Evaluation of cartilage, synovium and adipose tissue as cellular sources for osteochondral repair.

Osteochondral lesions are a major cause of pain and disability in several species including dogs, horses and human beings. The objective of this study was to assess three potential sources of canine cells for their osteochondral regenerative potential. Cartilage, synovium and adipose tissue cells were grown in pellet culture in chondrogenic or osteogenic media.

Variations in cell morphology in the canine cruciate ligament complex.

Cell morphology may reflect the mechanical environment of tissues and influence tissue physiology and response to injury. Normal cruciate ligaments (CLs) from disease-free stifle joints were harvested from dog breeds with a high (Labrador retriever) and low (Greyhound) risk of cranial cruciate ligament (CCL) rupture. Antibodies against the cytoskeletal components vimentin and alpha tubulin were used to analyse cell morphology; nuclei were stained with 4',6-diamidino-2-phenylindole, and images were collected using conventional and confocal microscopy.

Type IX collagen interacts with fibronectin providing an important molecular bridge in articular cartilage.

Type IX collagen is covalently bound to the surface of type II collagen fibrils within the cartilage extracellular matrix. The N-terminal, globular noncollagenous domain (NC4) of the α1(IX) chain protrudes away from the surface of the fibrils into the surrounding matrix and is available for molecular interactions. To define these interactions, we used the NC4 domain in a yeast two-hybrid screen of a human chondrocyte cDNA library.

The organisation of elastin and fibrillins 1 and 2 in the cruciate ligament complex.

Although elastin fibres and oxytalan fibres (bundles of microfibrils) have important mechanical, biochemical and cell regulatory functions, neither their distribution nor their function in cruciate ligaments has been investigated. Twelve pairs of cruciate ligaments (CLs) were obtained from 10 adult dogs with no evidence of knee osteoarthritis. Elastic fibres were identified using Verhoeff's and Miller's staining.

Biomarkers of cartilage turnover. Part 1: Markers of collagen degradation and synthesis.

Type II collagen is a major component of articular cartilage and its breakdown is a key feature of osteoarthritis. Products of cartilage collagen metabolism can be detected in the blood, synovial fluid and urine. Several biomarker assays have been developed which can be used to measure the synthesis and degradation of collagen, and therefore provide information regarding cartilage turnover.

Biomarkers of cartilage turnover. Part 2: Non-collagenous markers.

Osteoarthritis (OA) results in the destruction and breakdown of articular cartilage matrix. Breakdown of the cartilage proteoglycan component results in the generation of constituent fragments that can be detected in the blood, synovial fluid or urine. Non-collagenous, non-proteoglycan components of cartilage can also be detected following their release as a result of turnover and disease.

Development of an in vitro model of feline cartilage degradation.

Osteoarthritis is the most common arthropathy of mammalian species including cats. Cartilage degradation is central to the disorder and here we present, for the first time, an in vitro model of feline cartilage degradation which will be useful for further studies in this target species. Feline articular cartilage explant cultures were maintained for 28 days and in the presence of oncostatin M with and without interleukin (IL)-17, tumour necrosis factor (TNF), IL-1alpha, or IL-1beta.

MMP-mediated collagen breakdown induced by activated protein C in equine cartilage is reduced by corticosteroids.

The plasma serine protease activated protein C (APC) is synthesized by human chondrocytes at sites of pathological cartilage fibrillation. APC levels are increased in osteoarthritis (OA) synovial fluid, and in vitro APC has been shown to synergize with interleukin-1beta (IL-1) to promote degradation from ovine cartilage. A model of equine cartilage degradation was established and used to explore corticosteroid activities.

An exploration of the ability of tepoxalin to ameliorate the degradation of articular cartilage in a canine in vitro model.

Background: To study the ability of tepoxalin, a dual inhibitor of cyclooxygenase (COX) and lipoxygenase (LOX) and its active metabolite to reduce the catabolic response of cartilage to cytokine stimulation in an in vitro model of canine osteoarthritis (OA).Grossly normal cartilage was collected post-mortem from seven dogs that had no evidence of joint disease. Cartilage explants were cultured in media containing the recombinant canine interleukin-1beta (IL-1beta) at 100 ng/ml and recombinant human oncostatin-M (OSM) at 50 ng/ml.

Gene expression markers of tendon fibroblasts in normal and diseased tissue compared to monolayer and three dimensional culture systems.

Background: There is a paucity of data regarding molecular markers that identify the phenotype of the tendon cell. This study aims to quantify gene expression markers that distinguish between tendon fibroblasts and other mesenchymal cells which may be used to investigate tenogenesis.

Methods: Expression levels for 12 genes representative of musculoskeletal tissues, including the proposed tendon progenitor marker scleraxis, relative to validated reference genes, were evaluated in matched samples of equine tendon (harvested from the superficial digital flexor tendon), cartilage and bone using quantitative PCR (qPCR).

Modification of the composition of articular cartilage collagen fibrils with increasing age.

Recent studies have identified a range of interactions between type IX collagen and other cartilage matrix components. To determine the extent to which these interactions are important in maintaining the integrity of ageing articular cartilage, we analyzed an age range of normal healthy articular cartilage samples by Western blotting, immunohistochemical, and PCR analyses. Reduced levels of type IX collagen were detected in post adolescence cartilage.

Effects of hypoxia on glucose transport in primary equine chondrocytes in vitro and evidence of reduced GLUT1 gene expression in pathologic cartilage in vivo.

Articular chondrocytes exist in an environment lacking in oxygen and nutrients due to the avascular nature of cartilage. The main source of metabolic energy is glucose, which is taken up by glucose transporters (GLUTs). In diseased joints, oxygen tensions and glucose availability alter as a result of inflammation and changes in vascularisation.

High-intensity exercise induces structural, compositional and metabolic changes in cuboidal bones--findings from an equine athlete model.

Fatigue fracture of cuboidal bones occurs in the human foot as well as the equine carpus. The racehorse provides a naturally-occurring model to study the effects of high-intensity exercise on the morphology and metabolism of cuboidal bones. We studied both the mineral and the collagenous matrix of the third (C(3)) and radial (C(r)) carpal bones of raced and non-raced Thoroughbred (TB) horses.

MT3-MMP (MMP-16) is downregulated by in vitro cytokine stimulation of cartilage, but unaltered in naturally occurring equine osteoarthritis and osteochondrosis.

Matrix degradation by metalloproteinases is considered a key feature in the loss of articular cartilage seen in many joint diseases. Membrane-type matrix metalloproteinase-3 (MT3-MMP) expression is elevated in human cartilage in end-stage osteoarthritis. We investigated whether MT3-MMP is similarly regulated in cartilage in two naturally occurring arthropathies in vivo and whether proinflammatory cytokines regulate its expression in vitro.

Chondrocytes harvested from osteochondritis dissecans cartilage are able to undergo limited in vitro chondrogenesis despite having perturbations of cell phenotype in vivo.

Our objective was to characterize the variation in gene expression for key genes associated with chondrogenic phenotype of osteochondrosis (OC)-affected and normal chondrocytes, and to identify whether OC chondrocytes can redifferentiate and regain a phenotype similar to normal chondrocytes if appropriate chondrogenic signals are given. Equine articular cartilage removed at surgery to treat clinically significant OC lesions was collected (n = 10), and the gene expression evaluated and compared to aged-matched normal samples (n = 10). Cartilage was harvested from normal (n = 4) and OC (n = 3) joints from horses at necropsy.

Use of biochemical markers of osteoarthritis to investigate the potential disease-modifying effect of tibial plateau levelling osteotomy.

Objectives: To evaluate the hypothesis that the concentration of the 1/20/5D4 epitope of keratan sulphate, cartilage oligomeric matrix protein and total sulphated glycosaminoglycans in synovial fluids from dogs with cranial cruciate ligament disease would be affected by tibial plateau levelling osteotomy. In addition, to evaluate the hypothesis that medial meniscal release or meniscal injury would alter the expression of these candidate biomarkers.

Methods: Forty-one dogs with naturally occurring cranial cruciate ligament disease were recruited prospectively.

Tracheal organ cultures as a useful tool to study Felid herpesvirus 1 infection in respiratory epithelium.

Felid herpesvirus 1 (FeHV-1) is an important feline pathogen of the upper respiratory tract which can be identified in clinical cases by virus isolation and PCR. Studies on the effect and mode of spread of FeHV-1 in the respiratory epithelium, however, have previously only been performed in infected cats. Feline tracheal organ cultures which were inoculated with FeHV-1 at varying multiplicity of infection (MOI) were established.

Assessment of the use of RNA quality metrics for the screening of articular cartilage specimens from clinically normal dogs and dogs with osteoarthritis.

Objective: To assess 2 methods of RNA purification by use of different quality metrics and identify the most useful metric for quality assessment of RNA extracted from articular cartilage from dogs with osteoarthritis.

Sample Population: 40 articular cartilage specimens from the femoral heads of 3 clinically normal dogs and 37 dogs with osteoarthritis.

Procedures: RNA was extracted from articular cartilage by 2 purification methods.

The differentiation of bone marrow mesenchymal stem cells into chondrocyte-like cells on poly-L-lactic acid (PLLA) scaffolds.

While intervertebral disc (IVD) degeneration is associated with the majority of cases of low back pain, current treatments are symptomatic rather than curative. Tissue engineering offers a treatment that both cures the problem of disc degeneration and restores normal disc function. One of the major problems for any tissue engineering strategy, however, is ensuring that both the cells and matrices used are suitable for the target tissue.

Nutraceutical therapies for degenerative joint diseases: a critical review.

There is growing recognition of the importance of nutritional factors in the maintenance of bone and joint health, and that nutritional imbalance combined with endocrine abnormalities may be involved in the pathogenesis of osteoarthritis (OA) and osteochondritis dissecans (OCD). Despite this, dietary programs have played a secondary role in the management of these connective tissue disorders. Articular cartilage is critically dependent upon the regular provision of nutrients (glucose and amino acids), vitamins (particularly vitamin C), and essential trace elements (zinc, magnesium, and copper).

Type II collagen deposition in cruciate ligament precedes osteoarthritis in the guinea pig knee.

Objective: To examine the collagens in cruciate ligaments of young Dunkin-Hartley guinea pigs, to determine whether a change in specific collagen types is an early feature of the spontaneous osteoarthritis (OA), which consistently develops in the medial compartment of the knee in this strain.

Design: Collagen types I, II, III, IX, and XI were detected by immunofluorescence microscopy in the anterior and posterior cruciate ligaments of animals at 3, 4-5 and 12 weeks of age. Type II collagen in PCL was further analysed by confocal microscopy or biochemical assay after cyanogen bromide digestion, SDS-PAGE and immunoblotting.

The open reading frame of the Na(+)-dependent glutamate transporter GLAST-1 is expressed in bone and a splice variant of this molecule is expressed in bone and brain.

Previously we have reported expression of an mRNA with homology to the Na(+)-dependent glutamate transporter, GLAST-1, in bone. Here we demonstrate that the complete open reading frame of GLAST-1 mRNA and corresponding 69 kDa protein are expressed in rat bone in vivo. We have also discovered a novel splice variant (GLAST-1a), lacking exon 3, expressed in rat bone and brain.

Characterization of type XI collagen-glycosaminoglycan interactions.

Using competitive binding experiments, it was found that native type XI collagen binds heparin, heparan sulfate, and dermatan sulfate. However, interactions were not evident with hyaluronic acid, keratan sulfate, or chondroitin sulfate chains over the concentration range studied. Chondrocyte-matrix interactions were investigated using cell attachment to solid phase type XI collagen.

Elevated levels of proteolytic enzymes in the aging human vitreous.

Purpose: To identify whether aging of human vitreous is accompanied by an elevation in degradative enzymes within the tissue.

Methods: Human vitreous samples from donors aged 10 to 88 years were placed in two groups based on donor age of less than or more than 50 years. Homogenized samples were analyzed by gelatin substrate zymography for matrix metalloproteinases (MMP).