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Biomedical subjects

D E Ashhurst

Publications and source records attributed to D E Ashhurst.

At least 19 recordsLinked to original sources
PubMed2000

Tissues formed during distraction osteogenesis in the rabbit are determined by the distraction rate: localization of the cells that express the mRNAs and the distribution of types I and II collagens.

An experimental model of leg lengthening was used to study the morphology of, the collagenous proteins present, and the collagen genes expressed in the regenerating tissue following 20% lengthening at four different distraction rates. At a distraction rate of 0.3 mm/day (8 weeks distraction), the regenerate consists of intramembranous bone and localized areas of fibrocartilage. At rates of 0.7 (4 weeks) and 1.3 mm/day (2 weeks), the bone that grows from the cut ends of the cortical bone is separated by fibrous tissue and cartilage is present. At 2.7 mm/day (1 week), only fibrous tissue and sparse bone are present. Type I collagen is present in the matrices around the cells expressing its mRNA and similarly, type II collagen is located around the chondrocytes. Type I collagen mRNA is expressed predominantly by the fibroblasts in the fibrous tissue, the bone surface cells and to a reduced extent by the osteocytes. Type II collagen mRNA is expressed by chondrocytes. The results suggest that osteoblasts and chondrocytes within the regenerate originate from the same pool of progenitor cells, and the differentiation of these cells and the expression of types I and II collagen genes are altered by different rates of distraction. These observations suggest that the optimal rate of distraction in the model is 0.7 mm/day.

Animals↗
PubMed1999

Bone morphogenetic protein-2 increases the rate of callus formation after fracture of the rabbit tibia.

The effects of human recombinant bone morphogenetic protein-2 (rhBMP-2) on rabbit fractures healing under both stable and unstable mechanical conditions were investigated. rhBMP-2 was administered (1) on bioerodible particles, (2) in a collagen gel, and (3) by injection. rhBMP-2 on bioerodible particles has no effect as the particles prevent the migration of cells that produce the callus. The collagen gel is resorbed more rapidly; the development of the callus of mechanically unstable fractures is similar to controls at 14 days. When rhBMP-2 is injected, the callus of mechanically unstable fractures develops more rapidly so that cortical union occurs by 21 days, as compared with 28 days in control fractures. The effects on fractures healing under stable mechanical conditions are minimal. It is argued that mechanical factors influence the size of the callus of normally healing fractures and, although BMP-2 accelerates the rate of development of the callus and cortical union, it does not affect the amounts of bone and cartilage produced.

Animals↗
PubMed1999

The expression of the fibrillar collagen genes during fracture healing: heterogeneity of the matrices and differentiation of the osteoprogenitor cells.

The cells that express the genes for the fibrillar collagens, types I, II, III and V, during callus development in rabbit tibial fractures healing under stable and unstable mechanical conditions were localized. The fibroblast-like cells in the initial fibrous matrix express types I, III and V collagen mRNAs. Osteoblasts, and osteocytes in the newly formed membranous bone under the periosteum, express the mRNAs for types I, III and V collagens, but osteocytes in the mature trabeculae express none of these mRNAs. Cartilage formation starts at 7 days in calluses forming under unstable mechanical conditions. The differentiating chondrocytes express both types I and II collagen mRNAs, but later they cease expression of type I collagen mRNA. Both types I and II collagens were located in the cartilaginous areas. The hypertrophic chondrocytes express neither type I, nor type II, collagen mRNA. Osteocalcin protein was located in the bone and in some cartilaginous regions. At 21 days, irrespective of the mechanical conditions, the callus consists of a layer of bone; only a few osteoblasts lining the cavities now express type I collagen mRNA. We suggest that osteoprogenitor cells in the periosteal tissue can differentiate into either osteoblasts or chondrocytes and that some cells may exhibit an intermediate phenotype between osteoblasts and chondrocytes for a short period. The finding that hypertrophic chondrocytes do not express type I collagen mRNA suggests that they do not transdifferentiate into osteoblasts during endochondral ossification in fracture callus.

Animals↗
PubMed1999

Development and aging of the articular cartilage of the rabbit knee joint: Distribution of biglycan, decorin, and matrilin-1.

We determined the distributions of the small proteoglycans biglycan and decorin and the glycoprotein matrilin-1 (cartilage matrix protein) during development and aging of articular cartilage in the rabbit knee joint. Before cavitation, the matrices of the interzone and the adjacent epiphyseal cartilage do not contain biglycan or decorin, but some chondrocytes express their mRNAs. Matrilin-1 is found only in the deeper epiphyseal cartilage. After cavitation, biglycan and decorin are detected in the presumptive articular cartilage, but there is no matrilin-1. All are present in the underlying epiphyseal cartilage. In the neonate, the epiphyseal cartilage is ossified and the articular cartilage becomes a discrete layer. Biglycan and decorin accumulate in the articular cartilage, but matrilin-1 remains confined to the residual epiphyseal cartilage. In the adult, the distributions of biglycan and decorin are highly variable. Decorin tends to be confined to the central region; matrilin-1 is absent. The findings indicate that the articular and epiphyseal cartilages are different from the earliest developmental stages. The epiphyseal cartilage can be identified by its possession of matrilin-1. Epiphyseal cartilage is removed during development to leave the articular cartilage. The relationships between the distributions of decorin and matrilin-1 and the fibrillar collagens are discussed. (J Histochem Cytochem 47:1603-1615, 1999)

Aging↗
PubMed1998

Effect of depletion of glycosaminoglycans and non-collagenous proteins on interstitial hydraulic permeability in rabbit synovium.

1. The hydraulic resistance of synovial interstitium helps to retain a lubricating fluid within the joint cavity. The contributions of sulphated glycosaminoglycans to resistance were assessed by selective depletion by chondroitinase ABC, keratanase and heparinases I, II and III in vivo. Also, since glycosaminoglycans do not account fully for the resistance, the contribution of non-collagenous, structural proteins in interstitium was assessed by treatment with chymopapain, a collagen-sparing protease. 2. Ringer solution containing enzyme was injected into the synovial cavity of the knee in anaesthetized rabbits. After >= 30 min the intra-articular pressure was raised and the relation between pressure (Pj) and trans-synovial outflow (Qs) determined. The slope dQs/dPj at low pressures, i.e. below yield pressure, represents the hydraulic conductance of the lining, i.e. 1/resistance. The contralateral joint received Ringer solution without enzyme as a control. Action of enzymes on the tissue was confirmed by histochemical and immunohistochemical studies. 3. Treatment with chondroitinase ABC (5 joints) increased the hydraulic conductance of the lining by 2.3 times (control, 1.34 +/- 0.22 microliter l min-1 cmH2O-1; post-enzyme, 3.11 +/- 0.45 microliter l min-1 cmH2O-1). This was significantly less than the effects of leech, Streptomyces and testicular hyaluronidases, which caused an average 4.7 times increase (P < 0.001, ANOVA). Analogous findings were made above yield pressure. 4. Treatment with keratanase (3 joints) or heparinases I, II and III (3 joints) caused no significant increase in trans-synovial flows or conductance, even though the concentration of heparan sulphate in synovium is higher than that of chondroitin sulphates or hyaluronan. 5. Treatment with chymopapain (7 joints) caused the greatest increases in trans-synovial flow, which exceeded control flow by an order of magnitude in one case. After 0.1 U chymopapain the average conductance was 6.6 times the control conductance below yield pressure. Immunohistochemical studies confirmed that chymopapain treatment removed the synovial proteoglycans. 6. It is concluded that, despite their similar resistivities in vitro, the different glycosaminoglycans do not contribute equally, weight for weight, to interstitial resistance in vivo. Hyaluronan is the dominant glycosaminoglycan governing synovial interstitial resistance. In addition, non-collagenous structural proteins contribute significantly to interstitial resistance.

Animals↗
PubMed1998

Effect of depletion of interstitial hyaluronan on hydraulic conductance in rabbit knee synovium.

1. The hydraulic resistance of the synovial lining to fluid outflow from a joint cavity (Qs) is important for the retention of intra-articular lubricant. The resistance has been attributed in part to extracellular glycosaminoglycans, including hyaluronan and chondroitin sulphates. Increased permeability in joints infused with testicular hyaluronidase, which digests both chondroitin sulphates and hyaluronan, supports this view. In this study the importance of interstitial hyaluronan per se was assessed using leech and Streptomyces hyaluronidases, which degrade only hyaluronan. 2. Ringer solution was infused into the knee joint cavity of anaesthetized rabbits for 30 min, with or without hyaluronidase, after which intra-articular pressure (Pj) was raised and the relation between pressure and outflow determined. 3. Treatment with Streptomyces, leech or testicular hyaluronidases increased the fluid escape rates by similar factors, namely 4- to 6-fold. After Streptomyces hyaluronidase treatment the slope d 8d s/dPj, which at low pressures represents synovial hydraulic conductance, increased from a control of 0.90 +/- 0.20 microl min-1 cmH2O-1 (mean +/- s.e.m. , n = 6) to 4.52 +/- 0.70 microl min-1 cmH2O-1. The slope d 8d s/dPj increased to a similar level after testicular hyaluronidase, namely to 4.14 +/- 1.06 microl min-1 cmH2O-1 (control, 0.54 +/- 0.24 microl min-1 cmH2O-1). Streptomyces and leech hyaluronidases were as effective as testicular hyaluronidase (no statistically significant differences) despite differences in substrate specificity. 4. It was shown using histochemical and immunohistochemical techniques that hyaluronan was removed from the synovium by leech, Streptomyces and testicular hyaluronidases. The binding of antibodies 2-B-6 and 3-B-3 showed that the core proteins of the chondroitin sulphate proteoglycans remained intact after treatment with hyaluronidases, and the binding of 5-D-4 showed that keratan sulphate was unaffected. An azocasein digestion assay confirmed that the hyaluronidase preparations had no significant proteolytic activity. 5. The effect of the hyaluronidases was four times greater than predicted from the low concentration of interstitial hyaluronan and its resistivity. Factors that might amplify the effect of hyaluronan depletion include the matrix-organizing role of hyaluronan, and/or non-uniformity of hyaluronan distribution. It is concluded that interstitial hyaluronan makes a major contribution to synovial hydraulic resistance, but the mechanisms are as yet poorly understood.

Animals↗
PubMed1998

Osteogenic growth peptide enhances the rate of fracture healing in rabbits.

The discovery of growth factors, such as osteogenic growth peptide (OGP), that stimulate bone formation led to experiments to discover whether they can accelerate fracture healing. To determine whether OGP enhances the rate of healing in rabbits, fractures were made in the tibiae of New Zealand White rabbits and immobilized with either a plastic plate (unstable mechanical conditions), or a dynamic compression plate (stable mechanical conditions). OGP was administered to experimental animals by intravenous injection from day 4 until the day before sacrifice; control animals were not injected. After treatment with OGP, callus development under unstable mechanical conditions was accelerated. At 7 days, the cartilage in the centre of the callus was covered by bone and endochondral ossification had started; these events occur at 10 days in control fractures. Subsequently, endochondral ossification is completed earlier which allows the invasion of the fracture gap by cells, so that cortical union is complete by 21 to 28 days. In control fractures, bone is only beginning to form in the gaps at 28 days. There was no increase in the size of the callus in any of the experimental fractures compared to the untreated controls. Treatment with OGP has no observable effect on the rate of healing of fractures under stable mechanical conditions. These observations suggest that under unstable mechanical conditions only, the rate of callus formation and subsequent cortical healing is enhanced by treatment with OGP, but that the size of the callus is determined by mechanical and other factors.

Animals↗
PubMed1998

Is acid phosphatase activity present in bone matrix at sites of endochondral ossification in rabbit fracture callus?

It has been suggested that acid phosphatase activity is present in newly formed bone matrix at sites of endochondral ossification in rabbit fracture calluses. Because acid phosphatases are usually found intracellularly, it was decided to test this possibility more rigorously. Tissue from 10- and 14-day healing rabbit fractures was subjected to a series of critical tests for acid phosphatases with a pH optimum of 5.0. Fluoride, tartrate and molybdate were used as potential inhibitors of acid phosphatase activity. The effects of several counterstaining protocols were also investigated. A fluoride- and tartrate-resistant acid phosphatase is located in osteoclasts and mononuclear phagocytes. Diffuse staining of the bone matrix is seen, but it is dependent upon the length of incubation in the substrate medium and the distance from the acid phosphatase-reacting cells. It is concluded that the coloration of the bone matrix is probably caused by diffusion of the dye and reaction product and is, therefore, artifactual.

Acid Phosphatase↗
PubMed1998

The proteoglycans and glycosaminoglycan chains of rabbit synovium.

The synovial lining of joint capsules is important because it controls the flow of fluid into and out of the joint cavity. Physiological studies have shown that the glycosaminoglycans, particularly hyaluronan, have an important role in the control of fluid flow. The distribution of the glycosaminoglycans and proteoglycans in the synovium and subsynovium of rabbits (approximately 12 weeks old) was, therefore, determined immunohistochemically. Hyaluronan, chondroitin-4- and chondroitin-6-sulphates and keratan sulphate are present in the synovium and subsynovium; chondroitin-4-sulphate is at higher concentrations than chondroitin-6-sulphate. The core proteins of the chondroitin sulphate proteoglycans, biglycan and decorin, and of the keratan sulphate proteoglycan, fibromodulin, are also present. To date, fibromodulin has not been located in other synovial linings, and its presence corroborates that of keratan sulphate.

Animals↗
PubMed1997

Fetal and postnatal development of the patella, patellar tendon and suprapatella in the rabbit; changes in the distribution of the fibrillar collagens.

The development of the patella, its associated tendons, and suprapatella of the rabbit knee joint is described from the 17 d fetus to the mature adult. The patellar tendon (ligament) with the patella on its posterior surface is seen in the 17 d fetus and is fully developed by 1 postnatal wk. It is composed of bundles of types I and V collagens separated by endotenons of types III and V collagens. Anteriorly there is an epitenon of types III and V collagens while synovium and a fat pad cover its posterior surface. In the 25 d fetus, the patella is cartilaginous and is separated from the femoral condyles. The cartilage contains type II collagen, but types I, III and V collagens are found along the articular surface. Ossification starts 1 postnatal wk and at 6 wk only the articular cartilage remains. In addition to type II, types III and V collagens are located around the chondrocyte lacunae. The long anterior junction between the patella and its tendon is fibrocartilaginous at 1 wk, but as ossification proceeds this is replaced by bone. Types I and V collagens are found in this region. The suprapatella on the posterior surface of the quadriceps tendon is first seen 1 wk postnatally as an area of irregularly organised fibres and chondrocyte-like cells. Types I, II, III and V collagens are present from 3 wk onwards. It is compared with the fibrocartilage of other tendons that are under compression. The arrangement of the collagens in the patellar tendon is discussed in relation to its use as a replacement for injured anterior cruciate ligaments. It is suggested that the structural differences between the patellar tendon and anterior cruciate ligament preclude the translocated tendon acquiring mechanical strength similar to that of a normal cruciate ligament. The designation 'patellar ligament' as opposed to 'patellar tendon' is questioned. It is argued that the term patellar tendon reflects its structure more accurately than patellar ligament.

Animals↗
PubMed1996

Development and ageing of the articular cartilage of the rabbit knee joint: distribution of the fibrillar collagens.

The development of the articular cartilage of the rabbit knee joint from the 17-day fetus to the 2-year adult rabbit has been examined. At 17 days, the developing femur and tibia are separated by the interzone. Cavitation occurs around 25 days; the cells of the intermediate layer flatten and move onto those of the chondogenous layers to create the articular surfaces. After birth, growth of the cartilage is mainly the result of matrix production. Ossification of the epiphyses is complete by 6 weeks postpartum. Horizontal zones can be distinguished in the articular cartilage; the superficial cells are aligned parallel to the surface, but in the deep layers the cells are in columns. The tidemark is first seen at 12-14 weeks. The matrix of the interzone in the 17-day fetus contains types I, III and V collagens, but no type II. After cavitation at 25 days, the surface layer of the articular cartilage still contains type I, but no type II collagen. From 6 weeks postnatal onwards, type II collagen is present throughout the cartilage and type I disappears. Type III collagen is initially in the inter-territorial matrix, but later it is mainly pericellular. Type V collagen is pericellular both in the chondrogenous layers and later in the articular cartilage, but is not present in the epiphyseal cartilage below. From 6 weeks onwards, types III and V collagens create a capsule around all the chondrocytes above the tidemark. The relationship of types V and XI collagens is discussed. It is concluded that the articular chondrocytes form a unique subset of cells from the earliest stages of joint formation in the fetal rabbit.

Aging↗
PubMed1996

Changes in the distribution of fibrillar collagens in the collateral and cruciate ligaments of the rabbit knee joint during fetal and postnatal development.

The collateral ligaments can be clearly distinguished in the 25-day fetal rabbit knee joint. Type I and V collagens are present in the extracellular matrix between the cells of the lateral and medial collateral ligaments and this distribution persists until the rabbit is skeletally mature. From 8 months onwards type III collagen is also present, particularly around the cells. Type I collagen mRNA is expressed by the cells from the 25-day fetal to 8-month-old adult ligament. The ligament sheath is composed of types III and V collagens. The cruciate ligaments are present between the femur and tibia in the 20-day fetus. The matrix is composed of types I and V collagens from the 25-day fetus until at 12- to 14-weeks postnatal, type III collagen appears in the pericellular regions together with type V. At 8 months and 2 years the amount of type III collagen has increased. All the cells express the mRNA for type I collagen at 12- to 14-weeks, but only isolated cells express this mRNA at 8 months. Thus, both the collateral and cruciate ligaments undergo changes in their complement of collagens during postnatal development and ageing. The implications of these complex interactions of different types of collagen are discussed in relation to healing and the surgical replacement of torn ligaments by tendons.

Animals↗
PubMed1996

Changes in the content of the fibrillar collagens and the expression of their mRNAs in the menisci of the rabbit knee joint during development and ageing.

The menisci are first seen as triangular aggregations of cells in the 20-day rabbit fetus. At 25-days, a matrix that contains types I, III and V collagens has formed. These collagens are also found in the 1-week neonatal meniscus, but by 3 weeks, type II collagen is present in some regions. By 12 to 14 weeks, typically cartilaginous areas with large cells in lacunae are found and by 2 years, these occupy the central regions of the inner two-thirds of the meniscus. The surface layers of the meniscus contain predominantly type I collagen. From 12 to 14 weeks onwards, there is little overlap between the regions with types I or II collagens, that is, these are discrete regions of type I-containing fibrocartilage and type II-containing cartilage. Types III and V collagens are found throughout the menisci, particularly in the pericellular regions. All the cells in the fetal and early neonatal menisci express the mRNA for type I collagen. At 3 weeks postnatal, cells that express type I collagen mRNA are found throughout the meniscus, but type II collagen mRNA is expressed only in the regions of developing cartilage. At 12- to 14-weeks, only type II collagen mRNA is expressed, except at the periphery next to the ligament where a few cells still express type I collagen mRNA. Rabbit menisci, therefore, undergo profound changes in their content and arrangement of collagens during postnatal development.

Aging↗
PubMed1995

The effect of exogenous transforming growth factor-beta 2 on healing fractures in the rabbit.

The effects of exogenous TGF-beta 2 on normally healing fractures were investigated to see if healing can be accelerated; TGF-beta s stimulate bone and cartilage formation on calvariae and long bones. TGF-beta 2 (60 or 600 ng) was injected around the developing callus of rabbit tibial fractures healing under stable, or unstable, mechanical conditions 4 days after fracture. The fractures were examined 5, 7, 10, and 14 days after fracture. A large amount of edema developed around the injection sites. The callus of fractures healing under stable mechanical conditions consists almost entirely of bone. The effects of 60-ng injections of TGF-beta 2 are minimal, but 600-ng doses lead to a small increase in the size of the callus. The callus of fractures healing under unstable mechanical conditions has a large area of cartilage over the fracture site with bone on each side. The effects of TGF-beta 2 on unstable fractures are to retard and reduce bone and cartilage formation in the callus. The overall size of the callus is not affected. In conclusion, TGF-beta 2 does not stimulate fracture healing under either stable, or unstable, mechanical conditions during the initial healing phase. It is argued that agents which stimulate bone formation can retard remodeling when treatment is extended into the remodeling phase of healing.

Animals↗
PubMed1995

Exogenous fibroblast growth factors-1 and -2 do not accelerate fracture healing in the rabbit.

Both fibroblast growth factors-1 (acidic FGF) and -2 (basic FGF) increase the proliferation of osteoblasts and chondrocytes in vitro and FGF-2 stimulates angiogenesis and bone formation in vivo. To test their effects on rabbit tibial fracture-healing under stable and unstable mechanical conditions, 3 micrograms of either FGF-1 or FGF-2 was injected around rabbit tibial fractures on day 4 after fracture. Neither growth factor had a significant effect on either the size of, or the amounts of bone and cartilage in, the 10-day callus irrespective of the mechanical conditions under which the fracture was healing. The 10-day FGF-2-treated calluses were, however, more mature than FGF-1-treated calluses because the cartilage was separated from the periosteum by bone and endochondral ossification had progressed further. In conclusion, the application of FGF-1 or FGF-2 to normally healing fractures of the rabbit tibia does not have a significant effect on the rate of healing.

Animals↗
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↗