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D E Ashhurst

Publications and source records attributed to D E Ashhurst.

At least 37 records · Page 2Linked to original sources
PubMed1995

The expression of collagen mRNAs in normally developing neonatal rabbit long bones and after treatment of neonatal and adult rabbit tibiae with transforming growth factor-beta 2.

Normal transverse growth of long bones is by periosteal appositional bone formation, balanced by endosteal resorption. Changes in the distribution of cells that are expressing collagen mRNAs during growth were determined using digoxigenin-labelled riboprobes. In neonatal rabbit tibiae osteoblasts expressing type I collagen mRNA are found on periosteal, and at early stages on endosteal, bone surfaces and lining peripheral cavities. Occasional osteocytes express type I collagen mRNA very weakly. The pattern is disrupted when transforming growth factor-beta 2 (TGF-beta 2) is injected daily into the periosteum of neonatal animals; there is increased bone, and later cartilage, formation. Three injections of 20 ng TGF-beta 2 onto the tibia of 3-day-old rabbits led to an increase of periosteal osteoblasts that express the mRNA for type I collagen. Some endosteal osteoblasts and osteocytes in newly-formed peripheral woven bone also express the mRNA. After five injections chondrocytes expressing type II collagen mRNA are found around the injection site. Similar injections of TGF-beta 2 in old rabbits induce only fibrous tissue within which some cells express type I collagen mRNA. This precise localization of mRNAs shows that the expression of type I or II collagen mRNA is here restricted to osteoblasts and chondrocytes, respectively.

Animals↗
PubMed1994

The effects of age on the response of rabbit periosteal osteoprogenitor cells to exogenous transforming growth factor-beta 2.

Additional bone and cartilage are formed if transforming growth factor-beta is injected into the periosteum of calvariae or long bones. To investigate this further, transforming growth factor-beta 2 was injected into the periosteum of the tibia of 3-day-old, 3-month-old and 2-year-old rabbits. In all instances, there was an increase in proliferation of the cells of the cambial layer of the periosteum, that is, the osteoprogenitor cells, and breakdown of the fibrous layer. Oedema was induced in the surrounding connective tissues. Over the experimental period the normal neonatal tibia is undergoing rapid growth; there is periosteal bone formation and endosteal resorption. In the experimental neonatal tibiae, an increase in periosteal bone formation is seen after three injections of 20 ng of transforming growth factor-beta 2, which is accompanied by cartilage after five injections; the amounts of induced bone and cartilage increase with the number of injections. The chondrocytes hypertrophy after 4 days and the cartilage is replaced by bone endochondrally. In contrast, after seven injections of 20 ng transforming growth factor-beta 2, there is only a small amount of new bone on the 3-month-old tibia and none on the 2-year-old tibia. One day after seven injections of 200 ng transforming growth factor-beta 2, there is a small amount of bone formation, while seven days after cartilage is found as small discrete nodules on the 3-month-old tibia, but as small areas within the bone on the 2-year-old tibia. It is concluded that the primary effect of transforming growth factor-beta 2 in this experimental model is to increase the proliferative rate of the osteoprogenitor cells in the periosteum. It is argued that transforming growth factor-beta 2 does not initiate osteoblastic or chondrocytic differentiation of osteoprogenitor cells. It is suggested that their differentiation is controlled by the local environment, in particular, the vascularity and locally circulating growth factors.

Age Factors↗
PubMed1991

Digoxigenin as a probe label for in situ hybridization on skeletal tissues.

Non-specific staining was encountered using digoxigenin-labelled cDNA probes for in situ hybridization on sections of skeletal tissues. This staining was most pronounced in cartilaginous matrices. Experimental procedures indicate that the background staining is caused by antibody-binding to hydrophobic sites in the tissues revealed by proteolytic permeabilization. A protocol for minimizing this background is described.

Animals↗
PubMed1991

The transient expression of type II collagen at tissue interfaces during mammalian craniofacial development.

Using immunocytochemical techniques, the spatiotemporal distribution of the major collagen isoform of cartilage, type II collagen, has been investigated during early craniofacial development in the mouse embryo. Early and transient expression was associated with the otic and optic vesicles, the ventrolateral surfaces of the developing brain, olfactory conchi, endocardial and mesocardial tissues, the lateral and basal surfaces of the pharyngeal endoderm and beneath the ectoderm of the branchial arches. A number of these locations are sites of epithelial-mesenchymal tissue interaction believed to generate the component parts of the chondrocranium; here, type II collagen appears transiently in advance of overt chondrogenesis in the mesenchyme. At such sites, immunofluorescence is typically localised along the basal surface of the epithelial partner, with the strongest reaction detected between the basal aspects of the otic and rhombencephalic epithelia. Immunoelectron microscopy, using pre-embedding immunostaining and a protein G-gold technique, reveals that the type II collagen is adjacent to, but not integral with, the basal laminae. Gold particles are clearly associated with 10-15 nm fibrils of the extracellular matrix in the reticulate lamina region. The pattern of type II collagen expression in the mouse closely correlates with that demonstrated previously in the quail, indicating a high degree of phylogenetic conservation between these two vertebrate species. These findings are consistent with the hypothesis that the pattern of epithelial secretion of type II collagen, or a coexpressed matrix molecule, constitutes a morphogenetic signal, realised as a matrix-mediated tissue interaction, and specifying the form of the vertebrate chondrocranium. Three-dimensional reconstruction of early type II collagen distribution, and of the subsequent chondrocranial cartilages, reveals that chondrocranial form can be derived from a 'pre-pattern' of epithelially derived type II collagen expressed at epithelial-mesenchymal tissue interfaces.

Animals↗
PubMed1991

An immunohistochemical study of the collagens of rabbit synovial interstitium.

The interstitial pathway from joint cavity to synovial transport vessels contains a complex extracellular matrix. Rabbit knee synovial intima contains 3 fibrous elements--banded collagen fibrils, microfibrils and broad banded aggregates. The distribution of 4 types of collagen in rabbit knee synovium was investigated immunohistochemically. Types I and III collagens are both present, although the binding of anti-type I antibody was weak. Type V collagen, which forms thin fibrils, or is copolymerized with type I collagen, and type VI collagen, which forms broad banded aggregates and microfibrils, are widely distributed throughout the intimal and subintimal matrices.

Animals↗
PubMed1990

The collagens and glycosaminoglycans of the extracellular matrices secreted by bone marrow stromal cells cultured in vivo in diffusion chambers.

When rabbit bone marrow cells are cultured in a diffusion chamber implanted into the peritoneal cavity of an athymic mouse, the stromal cells proliferate, differentiate, and produce tissues that have the morphological features of loose fibrous tissue, woven or chondroid bone, and cartilage. The collagens synthesized during the development of the tissues from 7 to 28 days after implantation were identified using specific antibodies to rabbit types I, II, III, and V and rat type IX collagens, while the glycosaminoglycans were characterized histochemically using the dye, Alcian blue. Fibrous tissue forms in the first week and it contains types I, III, and V collagens and hyaluronan. Bone and cartilage develop within the fibrous tissue from about 12 days onwards. The bone matrix contains types I and V collagens, and chondroitin and keratan sulphates. The cartilaginous matrix contains types II and IX collagens, and chondroitin and keratan sulphates. Small amounts of type III collagen are found in the bone, and types I, III, and V collagens in the cartilage. These are thought to be the remnants of the fibrous matrix and decrease as the matrices mature. It is concluded that the tissue in diffusion chambers, formed by a small number of early precursor cells present in the soft tissues of the endosteum and marrow of young rabbits, contains extracellular matrix macromolecules similar to those found in bone and cartilage.

Animals↗
PubMed1990

Collagens synthesized by healing fractures.

Several extracellular matrices are formed by healing fractures, and the collagens within these matrices have been identified in rabbit and rat fracture models. Type III collagen is the major collagen of the fibrous matrix that forms along the periosteal surface. Type I collagen is secreted in large amounts as trabeculae of bone develop within the fibrous tissue. Type V collagen is found in both the fibrous tissue and bone; it is particularly associated with blood vessels. Type II collagen is the last of the major collagens to be synthesized; its synthesis is dependent on the mechanical conditions under which the fractures are healing. A large area of cartilage, and hence of Type II collagen, is formed only if the fracture is mechanically unstable. Of the minor collagens, only Types IX and X have been identified to date. Type IX collagen is present throughout the large areas of cartilage, and Type X is present only in calcified regions. The matrices are compared with those produced during embryonic limb development. Although cartilage in the embryo provides a rapidly growing model of the future bone, in healing fractures cartilage is produced only where the cellular environment precludes the differentiation of osteoblasts. The effects of mechanical stability on the matrices support and illustrate this conclusion.

Animals↗
PubMed1987

The effects of mechanical stability on the macromolecules of the connective tissue matrices produced during fracture healing. II. The glycosaminoglycans.

The glycosaminoglycans secreted into the matrices associated with fractures of the rabbit tibia healing under stable and unstable mechanical conditions have been characterized histochemically using the dye Alcian Blue at pH 5.7 in the presence of increasing concentrations of magnesium chloride, and after enzymatic extractions. These results are compared with those of immunohistochemical experiments using monoclonal antibodies which recognize epitopes specific to various glycosaminoglycans. The results indicate that the fibrous tissues, including those of the cavities of the cancellous bone and periosteum, possess hyaluronate and chondroitin sulphate, but the amounts present are small. The glycosaminoglycans detected in the cortical bone are located mainly around the osteocyte lacunae where chondroitin and keratan sulphates are found. The developing trabeculae of cancellous bone in the callus contain chondroitin and keratan sulphates, but as the trabeculae mature, these glycosaminoglycans are no longer present throughout the matrix; they are found particularly around the osteocyte lacunae. The cartilage in the callus of mechanically unstable fractures contains chondroitin, chondroitin-4- and 6-sulphates and keratan sulphate, through their distribution is variable. The small, transient areas of cartilage in the callus of mechanically stable fractures also contain those glycosaminoglycans, but they appear to be less highly sulphated. The mechanical stability of the fractures appears to affect the amount and degree of sulphation of the glycosaminoglycans, rather than the types of glycosaminoglycan produced. The glycosaminoglycans produced during fracture healing are compared with those produced during embryonic development and other healing processes.

Animals↗
PubMed1986

The effects of mechanical stability on the macromolecules of the connective tissue matrices produced during fracture healing. I. The collagens.

The distribution of types I, II, III, V and IX collagens in healing fractures of the rabbit tibia has been demonstrated by immunofluorescent techniques. It has also been shown that the mechanical stability of the healing fracture affects both the distribution and types of the collagens present. The initial fibrous matrix contains types III and V collagens; type I collagen was only located in this matrix if unfixed tissue was used. In mechanically stable fractures, cancellous bone forms over the entire periosteal surface by 5-7 days; type I collagen is laid down within the previous fibrous matrix. The trabeculae are heterogeneous in their collagen content. The cavities contain a matrix of types III and V collagens. Small nodules of cartilage may be present between 7 and 14 days; these contain types II and IX collagens. In mechanically unstable fractures, cancellous bone is initially formed away from the fracture gap. The fibrous tissue over the gap is replaced by cartilage; types II and IX collagens are laid down on the pre-existing fibrous matrix. The cartilage is replaced by endochondral ossification. At the ossification front, type I collagen is found around the chondrocyte lacunae of the spicules of cartilage. The new trabeculae contain a core of cartilage which is surrounded by a bone matrix of types I and V collagens. The fracture gaps are invaded by fibrous tissue, which contain types III and V collagens. this is later replaced by cancellous bone.

Animals↗
PubMed1983

Deposition of scar tissue in the central nervous system.

Standard parasagittal lesions were placed stereotactically in the cerebral hemispheres of neonatal and adult rats in order to compare scarring in the immature and mature animal. Lesions were examined by light and electron-microscopy and immunofluorescence to study the astrocyte reaction, collagen deposition, and the formation of the basement membrane of the glia limitans. Normal mature scarring characterized by the deposition of collagen, astrocyte end-feet alignment over a glia limitans, and the permanent presence of mesodermal cells (fibroblasts and macrophages) in the core of the lesion, does not occur in wounds before 8-10 days post-partum (dpp). Instead there is no deposition of collagen, and only a transitory astrocyte response occurs with the formation of an interrupted glia limitans. These latter features disappear with time so that the wound is ultimately obliterated by the growth of axons and dendrites through the lesion. Mature scarring is attained over 8-12 dpp when increasing amounts of collagen are deposited and a continuous permanent glia limitans is formed. The acquisition of the mature response to injury from 8-12 dpp may be correlated with the presence of increasing titres of a fibroblast growth factor (FGF), derived from autolytic digestion of injured brain tissue. We have investigated FGF activity using a 3 T 3 fibroblast tissue culture assay to detect mitogenic activity in brain extracts from rats lesioned at different ages and from leukodystrophic mice which have no myelin. Our results show that high titres of FGF are present in the developing brain long before myelination commences, and that normal levels of FGF are found in the brains of leukodystrophic mice which have no myelin. Scarring in brain lesions in these mutants is quite normal.

Animals↗
PubMed1982

A method for making reproducible experimental fractures of the rabbit tibia.

A method for making reproducible fractures of the tibia of the rabbit is described. The fractures may be stabilized by plating with either metallic compression, or plastic plates. If a stainless steel dynamic compression plate is used, the rigid immobilization of the fragments leads to direct Haversian remodelling across the fracture, i.e. primary healing, whereas when a plastic plate is used, the fixation is unstable and this leads to secondary healing with the development of a cartilaginous callus. Thus the method of healing may be determined predictably.

Animals↗
PubMed1980

Locust collagen: morphological and biochemical characterization.

Segment-long-spacing crystallites and reconstituted fibrils have been made from collagen extracted from the ejaculatory duct of the adult male locust, Locusta migratoria. These show the same banding pattern after positive staining as segment-long-spacing crystallites and fibrils made from mammalian type I collagen. Native fibrils show the same periodic pattern as type I fibrils, but it is not so distinct. Biochemical analysis of pepsin-digested locust collagen shows that there are two collagenous components. The alpha chains of the major component are similar to mammalian alpha 1 (I) chains, except that the number of hydroxylysine residues is elevated and the CNBr peptides differ. There are no alpha 2 chains; hence this locust collagen molecule is an alpha 1 trimer. The second component, which is present in only minute quantities, may be a type IV basement membrane collagen. It is concluded that the fibrous collagen molecule of the locust is very similar to that of mammalian type I trimers and to those of other invertebrates which have been examined.

Amino Acids↗
PubMed1979

Experimentally induced differentiation of slow tonic and fast twitch muscles in the chick.

The anterior latissimus dorsi (ALD) muscle of chickens is a slow tonic muscle, while the posterior latissimus dorsi (PLD) is a fast twitch muscle. These muscles on opposite sides of a 3-week-old chick were removed, minced and replaced in the site of the other muscle and left to regenerate. The regenerating muscles were examined at various periods from 4 days onwards and their contractile properties were found to resemble those typical of the muscle they replaced and not the original muscle. The regenerating muscles from 8 days onwards displayed the morphological features of the control muscles in the contralateral site. By 14 days, differentiation was almost complete and neuromuscular junctions were seen. It is suggested that the physiological and morphological characteristics of a muscle are determined by its position and possibly also by its innervation.

Animals↗
PubMed1976

The secretion of collagen by insects: uptake of [3H]proline by collagen-synthesizing cells in Locusta migratoria and Galleria mellonella.

The uptake of [H3]proline by collagen-secreting cells of the locust, Locusta migratoria, and wax-moth, Galleria mellonella, has been investigated by electron autoradiography. The locust cells are around the ejaculatory duct and they secrete collagen in the young adult male, while the wax-moth cells are those which produce the dorsal mass of connective tissue on the abdominal nerve cord during the late pupal stage. The cells were exposed to [H3]proline either by injection of the [3H]proline into the insect, or as a pulse while the tissue was maintained in a culture medium. The tissues were fixed at differeing experimental times after exposure to the [3H]proline. The resulting electron autoradiographs were subjected to quantitative analysis, and the silver grain distribution was determined as the relative number of grains per unit area over a series of tissue compartments. When the results of this analysis for the matrix, rough endoplasmic reticulum and Golgi complexes of the two tissues were plotted against experimental time, it was seen that the relative number of grains per unit area over the rough endoplasmic reticulum decreases while that over the matrix increases; statistical analysis has shown that these changes are significant. For the Golgi complexes, however, the theoretical variances are much greater, due to the small relative area occupied by this organelle. There is little evidence for anything other than random sampling fluctuations in the relative numbers of grains per unit area, and hence it is unlikely that the time course of the label over the Golgi complexes follows that over the rough endoplasmic reticulum. The conclusions drawn from these experiments are firstly that a large portion of the labelled protein passes straight from the rough endoplasmic reticulum to the matrix, but that a smaller portion of the labelled material might pass from the rough endoplasmic reticulum to the Golgi complexes and thence to the matrix. It is assumed that collagen comprises most of the protein which passes straight from the rough endoplasmic reticulum to the matrix, and while there is no evidence to exclude collagen from the material passing through the Golgi complexes, it is probable that other proteins and glycosaminoglycans are also present in this labelled material. These conclusions about the intracellular pathway for collagen secretion are similar to those derived from recent studies of some vertebrate fibroblasts. There is, however, conflicting opinion about the intracellular pathway of collagen and it is pointed out that there is diversity in collagen-synthesizing cells, which may account for the differences in the intracellular pathways for collagen secretion which have been proposed.

Animals↗