Acute and sublethal toxicity of thallium to aquatic organisms.
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Animals↗
Biomedical subjects
C A McDevitt
Publications and source records attributed to C A McDevitt.
Absolute concentrations of mRNA for type I and type VI collagen in the canine meniscus in normal and ACL-deficient knee joints obtained by RNase protection assay.
Relatively little is known about the cellular and molecular responses of the knee joint meniscus to joint injury, despite the functional importance of the tissue. We investigated how meniscus cells respond to joint injury in the early stages of post-traumatic osteoarthritis by characterizing the changes in matrix gene expression in menisci at 3 and 12 weeks post-surgery in dogs in which the anterior cruciate ligament (ACL) in one joint was transected and the other unoperated joint served as a control. Changes in the total RNA and DNA concentrations of the menisci were determined. Absolute concentrations of the mRNA of the COL1A1 gene of type 1 collagen, the major fibrillar collagen of the meniscus, and the COL6A3 gene of type VI collagen, a major repair molecule, were determined by quantitative ribonuclease (RNase) protection assay. The concentration of total RNA in medial and lateral menisci increased from 40 to 60 microg RNA/g wet wt in unoperated, control joints to 200-350 microg RNA/g wet wt in ACL-deficient joints. No significant changes were detected in the concentration of DNA (900-1200 microg DNA/g wet wt). Low concentrations of COL1A1 (2-3 pmol mRNA/g DNA) and COL6A3 (0.3-0.6 pmol mRNA/g DNA) mRNA transcripts were measured in normal menisci. ACL-deficiency induced a 20-38 fold increase in COL1A1 and COL6A3 mRNA concentration at 3 weeks, and an 11-19 fold increase at 12 weeks post-surgery. In general, the increase in COL1A1 and COL6A3 mRNA concentrations was greater in medial menisci than in lateral menisci. These results demonstrate that the menisci initiate a vigorous biosynthetic response to transection of the ACL.
Animals↗
Matrix protein mRNA levels in canine meniscus cells in vitro.
The resident cells of the meniscus synthesize a fibrocartilaginous extracellular matrix in vivo composed predominantly of type I collagen fibers. To increase our understanding of matrix biosynthesis by meniscus cells in vitro, we examined matrix protein mRNA levels in cultured meniscus cells isolated from skeletally mature dogs. The mRNA levels of five matrix protein genes (COL1A1, COL2A1, aggrecan, COL6A1, and fibronectin) were measured in meniscus cells by Northern blotting and compared with those of patellar tendon fibroblasts and femoral articular cartilage chondrocytes. In freshly isolated cells (Day 0 cells), COL1A1, COL2A1, and aggrecan mRNA levels were low or undetectable in both meniscus cells and tendon fibroblasts. In intact meniscus tissue, COL1A1 mRNA levels were also low or undetectable. COL2A1 and aggrecan mRNA transcripts were readily observed, however, in Day 0 articular chondrocytes. The levels of expression of COL6A1 and fibronectin mRNA transcripts in Day 0 meniscus cells were intermediate between higher articular chondrocyte levels and lower tendon fibroblast levels. After 1 week in monolayer culture (Day 7 cells), meniscus cells expressed readily detectable levels of COL1A1 mRNA transcripts, similar to that observed for cultured tendon fibroblasts. COL1A1 mRNA transcripts were either not detected or detected at very low levels in monolayer cultures of articular chondrocytes. COL2A1 and aggrecan mRNA transcripts were readily detected in cultured articular chondrocytes but not in meniscus cells or in tendon fibroblasts. All three types of cells continued to express COL6A1 and fibronectin mRNA transcripts after 1 week in culture. These results demonstrate that the patterns of expression of COL1A1 and COL2A1 mRNA transcripts by meniscus cells are similar to those of tendon fibroblasts and dissimilar to those of articular chondrocytes both in freshly isolated cells and in monolayer cultured cells. This mRNA expression pattern supports the idea that monolayer culture of meniscus cells results in the expression of a predominantly fibroblastic phenotype.
Aggrecans↗
Attachment of articular cartilage chondrocytes to the tissue form of type VI collagen.
Type VI collagen is composed of a short triple helix rich in RGD sequences with globular domains at each extremity of the helix. Disulfide-bonded tetramers of the monomeric molecule associate non-covalently to form networks of microfibrils in connective tissues, including cartilage. The disulfide-bonded tetramer can be extracted with 6 M guanidine HCl and purified without pepsin digestion and is referred to here as the tissue form of type VI collagen. Type VI collagen in mature articular cartilage appears to be concentrated pericellularly. We undertook a systematic investigation using solid phase assays to establish the nature of the attachment of bovine articular cartilage chondrocytes to the intact, tissue form of bovine type VI collagen. The tissue form of type VI collagen was extracted from bovine meniscus cartilage with 6 M guanidine HCl and purified by polyethylene glycol precipitation. When equal molar quantities were coated on microwells, the tissue form of type VI collagen attached more cells than the pepsin-digested form of the molecule that lacked the globular domains. The attachment to the intact, tissue form was dose-dependent and saturable and was not inhibited by heparin or type II collagen. A linear GRGDSP peptide failed to inhibit attachment of the chondrocytes to the intact, tissue or pepsin-digested forms of type VI collagen, but totally inhibited the interaction when the intact molecule was reduced and alkylated. In contrast, a cyclic C*GRGDSPC* peptide inhibited attachment to the tissue form of type VI collagen, but not to fibronectin. The attachment had a metal ion dependence that could be satisfied by MnCl2, slightly less by MgCl2, but not at all by CaCl2. A direct interaction between the tissue form of type VI collagen and a chondrocyte cell surface receptor or receptors is a structural feature of the pericellular matrix in cartilage.
Amino Acid Sequence↗
Thrombospondin 1 binds to the surface of bovine articular chondrocytes by a linear RGD-dependent mechanism.
Thrombospondin 1 is present in articular cartilage and is synthesized by chondrocytes. Adult bovine articular chondrocytes in serum-free medium were evaluated in a solid-phase assay for their ability to attach to thrombospondin 1 isolated from human platelets. The chondrocytes attached to the thrombospondin 1 by a mechanism that was inhibited by a synthetic linear GRGDSP but not a GRGESP peptide. The cells, however, did not spread on thrombospondin 1, but did spread on fibronectin and Pep-Tite 2000, a synthetic RGD-containing peptide. Preincubation of thrombospondin 1 with EDTA irreversibly inhibited its capacity to attach to chondrocytes. We conclude that thrombospondin 1 binds to chondrocytes by its RGD sequence.
Amino Acid Sequence↗
Role of heparan sulfate in the terminal differentiation of growth plate chondrocytes.
Recent studies have demonstrated that basic fibroblast growth factor (bFGF) plays a key role in the terminal differentiation of growth plate chondrocytes during endochondral ossification. In this paper we examined the potential role of heparan sulfate in the regulation of the action of basic fibroblast growth factor (bFGF) in the terminal differentiation of rat growth plate chondrocytes. As rat growth plate chondrocytes differentiate in vitro, the percentage of heparitinase-sensitive material decreases. Treatment of growth plate chondrocytes with sodium chlorate, a reversible inhibitor of glycosaminoglycan sulfation, resulted in terminal differentiation of growth plate chondrocytes even in the presence of bFGF at concentrations that normally repress their differentiation. Chlorate treatment in the presence of bFGF resulted in an increase in alkaline phosphatase activity and a decrease in cellular proliferation, both characteristics of the differentiated state. Chlorate treatment also reduced the binding of bFGF to growth plate chondrocytes and this effect could be reversed in a dose-dependent manner by the simultaneous addition of sodium sulfate. The reduced binding was a function of a reduced number of receptors and not a reduced affinity for the receptor. Pretreatment of the growth plate chondrocytes with heparitinase significantly reduced the binding of bFGF to both low- and high-affinity receptors, while pretreatment with chondroitinase ABC had no effect. Finally, addition of exogenous heparin restored bFGF binding to chlorate-treated chondrocytes in a concentration-dependent manner. These results provide evidence that a cell surface heparan sulfate is involved in the binding of bFGF to high-affinity receptors and that a downregulation of this glycosaminoglycan is part of the pathway that leads to terminal differentiation of growth plate chondrocytes.
Animals↗
Basic fibroblast growth factor binds to heparan sulfate in the extracellular matrix of rat growth plate chondrocytes.
Recent studies have demonstrated that basic fibroblast growth factor (bFGF) plays a key role in the terminal differentiation of growth plate chondrocytes during endochondral ossification. We therefore examined the binding of [125I]bFGF to an extract of extracellular matrix (ECM) of rat growth plate. Using a solid phase binding assay, binding of [125I]bFGF to ECM was demonstrated to be specific and saturable at 0.5-1 microgram/ml of bFGF. Scatchard analysis demonstrated the presence of a single class of binding sites with an apparent Kd of 14 nM. The binding of [125I]bFGF to rat growth plate ECM was inhibited by the addition of heparin, heparan sulfate, and dermatan sulfate. The binding was reversible as these glycosaminoglycans were also effective at displacement of bound [125I]bFGF from rat growth plate ECM. Chondroitin 4- or 6-sulfate had no effect on either binding or displacement when added at the same concentrations. Preincubation of the rat growth plate ECM with heparinase or heparitinase resulted in a reduction of the binding of [125I]bFGF to the ECM. Furthermore, these enzymes were able to significantly displace bound growth factor. In contrast, chondroitinase ABC or AC failed to displace bound [125I]bFGF from the ECM. Similar results were obtained when matrix derived from rat growth plate tissue was used for the binding studies. The results demonstrate that bFGF binds to a heparan sulfate in matrix produced by rat growth plate chondrocytes and matrix extracted from rat growth plate and suggest that this glycosaminoglycan may serve as a storage depot for this growth factor during endochondral ossification.
Alkaline Phosphatase↗
Comparison of collagen synthesis in the peripheral and central region of the canine meniscus.
A comparison of collagen synthesis in the peripheral and central regions of the normal medial meniscus, the normal lateral meniscus, and the medial meniscus, three months after reconstruction of the anterior cruciate ligament (ACL), was performed in a canine model. Triangular-shaped explants were labeled in vitro with tritiated proline to compare collagen synthesis. Regional cellularity was also quantified. In the normal medial and lateral meniscus, cellularity was similar. Total collagen synthesis, as assessed by collagenase digestible material, was significantly elevated in the peripheral one-third of the medial meniscus versus the central region (paired t-test, p = 0.026). However, in the medial meniscus after ACL reconstruction, cellularity and total collagen synthesis were significantly higher in the peripheral one-third versus the contiguous central region. They were also elevated relative to the peripheral and central region of the normal medial and lateral meniscus (ANOVA, p < 0.05; Student-Neuman-Kuel test, p < 0.05). Thus, in the normal medial meniscus, the peripheral one-third may have an increased healing potential based on its increased ability to synthesize collagen. Furthermore, after ACL reconstruction, only the peripheral, and not the central, region of the canine meniscus responds to the altered mechanical and synovial environment by significantly increasing cellularity and collagen synthesis.
Analysis of Variance↗
Interaction of intact type VI collagen with hyaluronan.
The capacity of non-pepsinyzed type VI collagen to bind to hyaluronan was investigated. Type VI collagen was extracted from bovine meniscal cartilage with 6 M GuHCl and purified by extraction of PEG precipitates and dissociative Sephacryl S-500 HR chromatography. Type VI collagen, detected with a monoclonal antibody, bound in 0.5 M NaCl to hyaluronan-coated micro-wells, the degree of binding being higher at 37 degrees C than 23 degrees C and 4 degrees C. Incubation of type VI collagen in competitive inhibition assays with testicular hyaluronidase digests of hyaluronan in liquid phase, reduced binding of the protein to hyaluronan-coated microwells to background levels. Thus, non-pepsinyzed type VI collagen binds to hyaluronan in vitro.
Animals↗
Thrombospondin in ligament, meniscus and intervertebral disc.
The presence of thrombospondin in extracts of ligament, meniscus and human nucleus pulposus was demonstrated by Western blot analysis using an anti-human thrombospondin antibody. Metabolic labeling of ligament fibroblast and meniscal fibrochondrocyte cultures followed by immunoprecipitation with anti-human thrombospondin revealed a single band corresponding to the molecular weight of thrombospondin when analyzed by SDS-PAGE and fluorography under reduced and non-reduced conditions. Thrombospondin, therefore, is a matrix constituent of ligament, knee joint meniscus and the nucleus pulposus and is synthesized by ligament fibroblasts and meniscal fibrochondrocytes in vitro.
Animals↗
A quantitative microwell assay for chondrocyte cell adhesion.
An assay for measuring the quantity of live articular chondrocytes attached to a substratum in microwell plates was established by measuring the absorbance of the blue formazan product generated from the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-dypenyl tetrazolium bromide (MTT). Blue formazan production was optimal at an MTT concentration of 1 mg/ml (100 microliters per microwell) and an incubation period of 3 h. The absorbance of the dye was linearly related to the quantity of cells added per microwell. The number of live chondrocytes attached to adhesive proteins can be quantitated using this technique.
Animals↗
Meniscal replacement using a cryopreserved allograft. An experimental study in the dog.
The medial menisci of 14 adult dogs were replaced using a cryopreserved meniscal allograft. The morphology and metabolic activity of the transplanted allografts were then evaluated using routine histology, a vascular-injection (Spalteholz) technique, and autoroentgenography (Na2(35)SO4 incorporation) at various intervals, from two weeks to six months postoperatively. After transplantation, the allografts retained their normal gross appearance and healed to the capsular tissues of the host by fibrovascular scar tissue. Histologically, the grafts demonstrated a decrease in the number of metabolically active cells after transplantation but had a normal cellular distribution and Na2(35)SO4 uptake by three months. The allografts appeared to function normally after transplantation. Although some degenerative changes were noted in the tibial articular cartilage not covered by the meniscus, the cartilage beneath the allograft appeared normal.
Animals↗
The ultrastructure and biochemistry of meniscal cartilage.
The meniscus is characterized at the light microscopic and ultrastructural levels by thick collagen fibers that are predominantly circumferential in orientation. The extracellular matrix of the meniscus is composed mainly of collagen, with smaller quantities of proteoglycans, matrix glycoproteins, and elastin. The collagen is predominantly Type I, with smaller quantities of Types II, III, and V. The proteoglycans are mainly large, aggregating proteoglycans with chondroitin sulfate as their dominant glycosaminoglycan. A small proportion of small dermatan sulfate proteoglycans is probably present. The matrix glycoproteins include the link proteins, the 116-k protein, and a group of adhesive or potentially adhesive proteins that includes Type VI collagen (here classified as a glycoprotein with a collagenous domain), fibronectin, and thrombospondin. The fibrochondrocytes of the meniscus appear to have considerable potential to respond to growth and other modulating factors in the repair or regeneration of the tissue.
Aging↗
Cigarette smoke degrades hyaluronic acid.
Cigarette smoke has been implicated in the pathogenesis of a variety of diseases that affect connective tissues. The effect of the gas phase of cigarette smoke on hyaluronic acid was investigated in vitro. The smoke markedly reduced the chain length of purified hyaluronic acid as assessed by both viscometry and gel chromatography. The hyaluronate exposed to smoke, nevertheless, could aggregate proteoglycans, although the viscosity of the aggregates was lower than in those formed with the unexposed material. The degradation of the hyaluronate was a rapid phenomenon and was inhibited by dimethylsulfoxide, a known scavenger of hydroxyl radicals. We conclude that free radicals in the gas phase of cigarette smoke degrade hyaluronic acid in vitro.
Chromatography, Gel↗
Biochemistry, cell biology, and immunology of osteoarthritis.
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Animals↗
Cartilage content of an immature dog.
The near total mineral-free and elastin-free cartilage content of a three-month-old mongrel dog was 0.73% of the dry body weight. Extraarticular (costal, tracheobronchial, thyroid, cricoid, nasal septum) cartilage constituted 0.44%; articular, 0.06; intervertebral disc, 0.10; elastic, 0.12 and meniscal fibrocartilage, 0.01%. Articular tissue accounted for only 7.25% of the total cartilage uronic acid. This is the first estimate of the amount and distribution of cartilage in a vertebrate species.
Aging↗
Experimental osteoarthritic articular cartilage is enriched in guanidine soluble type VI collagen.
Experimental osteoarthritis was surgically induced in the right knee joint of dogs; the left knee served as a control. Articular cartilage was extracted with 4 M guanidinium chloride, 0.05 M sodium acetate, pH 6.0, containing proteinase inhibitors and the proteins purified by associative CsCl density gradient centrifugation. Equal quantities of protein were electrophoresed in agarose-acrylamide gradient gels and the high molecular weight type VI collagen bands detected in immunoblots with a polyclonal antiserum. Type VI collagen bands between 185 and 220 kDa were evident in the pathological specimens of dogs sacrificed 3, 5, and 7 months after surgery and were either absent or only very weakly visible in the controls. These results demonstrate that experimental osteoarthritic cartilage is enriched in 4 M guanidine-soluble type VI collagen.
Animals↗
Superficial and deeper layers of dog normal articular cartilage. Role of hyaluronate and link protein in determining the sedimentation coefficients distribution of the nondissociatively extracted proteoglycans.
Proteoglycans were extracted under nondissociative conditions from superficial and deeper layers of dog normal articular cartilage. The purified a-A1 preparations were characterized by velocity gradient centrifugation. Superficial specimens exhibited an abundant population of slow sedimenting aggregates whereas the aggregates of deeper preparations sedimented as two well-defined families of molecules. These dissimilarities in the size distribution of the aggregates observed between superficial and deeper a-A1 preparations derived most of all from differences in their content of hyaluronate and link proteins: (a) superficial preparations contained twice as much hyaluronate as deeper specimens; (b) superficial aggregates were link-free and unstable at pH 5.0 whereas deeper preparations contained link-proteins and their faster sedimenting aggregates were stabilized against dissociation at pH 5.0. In these proteoglycan preparations from different cartilage layers, the monomers exhibited an identical capacity for aggregation and the hyaluronate molecules displayed quite similar molecular weight (Mr = 5 x 10(5] and aggregating capacity. These observations as well as aggregating studies conducted with highly purified link protein and purified hyaluronate specimens of different molecular weights support the following conclusions: (a) link protein not only stabilizes proteoglycan aggregates but also enhances the aggregating capacity of hyaluronate; (b) for all practical purposes, the slow sedimenting aggregates represent a secondary complex of hyaluronate and proteoglycan monomers whereas the fast sedimenting aggregates may be considered as a ternary complex wherein link protein stabilizes the hyaluronate-proteoglycans interaction; (c) the distinctive heterogeneity of articular cartilage can be related to structurally different proteoglycan aggregates. The structural dissimilarities observed between superficial and deeper aggregates could reflect the different macromolecular organization of the proteoglycan molecules in the territorial and interterritorial matrices, respectively.
Animals↗