Biomedical subjects

N Burton-Wurster

Publications and source records attributed to N Burton-Wurster.

At least 19 recordsLinked to original sources

PubMed2005

Genes in canine articular cartilage that respond to mechanical injury: gene expression studies with Affymetrix canine GeneChip.

The Affymetrix canine GeneChip with 23,836 probe sets was used to look for cartilage genes that are significantly altered in response to mechanical impact. The model using canine articular cartilage explants loaded in vitro has been described previously (Chen et al., J Orthop Res 19:703-711, 2001). It is our hypothesis that genes that are activated or repressed in articular cartilage after impact injury initiate cartilage degeneration, leading to osteoarthritis in dogs. Gene expression of known cartilage genes was generally consistent with cartilage biology. A total of 528 genes were significantly (P < .01) up- or down- regulated in response to mechanical damage. After applying the strict Bonferroni correction, 172 remained significantly affected. One of these genes, MIG-6/gene 33, was chosen for verification by real- time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). A 3.8- fold increase in expression was confirmed, consistent with the microarray chip data. Deficiencies in the current annotation of the canine chip are discussed. Gene expression studies with the Affymetrix canine GeneChip are potentially valuable, but await more complete annotation.

Animals↗

PubMed2003

TGF beta 1 and biglycan, decorin, and fibromodulin metabolism in canine cartilage.

OBJECTIVE: Small proteoglycans (PGs) may accumulate in late stage osteoarthritis even as aggrecan is lost. It is not clear what role transforming growth factor (TGF) beta has in this accumulation. Our goal was to investigate the ability of TGF beta 1 to modulate the synthesis and accumulation of decorin, biglycan, and fibromodulin in cartilage explants cultured under conditions in which aggrecan synthesis remains relatively constant. DESIGN: Articular cartilage was cultured in the presence or absence of 4 ng/ml TGF beta 1 for up to 16 days. Material extracted from cartilage was assayed for 35SO(4)-large and small PGs and for total endogenous decorin, biglycan and fibromodulin. RESULTS: The synthesis of 35SO(4)-small PGs increased during the 16 days in culture in response to TGF beta 1, but declined in control cultures. The difference in 35SO(4)-decorin between TGF beta 1 and control samples reached nine-fold after 16 days, while the difference in total endogenous decorin was less than 1.5-fold. 35SO(4)-decorin, which was present in TGF beta 1-treated cultures had an identical core protein, but a longer glycosaminoglycan chain than that of decorin in control cultures. No significant differences in endogenous biglycan were detected, but accumulation of fibromodulin in TGF beta 1 explants exceeded fibromodulin in controls, on average, by 3.8-fold. Fibromodulin was present in cartilage in both keratan sulfate- and non-sulfated oligosaccharide-substituted forms. CONCLUSIONS: The accumulation of each of the three small PGs was affected to a different extent in response to TGF beta 1. Of the three, fibromodulin content was most rapidly augmented in response to TGF beta 1.

Animals↗

PubMed2002

Characteristics of topographical heterogeneity of articular cartilage over the joint surface of a humeral head.

OBJECTIVE: To investigate the topographical variations among the morphological and physical properties of articular cartilage over a canine humeral head. DESIGN: Nine side-by-side specimens from a canine humeral head were used in the combined polarized light microscopy (PLM) and microscopic magnetic resonance imaging (muMRI). Sixteen images were acquired from each specimen in muMRI. Subsequently, ten to fourteen histological slices were prepared at the location of the muMRI slice from each specimen. Four optical images were acquired from each histological slice. Using these images, the images of the T(2) relaxation, the optical retardation, and the collagen-fiber orientation were constructed in two dimensions. RESULTS: Along the medial/lateral direction of the humeral head, we have observed a number of topographical variations. These include the total thickness of the cartilage tissue, the thickness of the individual histological zones, the depth of the minimum optical retardation in the tissue, and the depth of the maximum T(2) relaxation in the tissue. We found that the depth of the minimum retardation from PLM occurs at the geometric middle point of the transitional zone in histology, and that the depth of the maximum T(2) relaxation from muMRI is closely correlated with the minimum retardation depth in PLM. In addition, although the thickness of the total tissue increases approximately by the same amount on both the lateral and medial sides of the joint, a slight asymmetry exists between the measurements from the medial and lateral halves of the humerus. CONCLUSIONS: Cartilage from different sites over a single joint could have different properties, possibly the consequence of the variation in load-bearing status. Because of these topographical variations, one must keep in mind that a different sampling site within a relatively small area of the same joint may significantly influence the results of the study.

Animals↗

PubMed2001

Corticosteroids alter the differentiated phenotype of articular chondrocytes.

Experimental evidence suggests that recommended dosages of some corticosteroids used clinically as antiinflammatory agents for treating arthropathies damage articular cartilage, but low dosages may be chondroprotective. The purpose of this study was to evaluate how different concentrations of methylprednisolone affect chondrocyte function and viability. Articular cartilage and chondrocytes were obtained from young adult horses, 1.5-3.5 years of age. Corticosteroid-induced changes in collagen expression were studied at the transcriptional level by Northern blot analyses and at the translational level by measuring [3H]-proline incorporation into [3H]-hydroxyproline. Fibronectin mRNA splicing patterns were evaluated with ribonuclease protection assays. Cytotoxicity was studied using erythrosin B dye exclusion. Steady-state levels of type II procollagen mRNA decreased without concurrent changes in type I procollagen expression as the medium methylprednisolone concentrations were increased from 1 x 10(1) to 1 x 10(8) pg/ml, dropping below 10% of control values by 1 x 10(5) pg/ml. Cytotoxicity occurred as methylprednisolone levels were increased further from 1 x 10(8) to 1 x 10(9) pg/ml. Changes in total collagen (protein) synthesis were less pronounced, but also demonstrated significant suppression between 1 x 10(4) and 1 x 10(8) pg/ml. Corticosteroid-induced changes in fibronectin isoform levels were evaluated in articular cartilage samples without in vitro culture. The cartilage-specific (V + C)(-) isoform was suppressed in both normal and inflamed joints by a single intraarticular injection (0.1 mg/kg) of methylprednisolone. Combined, these data indicate that methylprednisolone suppresses matrix protein markers of chondrocytic differentiation. Decreased and altered chondrocyte expression of matrix proteins likely contributes to the pathogenesis of corticosteroid-induced cartilage degeneration.

Animals↗

PubMed2001

Chondrocyte necrosis and apoptosis in impact damaged articular cartilage.

A decrease in chondrocyte numbers is one characteristic of osteoarthritic cartilage. This decrease may be the result of apoptosis or other forms of cell death induced by mechanical damage. Furthermore, cell death may contribute to the structural and metabolic changes found in osteoarthritic cartilage. Therefore, we investigated cell viability and the mode of cell death in cartilage subjected to an increasing severity of impact loads expected to cause compositional damage and osteoarthritic-like metabolic alterations. Canine cartilage explants were subjected to cyclic indentation impacts of 5 megapascals at 0.3 Hz for 0, 2, 20, and 120 min and then kept in culture for 2, 4, 48, and 144 h. Cell death was assessed by the TUNEL assay and by uptake of propidium iodide. Viable cells were detected by the ability to metabolize fluorescein diacetate. Nuclear morphology and ultrastructure of the cell were examined using Hoechst 33342 fluorescent staining and transmission electron microscopy (TEM). As controls for necrosis and apoptosis, cartilage was, respectively, frozen and thawed or incubated with mitomycin-C, an apoptosis inducer. In cartilage that had been loaded for 2 h, 32% of the chondrocytes in the loaded core took up propidium iodide within 2 h after loading. Most of these were in the middle to superficial zones and reflected leaky cell membranes usually characteristic of necrosis. Less than 1% of these chondrocytes were positive in the TUNEL assay after 4 h. After additional culture for 2 days, however, the proportion of chondrocytes which were positive in the TUNEL assay reached 73%. A dose dependent response to duration of loading was detected with the TUNEL assay at this time. The TUNEL assay was not specific for apoptosis since 92% of chondrocytes in freeze/thawed cartilage were TUNEL positive. However, some cells with apoptotic bodies and chromatin condensation characteristic of apoptosis were found in the transition zone between necrotic and normal chondrocytes, but not in the superficial and upper zones, in impact damaged cartilage. We concluded that in this study, necrosis occurred first, followed by apoptosis.

Animals↗

PubMed2001

Quantitative in situ correlation between microscopic MRI and polarized light microscopy studies of articular cartilage.

OBJECTIVE: To establish the correlation between the non-invasive imaging by magnetic resonance microscopy (microMRI) and the histological imaging by polarized light microscopy (PLM) accurately, quantitatively, at the highest possible MRI resolution (13.7 microm), and based on the same piece of tissue (articular cartilage from canine shoulder joint). DESIGN: In microMRI experiments, the laminar appearance (the magic angle effect) of the proton intensity images and the anisotropic characteristics of the T(2)relaxation images were analysed. In PLM experiments, the images of the optical retardation and collagen-fibre orientation in cartilage were constructed in two dimensions. RESULTS: The T(2)profile has a distinctly asymmetric bell-shaped curve and three featured zones. The retardation profile has a non-zero minimum at the middle of the transitional zone of the tissue. The angle profile has a smooth variation across the transitional zone. These facts suggest that the collagen fibres in the transitional zone are not entirely random but have a residual order. In addition, the peak of the T(2)profile coincides with the minimum of the retardation profile, both represent the most isotropic region of the tissue. A hyperbolic tangent function was found to best describe the transition of the collagen fibres in cartilage. A set of criteria was developed for each technique to define the features in the quantitative measurements. CONCLUSIONS: The criteria offer, for the first time, a set of quantitative and objective means to subdivide the tissue thickness into the zones in histology and in MRI. It is shown that the microMRI zones based on the T(2)characteristics are statistically equivalent to the histological zones based on the collagen fibre orientation (t-probabilities of 0.730, 0.973, 0.647, 0.850 for the superficial, transitional, radial zones and the total thickness).

Animals↗

PubMed2001

Intercellular signaling as a cause of cell death in cyclically impacted cartilage explants.

UNLABELLED: Recently, in vitro cartilage studies have shown that impact loading can produce structural damage and osteoarthritis-like changes, including tissue swelling, collagen denaturation, and cell death. OBJECTIVE: This study was to determine whether a signal for cell death moves through the cartilage matrix, resulting in the spread of cell death over time from impacted to unimpacted regions. DESIGN: Cyclic impacts were applied to the 2 mm core of 4 mm cartilage discs. Post-impact culturing extended for 3, 6 or 21 days and occurred in one of two ways. In one, discs were cultured intact. In the second, cores were removed immediately after cessation of impact and cores and rings cultured separately. Cells in apoptosis and later stage necrosis were monitored using the TUNEL assay. RESULTS: The extent of cell death in impacted samples increased with increased duration of post-impact culturing. At the early time, the majority of cell death was located in the regions of direct impact whereas after extended culture, the extent of cell death was similar in the surrounding unimpacted regions and in the impacted core region. However, the physical separation of the impacted core from the surrounding, non-impacted ring regions immediately after impact, and prior to independent culture, kept the level of cell death in the surrounding ring close to control levels, even after 21 days of incubation. DISCUSSION: These findings indicate that soluble intercellular signalling is involved in the spreading of cell death through the cartilage matrix, and that its effects can be prevented by physical isolation of the surrounding ring from the impacted core.

Analysis of Variance↗

PubMed2000

Phenotypic stability of articular chondrocytes in vitro: the effects of culture models, bone morphogenetic protein 2, and serum supplementation.

Numerous in vitro culture models have been developed for the investigation of chondrocyte and cartilage biology. In this study, we investigated the stability of the chondrocytic phenotype in monolayer, aggregate, pellet, and explant culture models and assessed the effects of recombinant human bone morphogenetic protein 2 (rhBMP-2) and serum supplementation on the phenotype in each model. Phenotypic effects were assessed by analyses of procollagen type II, aggrecan, (V + C)- fibronectin, and procollagen type I messenger RNA expression. In monolayer cultures, we noted a characteristic loss of procollagen type II and induction of procollagen type I expression. The aggregate and pellet culture models supported matrix protein gene expression profiles more reflective of in vivo levels. In explant cultures, expression of matrix protein genes was consistently depressed. Treatment with rhBMP-2 significantly increased the expression of procollagen type II and aggrecan in monolayer cultures; however, other models showed comparatively little response. Similarly, serum supplementation significantly down-regulated procollagen type II and aggrecan expression in monolayer cultures but had less effect on gene expression in the other models. Serum supplementation increased procollagen type I expression in monolayer and aggregate cultures. These results suggest that the influence of exogenous BMP-2 and serum on expression of chondrocyte-specific matrix protein genes is influenced by aspects of substrate attachments, cellular morphology, and/or cytoskeletal organization. Finally, the analyses of fibronectin expression suggest that V and C region alternative splicing in chondrocytes is linked to the establishment of a three-dimensional multicellular complex.

Aggrecans↗

PubMed1999

The cartilage-specific (V+C)- fibronectin isoform exists primarily in homodimeric and monomeric configurations.

Fibronectin is an extracellular-matrix glycoprotein encoded by a single gene, but with significant protein heterogeneity introduced through alternative RNA splicing and post-translational modifications. The (V+C)(-) splice variant, in which nucleotides encoding protein segments III-15 and I-10 are deleted along with the entire variable region, is unique in that expression is restricted to cartilaginous tissues. All known fibronectin splice variants retain the two C-terminal cysteine residues essential for dimerization, but cellular and/or structural constraints appear to influence homo- and heterodimerization patterns. Dimerization patterns of the (V+C)(-) isoform were studied under native conditions within canine articular cartilage and experimentally in COS-7, NIH-3T3 and CHO-K1 cell cultures. In all systems, (V+C)(-) fibronectin secretion was predominantly in a homodimeric configuration. Lower levels of (V+C)(-) monomers were also present. Heterodimers of (V+C)(-) with V(+),C(+) (V120) isoforms were not detected. Heterodimers of (V+C)(-) with V(-),C(+) (V0) subunits were detected only at low levels. Functional properties may differ significantly among monomers, homodimers and heterodimers. The unique dimerization pattern of (V+C)(-) fibronectin is consistent with this isoform having specialized functional properties in situ that are important for either the structural organization and biomechanical properties of cartilage matrix or regulation of a chondrocytic phenotype.

Animals↗

PubMed1999

Compositional and metabolic changes in damaged cartilage are peak-stress, stress-rate, and loading-duration dependent.

The first objective of this study was to determine if the cumulative effects of impact or smoothly arising compression would damage the matrix of articular cartilage. Canine cartilage explants were subjected to repeated impacts or to smoothly arising compressions of as much as 20 MPa at 0.3 Hz for as long as 120 minutes. An increase in the water content of the loaded core compared with the surrounding ring was considered indicative of matrix damage. The results showed that damage to cartilage required repeated impacts with a peak stress of at least 2.5 MPa and a stress rate of at least 30 MPa/sec for 2 minutes or longer. This suggested that impact damage is cumulative and stress-rate dependent. The second objective was to identify biosynthetic and compositional changes in impact-damaged cartilage over a period of time after loading. Accordingly, canine cartilage explants were subjected to repetitive impacts of 5 MPa at 0.3 Hz for 2, 20, and 120 minutes. The loaded explants were then cultured for as long as 10 days. The increase in water content (1.9-3.8%) in the core region relative to the surrounding ring persisted during the 10-day culture. A significant increase in fibronectin synthesis (22-47%) was found in the core region of impact-damaged cartilage. Proteoglycan synthesis was increased by 41-104%. An increase in denatured collagens (11-70%) in the loaded cores substantiated damage to the collagen network. Denatured collagens stained with COL2-3/4m monoclonal antibody were consistent with the compositional findings and were mainly located near the articular surface and in the deep zone. These changes were consistent with early osteoarthritis and suggested the induction of the initial stages of osteoarthritis in the impact-damaged cartilage.

Animals↗

PubMed1999

Site-specific variation in femoral head cartilage composition in dogs at high and low risk for development of osteoarthritis: insights into cartilage degeneration.

OBJECTIVE: To determine which characteristics of cartilage lesion pathology are detected in dogs at high risk to develop osteoarthritis prior to diagnosis by standard radiographs or macroscopic cartilage abnormality on necropsy. METHODS: Fourteen disease-free dogs were assigned to risk groups based on hip distraction index. For seven dogs, three dimensional images of hip joints from computed tomography were available. At necropsy, ligamentum capitis femoris volumes were measured and articular cartilage was harvested and analyzed for percent water, swelling, glycosaminoglycan, and fibronectin. Comparisons were made with nine dogs with macroscopic cartilage lesions (OA group). RESULTS: Ligament volumes were greater in the high distraction index group (P=0.000). Water content was elevated in the lesion area in both low and high risk dogs (P=0.000); no additional increase was noted in the high risk group. Glycosaminoglycan content was slightly elevated in the surrounding area in both groups (P< 0.02) but loss was noted histologically in the lesion area of the high risk group. Fibronectin was increased in the lesion area and in the high risk group (P=0.000). The magnitude of this increase was greatest in the lesion area (P=0.000) in explants. Computed tomography indicated dorsal acetabular rim impingement on the lesion area in high risk dogs. CONCLUSIONS: Water content and swelling suggest matrix structure is weaker at the site of lesion predilection in all dogs regardless of risk status. Computed tomography imaging is consistent with site specific initiation of lesions by mechanical factors.

Animals↗

PubMed1998

Differentiated cellular function in fetal chondrocytes cultured with insulin-like growth factor-I and transforming growth factor-beta.

This study examined fetal chondrocyte proliferation and function following exposure to transforming growth factor-beta and insulin-like growth factor-I. Fetal equine articular chondrocytes of the early third-trimester were isolated and cultured in monolayer conditions, then exposed to 0, 1, 5, or 10 ng/ml transforming growth factor-beta or 0, 10, 50, or 100 ng/ml insulin-like growth factor-I for 48 hours. Proliferative responses were assessed by cell counts and [3H]thymidine uptake into precipitable DNA. Differentiated chondrocyte metabolic activity was determined by sulfated glycosaminoglycan quantitation, 35[SO4] incorporation into precipitable glycosaminoglycan, and proteoglycan molecular sizing by CL-2B column chromatography. Morphological changes seen on phase-contrast microscopy included a larger proportion of rounded cells in monolayer cultures supplemented with insulin-like growth factor-I and cytotoxic changes in cells treated with transforming growth factor-beta. Both insulin-like growth factor-I and transforming growth factor-beta resulted in significant elevations of [3H]thymidine uptake; however, cell numbers did not rise sufficiently over the 48-hour culture period to reach significant levels. Maximum mitogenic responses were evident at 50 and 100 ng/ml insulin-like growth factor-I and 5 ng/ml transforming growth factor-beta. The production of proteoglycan was also enhanced (435%) by exposure to 50 ng/ml insulin-like growth factor-I, and an increased proportion of larger proteoglycan monomer species was evident in cultures treated with 50 and 100 ng/ml insulin-like growth factor-I. A similar dose-response was also evident in cultures treated with transforming growth factor-beta (maximal 164% increase with 5 ng/ml), although the presence of serum in the culture medium altered the pattern of enhanced proteoglycan synthesis to favor the lower concentration of 1 ng/ml (191%). Additionally, larger proteoglycan molecules were synthesized in response to high concentrations of transforming growth factor-beta in serum-free cultures. Significant biochemical changes resulted from the addition of transforming growth factor-beta to fetal chondrocyte cultures; however, monolayer cultures that were treated with transforming growth factor-beta and supplemented with serum began to develop cellular toxicity, including nuclear pyknosis and cytoplasmic fragmentation. Degenerative cellular changes were not evident in cultures treated with insulin-like growth factor-I, and significant differentiated metabolic activity resulted from the presence of insulin-like growth factor-I in the culture medium. These data suggest that the responses of fetal chondrocytes to insulin-like growth factor-I and transforming growth factor-beta were enhanced compared with the responses of chondrocytes derived from postnatal animals and that these metabolically active cells can be primed by endogenous or exogenous growth factors to provide enhanced articular function and repair.

Animals↗

PubMed1998

Expression of the (V+C)- fibronectin isoform is tightly linked to the presence of a cartilaginous matrix.

Fibronectin is encoded by a single gene, but heterogeneity is introduced by alternative splicing of the pre-mRNA. An unique splice variant, designated (V+C)-, which deletes nucleotides encoding the V, III-15 and I-10 segments, has been identified in articular cartilage. In this study, a ribonuclease protection assay was used to quantitate expression of the (V+C)- isoform in eight canine cartilaginous tissues and in chondrocytes cultured as monolayers or in alginate beads. The (V+C)- fibronectin isoform was detected in all cartilaginous tissues examined, ranging from a low of 11% of steady-state fibronectin mRNA in the nucleus pulposus to 71% in the rib. An age dependent increase, from 18% in the epiphyseal cartilage of a newborn to 54% in the articular cartilage of dogs over 10 months of age, was observed. The ubiquitous presence of this isoform in cartilaginous tissues and its absence in all non-cartilaginous tissues examined to date is consistent with a very strong association of the (V+C)- fibronectin isoform with the cartilaginous phenotype. Results from a ribonuclease protection assay using a probe extending into the V region from III-14 were combined with the quantitative information about (V+C)- fibronection expression to develop an over-all profile of splicing within the V region in cartilage. Monolayer culture of articular chondrocytes altered fibronectin splicing patterns. The (V+C)- isoform was rapidly lost and ED-A(+) fibronectin was induced. Three-dimensional culture in alginate beads prevented induction of ED-A(+) fibronection, but failed to sustain expression of the (V+C)- isoform. Thus, some matrix component or structure, lost in cell culture, may be essential to maintain expression of the (V+C)- isoform. The possible relationship of changing patterns of fibronectin isoforms in cultured chondrocytes to maintenance of the differentiated phenotype is discussed.

Alternative Splicing↗

PubMed1997

Onset of epiphyseal mineralization and growth plate closure in radiographically normal and dysplastic Labrador retrievers.

OBJECTIVE: To determine whether onset of mineralization of the femoral and proximal tibial epiphyses and age at closure of the femoral and acetabular triradiate growth plates was different for Labrador Retrievers that were radiographically normal or that had canine hip dysplasia (CHD). DESIGN: Cohort study. ANIMALS: 36 Labrador Retriever puppies. PROCEDURE: Puppies were radiographed every other day from the time they were 8 to 10 days old until ossification of the femoral heads was apparent. Radiographs were then obtained weekly until puppies were 1 month old and then monthly until puppies were 8 to 12 months old. Age at which mineralization was first observed in the proximal and distal femoral and proximal tibial epiphyses and at which the femoral capital, triradiate acetabular, and distal femoral growth plates were no longer radiographically visible were recorded. Fifteen dogs were euthanatized and necropsied to determine whether dogs had CHD. RESULTS: There were 26 radiographically normal left and right hip joints and 10 dysplastic left and right hip joints. Onset of mineralization of the proximal femoral epiphyses and of the right proximal tibial epiphysis was significantly later in dysplastic than in radiographically normal puppies. The left femoral capital growth plates closed significantly later in dysplastic than in radiographically normal joints, but other differences in growth plate closure were not detected. CLINICAL IMPLICATIONS: Endochondral ossification may be abnormal in dogs with CHD. The disease appears to affect multiple joints, even though it is most evident clinically in the hip joint.

Aging↗

PubMed1997

Variations in composition of cartilage from the shoulder joints of young adult dogs at risk for developing canine hip dysplasia.

OBJECTIVE: To determine whether the composition of cartilage from the shoulder joints of dogs varied with the risk of developing canine hip dysplasia (CHD). DESIGN: Observational study. ANIMALS: 12 skeletally mature (approx 1 year old) Labrador Retrievers. PROCEDURE: Dogs were classified as having a low, moderate, or high risk of developing CHD on the basis of distraction indices. Cartilage was harvested from the craniolateral and weight-bearing regions of the humeral heads, and wet weight per unit area and dry, glycosaminoglycan, and fibronectin contents were determined. RESULTS: Glycosaminoglycan and dry contents did not vary among risk groups. For cartilage from the craniolateral region of the humeral head, wet weight per unit area and fibronectin content increased as risk of developing CHD increased. Wet weight and fibronectin content of cartilage from the weight-bearing region of the humeral head did not vary among risk groups. CLINICAL IMPLICATIONS: Dogs that have a high risk of developing CHD are also more likely to develop osteoarthritis of the shoulder joint. The observed increases in wet weight per unit area and fibronectin content in cartilage from the craniolateral region of the humeral head in dogs at a high risk of developing CHD may be early sings of incipient osteoarthritis.

Animals↗

PubMed1997

Cartilage fibronectin isoforms: in search of functions for a special population of matrix glycoproteins.

Fibronectins are a part of the repertoire of matrix molecules produced by the chondrocyte in order to assemble a functional cartilage matrix. They are encoded by a single gene, but significant protein heterogeneity results from alternative RNA splicing. The population of fibronectin isofroms in adult cartilage is significantly different from fibronectins in other tissues and includes relatively high levels (20-30%) of ED-B(+) fibronectins and high levels (50-80%) of the cartilage specific (V + C)- isoform which lacks the V, III-15 and I-10 segments. Less than 4% of the fibronectins in cartilage are ED-A(+). The synthesis and accumulation of cartilage fibronectins are modulated in response to matrix pathology and to biochemical and mechanical mediators. In addition, alternative splicing patterns are altered when chondrocytes are allowed to dedifferentiate in monolayer culture such that the (V + C)- isoform is lost but the ED-A(+) isoform is reexpressed at high levels. Cartilage fibronectins have the potential to participate in cell signalling via integrin mediated pathways and to interact with other cartilage matrix macromolecules. The tissue-specific splicing pattern gives rise to a unique population of fibronectins within the cartilage. Together, this points to a critical role for cartilage fibronectins in chondrocyte cell biology and the organization of a biomechanically sound matrix. However, the precise function (or functions) of the cartilage fibronectins has (or have) not been defined. This minireview examines current information about the structure, synthesis and interactions of cartilage fibronectins. When possible, potential consequences of the inclusion of the ED-B segment or the exclusion of the V, III-15 and I-10 segments are discussed. The goal is to stimulate critical thought and discussion in the field about cartilage fibronectin isoforms, their function(s) in normal cartilage, and their role(s) in the pathogenesis of cartilage diseases.

Alternative Splicing↗

PubMed1996

Fibronectin mRNA splice variant in articular cartilage lacks bases encoding the V, III-15, and I-10 protein segments.

Fibronectin is an extracellular matrix glycoprotein encoded by a single gene. Alternative RNA splicing has been reported at three sites, ED (extra type III domain)-A, ED-B, and the variable or V region. Articular cartilage fibronectin monomers are rarely (ED-A)+, but approximately 25% are (ED-B)+. RNA gel electrophoresis and Northern blot analysis identified two (ED-B)+ and two (ED-B)- fibronectin transcripts in cartilage, each pair differing by approximately 750 bases. This difference results from a previously unreported RNA splicing pattern that eliminates not only the V region but also nucleotides encoding protein segments III-15 and I-10. This new splice variant, which we designate (V+C)-, represents the majority of fibronectin transcripts in equine, canine, and rabbit articular cartilage but is absent in the liver. Reverse transcriptase-polymerase chain reaction analyses of 11 additional equine tissues failed to detect the (V+C)- splice variant, except for very low levels in lymph node, bone, aorta, and skin. Furthermore, chondrocytes grown in monolayer culture maintain high levels of fibronectin expression but stop expressing (V+C)- transcripts over time. The tissue-specific expression pattern of this novel fibronectin isoform suggests that it may have an important function in the matrix organization of cartilage.

Alternative Splicing↗

PubMed1996

Swelling and fibronectin accumulation in articular cartilage explants after cyclical impact.

The objective of this study was to determine if repeated impact could damage living cartilage and lead to osteoarthritis-like changes in its biology. Canine cartilage explants were subjected to impacts of as much as 50 MPa once every 5 seconds for 30 minutes. On each impact cycle, the loading rate was 100 MPa/sec to the assigned peak stress, which was held for 1 second. After impact testing, the cartilage was kept in defined culture for as long as 10 days. Radiosulfate incorporation in the region that received direct impact varied with load 0-4 hours after impact, but it did not vary with load at 20-24 hours after impact. Even so, most explants were visibly damaged by 20 or 50 MPa, and there was subtle evidence of damage from impacts of 5 or 10 MPa. For example, ion-induced swelling in 0.01 M NaCl was increased, suggesting that the physical integrity of the matrix was reduced relative to controls. Self-diffusion of water, measured by proton magnetic resonance imaging was also increased in the deeper zones of the explant, consistent with changes in structure at the molecular level. Ten days after impact, the water content and the fibronectin content of the loaded region of the explant were both increased. In combination, these osteoarthritis-like changes suggested that the physical strength of normal cartilage limits its ability to withstand cyclical impact.

Animals↗