis, but division was noted when PG depletion was accompanied by matrix disruption and cell shape changes. Cell shape appears to alter the chondrocytes ability to respond to IGF-1 as a mitogen.

Animals↗
PubMed1993

Novel articular cartilage structure in the South American opossum, Monodelphis domestica.

Articular cartilage provides smooth surfaces for low-friction, unrestricted movement of opposing skeletal elements. The surface topography of articular cartilage has been the subject of numerous studies and, with few exceptions, is considered to be smooth (at least at the light microscopic level). Some studies have reported 'humps' on the articular surface which have been related to underlying chondrocytes residing very close to the surface. Here we report on a highly nodular form of articular cartilage in the distal limb joints of the South American opossum, Monodelphis domestica. Unlike previous reports, these articular 'humps' are visible under a dissecting microscope. Each 'hump' or 'nodule' represents the surrounding matrix of single or sometimes paired rounded chondrocytes. Flattened chondrocytes normally associated with mammalian articular cartilage were absent from these joints. Interestingly, the articular cartilage of the more proximal limb joints such as the knee showed more typical features of articular cartilage including flattened superficial chondrocytes.

Animals↗