Nancy Burton-Wurster

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.

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.

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 ~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.

Quantitative trait loci for hip dysplasia in a crossbreed canine pedigree

Canine hip dysplasia is a common developmental inherited trait characterized by hip laxity, subluxation or incongruity of the femoral head and acetabulum in affected hips. The inheritance pattern is complex and the mutations contributing to trait expression are unknown. In the study reported here, 240 microsatellite markers distributed in 38 autosomes and the X chromosome were genotyped on 152 dogs from three generations of a crossbred pedigree based on trait-free Greyhound and dysplastic Labrador Retriever founders. Interval mapping was undertaken to map the QTL underlying the quantitative dysplastic traits of maximum passive hip laxity (the distraction index), the dorsolateral subluxation score, and the Norberg angle. Permutation testing was used to derive the chromosome-wide level of significance at p < 0.05 for each QTL. Chromosomes 4, 9, 10, 11 (p < 0.01), 16, 20, 22, 25, 29 (p < 0.01), 30, 35, and 37 harbor putative QTL for one or more traits. Successful detection of QTL was due to the crossbreed pedigree, multiple-trait measurements, control of environmental background, and marked advancement in canine mapping tools.

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.

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 × 101 to 1 × 108 pg/ml, dropping below 10% of control values by 1 × 105 pg/ml. Cytotoxicity occurred as methylprednisolone levels were increased further from 1 × 108 to 1 × 109 pg/ml. Changes in total collagen (protein) synthesis were less pronounced, but also demonstrated significant suppression between 1 × 104 and 1 × 108 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.

Fibronectin and proteoglycan synthesis in long term cultures of cartilage explants in Ham's F12 supplemented with insulin and calcium : effects of the addition of TGF-β

Canine cartilage explants were maintained in a basal medium supplemented with a commercially available supplement (ITSCR+) which includes insulin for up to 12 days in culture. During this time it was found that proteoglycan synthesis, as measured by 35SO4 incorporation into high molecular weight proteoglycans, was maintained at levels comparable to those at Day O. This is in substantial agreement with the results of McQuillan et al. (1) for bovine cartilage explants. Since the basal medium which we used, Hams F12, is low in calcium, we found that supplementation with additional calcium also was needed for maintenance of proteoglycan synthesis. This defined medium was not adequate to prevent a decrease in fibronectin, total protein, and collagen synthesis relative to Day O levels. The addition of transforming growth factor-beta (TGF-beta) at 2 and 10 ng/ml to the defined medium not only prevented the decline in fibronectin synthesis but progressively increased the rate of fibronectin synthesis until the Day O levels were exceeded by an average of fourfold. This TGF-beta-induced increase in fibronectin synthesis was contrasted with the increase in fibronectin synthesis previously reported for degenerated cartilage of osteoarthritic joints (2,3), and possible implications for understanding the disease were discussed.

Expression of the ED B fibronectin isoform in adult human articular cartilage

The most recently described region of alternate splicing of fibronectin mRNA results in expression of an isoform which includes the type III repeat termed ED B (EIII B). To date this isoform has been detected in transformed cells in culture, in the synovial membrane and ovary but in no other adult tissue, and in embryonic chick cartilage to a much greater extent than in other cells of mesenchymal origin. Monoclonal antibody BC-1, which recognizes an epitope within the ED B segment, was used in two different assays and provided evidence that adult human articular cartilage contains ED B fibronectin. The extent of expression of this isoform, however, was variable and less than that found in fibronectin produced by the transformed fibroblast cell line WI-38VA13.

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.

Immunohistochemical localization of fibronectin and chondronectin in canine articular cartilage.

We compared the distribution of fibronectin and chondronectin within the matrix of canine articular cartilage. Fibronectin was found throughout the matrix as well as pericellularly. In contrast, chondronectin was observed predominantly associated with the cell or pericellular matrix. Interactions of these molecules with matrix components in the pericellular matrix probably differs, however, since concentrations of hyaluronidase which prevented detection of pericellular fibronectin allowed detection of chondronectin. Chondronectin and fibronectin were detected in osteoarthritic cartilage as well as in disease-free cartilage. Penetration of biotinylated fibronectin into cartilage from the external medium occurred only in osteoarthritic cartilage and proceeded only from the articular surface. Disease-free cartilage appeared to maintain a barrier to fibronectin penetration from the articular surface which was sustained even after the proteoglycan content was markedly depleted by incubation of cartilage with catabolin or lipopolysaccharide. In cartilage that was proteoglycan-depleted, the only detectable penetration of external fibronectin was from the cut surface.