Long term outcomes of biomaterial-mediated repair of focal cartilage defects in a large animal model.

The repair of focal cartilage defects remains one of the foremost issues in the field of orthopaedics. Chondral defects may arise from a variety of joint pathologies and left untreated, will likely progress to osteoarthritis. Current repair techniques, such as microfracture, result in short-term clinical improvements but have poor long-term outcomes. Emerging scaffold-based repair strategies have reported superior outcomes compared to microfracture and motivate the development of new biomaterials for this purpose. In this study, unique composite implants consisting of a base porous reinforcing component (woven poly(ε-caprolactone)) infiltrated with 1 of 2 hydrogels (self-assembling peptide or thermo-gelling hyaluronan) or bone marrow aspirate were evaluated. The objective was to evaluate cartilage repair with composite scaffold treatment compared to the current standard of care (microfracture) in a translationally relevant large animal model, the Yucatan minipig. While many cartilage-repair studies have shown some success in vivo, most are short term and not clinically relevant. Informed by promising 6-week findings, a 12-month study was carried out and those results are presented here. To aid in comparisons across platforms, several structural and functionally relevant outcome measures were performed. Despite positive early findings, the long-term results indicated less than optimal structural and mechanical results with respect to cartilage repair, with all treatment groups performing worse than the standard of care. This study is important in that it brings much needed attention to the importance of performing translationally relevant long-term studies in an appropriate animal model when developing new clinical cartilage repair approaches.

Recombinant human FGF18 preserves depth-dependent mechanical inhomogeneity in articular cartilage.

Articular cartilage is a specialised tissue that has a relatively homogenous endogenous cell population but a diverse extracellular matrix (ECM), with depth-dependent mechanical properties. Repair of this tissue remains an elusive clinical goal, with biological interventions preferred to arthroplasty in younger patients. Osteochondral transplantation (OCT) has emerged for the treatment of cartilage defects and osteoarthritis. Fresh allografts stored at 4 °C have been utilised, though matrix and cell viability loss remains an issue. To address this, several studies have developed media formulations to maintain cartilage explants in vitro. One promising factor for these applications is sprifermin, a human-recombinant fibroblast growth factor-18, which stimulates chondrocyte proliferation and matrix synthesis and is in clinical trials for the treatment of osteoarthritis. The study hypothesis was that addition of sprifermin during storage would maintain the unique depth-dependent mechanical profile of articular cartilage explants, a feature not often evaluated. Explants were maintained for up to 6 weeks with or without a weekly 24 h exposure to sprifermin (100 ng/mL) and the compressive modulus was assessed. Results showed that sprifermin-treated samples maintained their depth-dependent mechanical profile through 3 weeks, whereas untreated samples lost their mechanical integrity over 1 week of culture. Sprifermin also affected ECM balance by maintaining the levels of extracellular collagen and suppressing matrix metalloproteinase production. These findings support the use of sprifermin as a medium additive for OCT allografts during in vitro storage and present a potential mechanism where sprifermin may impact a functional characteristic of articular cartilage in repair strategies.

A large animal model that recapitulates the spectrum of human intervertebral disc degeneration.

OBJECTIVE: The objective of this study was to establish a large animal model that recapitulates the spectrum of intervertebral disc degeneration that occurs in humans and which is suitable for pre-clinical evaluation of a wide range of experimental therapeutics. DESIGN: Degeneration was induced in the lumbar intervertebral discs of large frame goats by either intradiscal injection of chondroitinase ABC (ChABC) over a range of dosages (0.1U, 1U or 5U) or subtotal nucleotomy. Radiographs were used to assess disc height changes over 12 weeks. Degenerative changes to the discs and endplates were assessed via magnetic resonance imaging (MRI), semi-quantitative histological grading, microcomputed tomography (µCT), and measurement of disc biomechanical properties. RESULTS: Degenerative changes were observed for all interventions that ranged from mild (0.1U ChABC) to moderate (1U ChABC and nucleotomy) to severe (5U ChABC). All groups showed progressive reductions in disc height over 12 weeks. Histological scores were significantly increased in the 1U and 5U ChABC groups. Reductions in T2 and T1ρ, and increased Pfirrmann grade were observed on MRI. Resorption and remodeling of the cortical boney endplate adjacent to ChABC-injected discs also occurred. Spine segment range of motion (ROM) was greater and compressive modulus was lower in 1U ChABC and nucleotomy discs compared to intact. CONCLUSIONS: A large animal model of disc degeneration was established that recapitulates the spectrum of structural, compositional and biomechanical features of human disc degeneration. This model may serve as a robust platform for evaluating the efficacy of therapeutics targeted towards varying degrees of disc degeneration.

A high-throughput model of post-traumatic osteoarthritis using engineered cartilage tissue analogs.

OBJECTIVE: A number of in vitro models of post-traumatic osteoarthritis (PTOA) have been developed to study the effect of mechanical overload on the processes that regulate cartilage degeneration. While such frameworks are critical for the identification therapeutic targets, existing technologies are limited in their throughput capacity. Here, we validate a test platform for high-throughput mechanical injury incorporating engineered cartilage. METHOD: We utilized a high-throughput mechanical testing platform to apply injurious compression to engineered cartilage and determined their strain and strain rate dependent responses to injury. Next, we validated this response by applying the same injury conditions to cartilage explants. Finally, we conducted a pilot screen of putative PTOA therapeutic compounds. RESULTS: Engineered cartilage response to injury was strain dependent, with a 2-fold increase in glycosaminoglycan (GAG) loss at 75% compared to 50% strain. Extensive cell death was observed adjacent to fissures, with membrane rupture corroborated by marked increases in lactate dehydrogenase (LDH) release. Testing of established PTOA therapeutics showed that pan-caspase inhibitor [Z-VAD-FMK (ZVF)] was effective at reducing cell death, while the amphiphilic polymer [Poloxamer 188 (P188)] and the free-radical scavenger [N-Acetyl-L-cysteine (NAC)] reduced GAG loss as compared to injury alone. CONCLUSIONS: The injury response in this engineered cartilage model replicated key features of the response of cartilage explants, validating this system for application of physiologically relevant injurious compression. This study establishes a novel tool for the discovery of mechanisms governing cartilage injury, as well as a screening platform for the identification of new molecules for the treatment of PTOA.

Glucosamine sulfate modulates the levels of aggrecan and matrix metalloproteinase-3 synthesized by cultured human osteoarthritis articular chondrocytes.

OBJECTIVE: The functional integrity of articular cartilage is determined by a balance between chondrocyte biosynthesis of extracellular matrix and its degradation. In osteoarthritis (OA), the balance is disturbed by an increase in matrix degradative enzymes and a decrease in biosynthesis of constitutive extracellular matrix molecules, such as collagen type II and aggrecan. In this study, we examined the effects of the sulfate salt of glucosamine (GS) on the mRNA and protein levels of the proteoglycan aggrecan and on the activity of matrix metalloproteinase (MMP)-3 in cultured human OA articular chondrocytes. DESIGN: Freshly isolated chondrocytes were obtained from knee cartilage of patients with OA. Levels of aggrecan and MMP-3 were determined in culture media by employing Western blots after incubation with GS at concentrations ranging from 0.2 to 200 microM. Zymography (casein) was performed to confirm that effects observed at the protein level were reflected at the level of enzymatic activity. Northern hybridizations were used to examine effects of GS on levels of aggrecan and MMP-3 mRNA. Glycosaminoglycan (GAG) assays were performed on the cell layers to determine levels of cell-associated GAG component of proteoglycans. RESULTS: Treatment of OA chondrocytes with GS (1.0-150 microM) resulted in a dose-dependent increase in aggrecan core protein levels, which reached 120% at 150 microM GS. These effects appeared to be due to increased expression of the corresponding gene as indicated by an increase in aggrecan mRNA levels in response to GS. MMP-3 levels decreased (18-65%) as determined by Western blots. Reduction of MMP-3 protein was accompanied by a parallel reduction in enzymatic activity. GS caused a dose-dependent increase (25-140%) in cell-associated GAG content. Chondrocytes obtained from 40% of OA patients failed to respond to GS. CONCLUSIONS: The results indicate that GS can stimulate mRNA and protein levels of aggrecan core protein and, at the same time, inhibit production and enzymatic activity of matrix-degrading MMP-3 in chondrocytes from OA articular cartilage. These results provide a cogent molecular mechanism to support clinical observations suggesting that GS may have a beneficial effect in the prevention of articular cartilage loss in some patients with OA.

Dose-dependent effects of corticosteroids on the expression of matrix-related genes in normal and cytokine-treated articular chondrocytes.

OBJECTIVE AND DESIGN: To assess the effects of glucocorticoids on the expression of multiple matrix-related genes in normal and cytokine-treated cultured equine articular chondrocytes in a phenotypically correct suspension culture. MATERIAL OR SUBJECTS: Articular cartilage harvested from the joints of 15 foals, 7 yearling horses, and 16 adult horses. TREATMENT: Glucocorticoids (dexamethasone, prednisolone, triamcinolone) at 10(-10) to 10(-4) M. METHODS: Equine articular chondrocytes maintained in suspension cultures were treated with glucocorticoids with and without human recombinant interleukin 1-beta (IL1-beta) and tumor necrosis factor-alpha (TNF-alpha). Northern blots of total RNA from the treated cells were probed with equine specific cDNA probes for a number of cartilage matrix-related genes. Zymography, Western blotting, and fluorography were also performed to study the effects on protein synthesis. RESULTS: The glucocorticoids, dexamethasone, triamcinolone, and prednisolone, markedly decreased MMP1, MMP3, MMP13, TIMPI, and ferritin steady-state mRNA levels. There were no qualitative differences seen among the tested corticosteroids although dexamethasone and triamcinolone appeared to be slightly more potent than prednisolone. The effects of the glucocorticoids on MMP transcription occurred consistently at lower doses than those required to similarly downregulate type II collagen and aggrecan. Link protein and fibronectin mRNA were increased by the glucocorticoids, and biglycan and decorin were minimally affected. Fluorography of [14-C] proline-labeled media demonstrated that the decrease in type II collagen transcription (mRNA levels) was paralleled at the protein level. Zymography and Western blotting confirmed the decrease in functional metalloproteinases found in chondrocyte cultures following glucocorticoid treatment. CONCLUSIONS: The effects of glucocorticoids are complex inasmuch as they differentially affect numerous genes involved in the composition of cartilage matrix and the degradation of that matrix. This study provides new insight into the effects of glucocorticoids on the regulation of extra-cellular matrix and matrix-related genes by demonstrating that low doses of glucocorticoids can inhibit the degradative metalloproteinases with minimal negative effects on the transcription of extracellular matrix genes.

Preferential incorporation of glucosamine into the galactosamine moieties of chondroitin sulfates in articular cartilage explants.

OBJECTIVE: To determine the metabolic fate of glucosamine (GlcN) in intact articular cartilage tissue. METHODS: Intact articular cartilage explants were cultured for up to 13 days in Dulbecco's modified Eagle's medium supplemented with 1) 1-13C-labeled GlcN, 2) 1-13C-labeled glucose (Glc), or 3) no labeling. Every 3-4 days, samples were removed and frozen in liquid nitrogen for carbon-13 magnetic resonance spectroscopic (MRS) analysis. The metabolic products of the labeled precursors were determined from the MRS data based on resonance positions and comparison with known standards and published values. RESULTS: GlcN was taken up by the chondrocytes and incorporated selectively into the hexosamine, but not the hexuronic acid, components of the glycosaminoglycan chains of articular cartilage proteoglycan. The data also demonstrated that GlcN is the substrate of choice for the galactosamine moieties of the chondroitin sulfates, incorporating at levels 300% higher than with an equivalent amount of labeled Glc. CONCLUSION: The results indicate that GlcN facilitates the production of proteoglycan components that are synthesized through the hexosamine biochemical pathway.

Characterization of a biomaterial with cartilage-like properties expressing type X collagen generated in vitro using neonatal porcine articular and growth plate chondrocytes.

OBJECTIVE: The availability of cartilage with or without the potential to ossify and suitable for surgical restoration and resurfacing of joints is an important clinical problem in arthritis-related pathology, trauma and reconstructive surgery. Here, we designed experiments to generate a biomaterial with cartilage-like properties by culturing neonatal porcine articular and growth plate chondrocytes on a hydrogel substrate and to examine the biochemical and histological characteristics of the resulting tissue. DESIGN: Neonatal porcine epiphyseal and growth plate chondrocytes were cultured on poly(2-hydroxyethyl methacrylate) (polyHEMA)-coated dishes to prevent their adherence to plastic. We previously described that this procedure allows the maintenance of the chondrocyte-specific phenotype for > or = 8 months. Chondrocytes were isolated by successive enzymatic digestions and cultured at high density (>2.0 x 10(7) cells/ml) in DMEM with 10% FBS, 50 microg/ml ascorbic acid, glutamine, vitamins, and antibiotics for up to 10 weeks on 60 mm plastic culture dishes coated with polyHEMA. The tissues produced during culture were studied histologically and biochemically and were examined for cellular proliferation employing(3)H-thymidine incorporation and for their collagen production employing biosynthetic labeling with(14)C-proline and Western blot with specific antibodies. The expression of relevant collagen genes was examined employing RT-PCR. RESULTS: Within 24 h of culture, isolated chondrocytes organized into well-formed clusters and in 2 weeks formed structures with gross appearance and consistency similar to those of natural cartilage. The wet weight of the tissue formed in vitro increased six-fold during the 10-week period of study. Cell proliferation measured by the incorporation of(3)H-thymidine increased during the first 3 weeks and reached a plateau in subsequent weeks. Histological examination showed that the cultures contained rounded chondrocytes embedded in an abundant cartilaginous extracellular matrix. The cartilage formed contained large amounts of collagen and sulfated proteoglycans as examined by staining with Masson's Trichrome and Alcian blue, respectively. Deposition of calcium in the deeper layers of the tissue was demonstrated with the von Kossa stain. Western analyses with specific antibodies showed that type II collagen was present from the first week and progressively increased in the cultures, whereas type X collagen was first detected at 4 weeks and increased with length of culture. When chondrocytes isolated from the growth plate were included, small amounts of type I collagen were detected in the medium of cultured biomaterial as expected. Type III collagen was not detected by Western blot over the 10-week period. High levels of type II and type X collagen gene expression were demonstrated by RT-PCR. CONCLUSION: These studies demonstrate the production in vitro of cartilage-like tissue with similar morphological, histochemical and biochemical characteristics to those of natural growth plate cartilage. The cartilage generated in vitro has the potential to be used in reconstructive surgery and in joint resurfacing and restoration of skeletal defects.

Effects of interleukin-1beta and tumor necrosis factor-alpha on expression of matrix-related genes by cultured equine articular chondrocytes.

OBJECTIVE: To determine the effects of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on expression and regulation of several matrix-related genes by equine articular chondrocytes. SAMPLE POPULATION: Articular cartilage harvested from grossly normal joints of 8 foals, 6 yearling horses, and 8 adult horses. PROCEDURE: Chondrocytes maintained in suspension cultures were treated with various doses of human recombinant IL-1beta or TNF-alpha. Northern blots of total RNA from untreated and treated chondrocytes were probed with equine complementary DNA (cDNA) probes for cartilage matrix-related genes. Incorporation of 35S-sulfate, fluorography of 14C-proline labeled medium, zymography, and western blotting were used to confirm effects on protein synthesis. RESULTS: IL-1beta and TNF-alpha increased steady-state amounts of mRNA of matrix metalloproteinases 1, 3, and 13 by up to 100-fold. Amount of mRNA of tissue inhibitor of metalloproteinase-1 also increased but to a lesser extent (1.5- to 2-fold). Amounts of mRNA of type-II collagen and link protein were consistently decreased in a dose-dependent manner. Amount of aggrecan mRNA was decreased slightly; amounts of biglycan and decorin mRNA were minimally affected. CONCLUSIONS AND CLINICAL RELEVANCE: Treatment of cultured equine chondrocytes with IL-1beta or TNF-alpha resulted in marked alterations in expression of various matrix and matrix-related genes consistent with the implicated involvement of these genes in arthritis. Expression of matrix metalloproteinases was increased far more than expression of their putative endogenous inhibitor. Results support the suggestion that IL-1beta and TNF-alpha play a role in the degradation of articular cartilage in arthritis.

Cartilage-reactive T cells in rheumatoid synovium.

Rheumatoid arthritis (RA) is an inflammatory polyarthritis genetically linked to HLA-DR4 and related haplotypes. RA synovial tissue is characterized by T cell infiltration and activation of macrophage-like cells, strongly implicating a T cell-antigen-presenting cell (APC) interaction in RA pathogenesis. To investigate the nature of the antigens driving the T cell response, synovial tissue was obtained from a patient with chronic RA and T cells were enriched. These T cells were stimulated by endogenous APC from the same synovial tissue. The T cell lines were subsequently evaluated for responsiveness to autologous APC and cartilage antigens. Specific proliferative responses to autologous APC which were enhanced by cartilage extract were seen. Immunomagnetic bead selection and RT-PCR was used to identify TCR alphabeta pairs which appeared to respond to antigen(s) in the cartilage extract. T cell clones derived from the same joint were shown to release IL-2 in response to the cartilage extract and expressed a related TCR. With these experiments we have shown direct evidence that autoreactive T cells are found within the inflamed rheumatoid synovium and, further, that the antigens driving these T cells are cartilage derived. Since the antigens recognized by these populations of T cells are found within cartilage our data provides evidence that RA pathology could be related to a self-driven autoimmune response to cartilage proteins.

Molecular characteristics of equine stromelysin and the tissue inhibitor of metalloproteinase 1.

OBJECTIVE: To clone the entire coding sequence of equine matrix metalloproteinase-3 (MMP-3, stromelysin) and tissue inhibitor of metalloproteinase-1 (TIMP-1) and compare their nucleotide and amino acid sequences with those of MMP-3 and TIMP-1 from other species. SAMPLES: Articular cartilage harvested from the joints of 4 foals, 2 yearlings, and 3 adult horses. PROCEDURE: A cDNA library was constructed from mRNA extracted from equine chondrocytes. The library was screened and clones selected that contained the cDNA for MMP-3 and TIMP-1. The cDNA was sequenced and the nucleotide and deduced amino acid sequences compared with known sequences in other species. Northern blot analysis was performed, using the resulting cDNA clones. RESULTS: An 1803-bp cDNA for MMP-3 including the entire coding sequence of 1434 bases was cloned and sequenced. A 744-bp cDNA for TIMP-1 including the entire coding sequence of 624 bases was cloned and sequenced. Northern analysis revealed MMP-3 to hybridize to a single mRNA species at approximately 2.1 kb. TIMP-1 hybridized to a single mRNA species at approximately 0.8 kb. CONCLUSIONS: MMP-3 and TIMP-1 were highly homologous to that of other species at the nucleotide and amino acid level although each had unique residues in part of the peptide that is generally conserved. CLINICAL RELEVANCE: Understanding the molecular structure of MMP-3 and TIMP-1 and the availability of their cDNA should allow a more detailed understanding of their balance in cartilage and the degradative processes in joint disease.

Effects of interferon-gamma and tumor necrosis factor alpha on the expression of the genes encoding aggrecan, biglycan, and decorin core proteins in cultured human chondrocytes.

OBJECTIVE: To determine the effects of interferon-gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha), alone or in combination, on the expression of aggrecan, biglycan, and decorin core protein genes in human chondrocytes. METHODS: Isolated human chondrocytes were cultured on poly(2-hydroxyethyl methacrylate)-coated plastic dishes to prevent the loss of cartilage-specific phenotype, and the effects of IFN gamma and TNF alpha, alone or in combination, on aggrecan, biglycan, and decorin core protein gene transcription and steady-state messenger RNA (mRNA) levels were examined. RESULTS: The addition of IFN gamma (1.5 pM) or TNF alpha (0.3 pM) caused a decrease in the steady-state level of aggrecan mRNA (-25% and -15%, respectively), and the combination of these low-concentration cytokines caused a potent inhibition (-66%). These effects were the result of a decrease (-50%) in the transcription rate of the corresponding gene. At the concentrations used, IFN gamma did not alter the levels of biglycan mRNA or the transcription rates of the biglycan core protein gene. In contrast, TNF alpha decreased biglycan steady-state mRNA levels (-62%) and the biglycan core protein gene transcription rate (-18%). The combination of IFN gamma and TNF alpha resulted in a potentiation of the inhibitory effects of TNF alpha on biglycan mRNA levels (-79%) and transcription rate of the biglycan core protein gene (-46%). IFN gamma produced a modest decrease in decorin mRNA levels (-23%) and decorin core protein gene transcription rate (-17%). In contrast, TNF alpha resulted in a marked increase in decorin mRNA levels (+260%) that was not the result of transcriptional regulation. Notably, the combination of IFN gamma and TNF alpha potentiated the inhibitory effects of IFN gamma on decorin mRNA (-80%) and on the transcription of the corresponding gene (-43%). Similar results were obtained in fetal and adult articular chondrocytes. CONCLUSION: These data demonstrate that 1) the expression of the core protein genes encoding the cartilage proteoglycans aggrecan, biglycan, and decorin is differentially regulated by IFN gamma and TNF alpha; 2) these effects are mediated by transcriptional and posttranscriptional mechanisms; and 3) the combination of the 2 cytokines causes a potent inhibitory effect on the expression of the genes for the core proteins of these 3 proteoglycans, which occurs largely at the transcriptional level. The inhibition of aggrecan, decorin, and biglycan core protein gene expression by the combination of IFN gamma and TNF alpha may contribute to the cartilage destruction that is characteristic of inflammatory joint diseases.

Production of cartilage oligomeric matrix protein (COMP) by cultured human dermal and synovial fibroblasts.

OBJECTIVE: Cartilage oligomeric matrix protein (COMP) is a large disulfide-linked pentameric protein. Each of its five subunits is approximately 100,000 Da in molecular weight. COMP was originally identified and characterized in cartilage and it has been considered a marker of cartilage metabolism because it is currently thought not to be present in other joint tissues, except for tendon. To confirm the tissue specificity of COMP expression we examined cultured human dermal fibroblasts, human foreskin fibroblasts, and normal human synovial cells for the synthesis of COMP in culture. METHOD: Normal synovial cells and normal human dermal foreskin fibroblasts were isolated from the corresponding tissues by sequential enzymatic digestions and cultured in media containing 10% fetal bovine serum until confluent. During the final 24 h of culture, the cells were labeled with 35S-methionine and 35S-cysteine in serum- and cysteine/methionine-free medium. The newly synthesized COMP molecules were immunoprecipitated from the culture media with a COMP-specific polyclonal antiserum, or with monoclonal antibodies or affinity-purified COMP antibodies. The immunoprecipitated COMP was analyzed by electrophoresis in 5.5% polyacrylamide gels. For other experiments, synovial cells cultured from the synovium of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) were similarly examined. RESULTS: A comparison of the amounts of COMP produced by each cell type (corrected for the DNA content) revealed that synovial cells produced > or = 9 times more COMP than chondrocytes or dermal fibroblasts. COMP could be easily detected by immunoprecipitation in all cell types. Electrophoretic analysis revealed a distinct band with an apparent MW of 115-120 kDa in samples from each of the three cell types, regardless of the antibody used. COMP expression in cultures of synoviocytes derived from OA and RA patients showed that OA and RA synovial cells produced similar amounts of monomeric COMP of identical size to those COMP monomers produced by normal synovial cells. The addition of TGF-beta to these cultures resulted in an increase in COMP production in normal, OA and RA synovial cells (45, 116 and 115% respectively). CONCLUSION: These studies demonstrate that substantial amounts of COMP are produced by several mesenchymal cells including synoviocytes and dermal fibroblasts. These findings raise important concerns regarding the utility of measurements of COMP levels in serum or in synovial fluid as markers of articular cartilage degradation because of the likelihood that a substantial proportion of COMP or COMP fragments present in serum or synovial fluid may be produced by cells other than articular chondrocytes.

Reduction in fibronectin expression and alteration in cell morphology are coincident in NIH3T3 cells expressing a mutant E2F1 transcription factor.

Fibronectin within the extracellular matrix plays a role in cell attachment, spreading, and shape, while it also affects aspects of cell proliferation. Transcription factors such as E2F1 are also known to regulate cell shape and cell proliferation. Yet, to date no linkage has been established between fibronectin expression and E2F1. We show here that cells constitutively expressing a mutant E2F1 protein (E2F1d87) produce reduced amounts of fibronectin mRNA and protein. The altered expression of fibronectin seen in the E2F1d87 expressing cells is due, in part, to a reduction in transcription from the fibronectin promoter. Providing exogenous fibronectin, but not Type I collagen or laminin, as a substrate for cell adhesion is sufficient to revert the altered morphology and reestablish actin-containing microfilaments lost in the mutant cell line. An additional characteristic of the cells expressing the mutant E2F1 is that they demonstrate slow growth and a doubling in S phase duration. While providing exogenous fibronectin as an adhesion substrate did not shorten the S phase duration in the mutant line, it did significantly shorten the S phase duration in the parental NIH3T3 cell line, implicating a role for the extracellular matrix in regulating S phase transit in normal cells.

Cloning of equine type II procollagen and the modulation of its expression in cultured equine articular chondrocytes.

The complete nucleotide sequence of equine type II procollagen has not been previously reported, and equine-specific probes have not been available. We report the complete sequence and discuss the molecular characteristics of equine type II procollagen mRNA which was cloned from a cDNA library prepared from mRNA isolated from equine articular chondrocytes. The coding sequence (4257 bp) was 92.4% homologous to the cDNA of the human sequence, and the propeptide was 97% identical to the human sequence. We demonstrated that when equine chondrocytes are grown in phenotypically-maintained cultures, the expression of type II procollagen is linked to the age of the animal. Additionally, in cultures of young equine chondrocytes, IL1-beta and TNF-alpha both reduced the expression of type II procollagen mRNA in a dose responsive manner.

Characterization of human type II procollagen and collagen-specific antibodies and their application to the study of human type II collagen processing and ultrastructure.

Type II collagen is the most abundant collagen in articular cartilage and, together with other tissue-specific collagens and proteoglycans, provides the tissue with its shock-absorbing properties and its resiliency to stress. Specific antibodies which recognize various collagen types have been very useful in the study of collagen biosynthesis, structure and metabolism in normal and pathological conditions. Antibodies which recognize epitopes of type II collagen have been described previously; however, many of these antibodies display cross-reactivity with other collagens or with type II collagen from other species, reflecting the high degree of homology of the helical domains of fibrillar collagens. In this study, we prepared antibodies to sequential determinants of human type II procollagen employing synthetic peptides with sequences deduced from the nucleotide sequence of the human alpha 1 (II) procollagen cDNA. The antibodies were highly specific for epitopes in either the C-terminal propeptide or the telopeptide of the human type II collagen and did not cross-react with other human interstitial collagens or with murine type II collagen. These antibodies were used in conjunction with biosynthetic labeling to study the secretion and processing of human type II procollagen and collagen in human chondrocytes in vitro. The results indicated that a lag period of about 90 min was required for the secretion of newly synthesized type II procollagen. Conversion of the secreted procollagen into fully processed alpha-chains and their deposition in the cell layer were first apparent 240 min following the initiation of biosynthetic labeling. The antibodies were also used to examine, by immunoelectron microscopy, the structure of the extracellular matrix produced by human chondrocytes maintained in long-term cultures under conditions which permit the preservation of the cartilage-specific phenotype. These highly specific antibodies provide valuable tools to study the metabolism and structure of human type II procollagen and collagen in normal and pathologic conditions.

Expression of the basement membrane heparan sulfate proteoglycan (perlecan) in human synovium and in cultured human synovial cells.

BACKGROUND: Perlecan is a heparan sulfate proteoglycan that has a core protein of 466 kDa and is composed of five modules, four of which share a high degree of homology with a variety of biologically important molecules including the low-density lipoprotein receptor, laminin, and the neural cell adhesion molecule. Previously, this specialized proteoglycan, characteristic of the basement membrane, was not identified as a constitutive extracellular component of human synovium. EXPERIMENTAL DESIGN: Using human synovium and cultured human synovial cells, we examined perlecan gene expression in human synovium, a tissue lacking a classic basement membrane. The modulation of gene expression of the perlecan core protein by transforming growth factor-beta (TGF-beta) and basic fibroblast growth factor (FGF) was examined in cultures of normal human synovial cells, and by metabolic labeling using radioactive sodium sulfate, the question of whether this specialized protein is produced by synovial cells as a proteoglycan was addressed. RESULTS: Cultures of normal human synovial cells were shown to contain the large 14.5 kb perlecan mRNA and produced substantial amounts of perlecan core protein as shown by immunohistochemistry employing specific human perlecan Ab. Immunohistochemical detection showed intense staining in the intimal and subintimal layers of human synovial membrane. Moreover, the perlecan core protein was shown to be up-regulated by TGF-beta and down-regulated by basic FGF. In addition, a sizable portion (approximately 25%) of the synovial cell-produced proteoglycan was shown to contain heparan sulfate, providing evidence that synovial cell perlecan is produced as a proteoglycan. CONCLUSIONS: The presence of perlecan in human synovium and the modulation of the biosynthesis of its core protein by TGF-beta and FGF suggest that, in addition to its structural role, this unique heparan sulfate proteoglycan may be involved in normal synovial membrane function and in the pathogenesis of arthritis.

Transcatheter delivery of c-myc antisense oligomers reduces neointimal formation in a porcine model of coronary artery balloon injury.

BACKGROUND: Smooth muscle cell proliferation and extracellular matrix accumulation are the principal mechanisms leading to vascular restenosis. We have previously demonstrated the growth-inhibitory effect of antisense oligomers targeting the c-myc proto-oncogene in human smooth muscle cells. The goal of this study was to investigate whether c-myc antisense oligomers reduce neointimal formation in balloon-denuded porcine coronary arteries. METHODS AND RESULTS: First, type I collagen synthesis, which reflects synthetic function, was markedly reduced following c-myc antisense oligomers in porcine vascular smooth muscle cells independent of the growth inhibition. These effects in vitro provided the rationale for assessing c-myc antisense oligomers in the prevention of neointima in vivo. Second, the efficiency of single transcatheter delivery of oligomers into denuded porcine coronary arteries was determined. Despite rapid plasma clearance following local delivery, oligomers persisted at the site of injection for at least 3 days, exceeding by severalfold their concentration in peripheral organs. Third, morphometric analyses were carried out in balloon-denuded coronary arteries at 1 month after transcatheter c-myc antisense oligomer administration. Maximal neointimal area was reduced from 0.80 +/- 0.17 mm2 in the control group (n = 12) to 0.24 +/- 0.06 mm2 in the antisense-treated group (n = 13, P < .01). Likewise, a significant reduction in maximal neointimal thickness was observed in the antisense-treated group (P < .01). These changes in vascular remodeling following denuding injury resulted in an increase in residual lumen from 64 +/- 6% in the control group to 81 +/- 5% in the antisense-treated group (P < .05). CONCLUSIONS: (1) Single transcatheter administration allowed for endoluminal delivery of oligomers to the site of coronary arterial injury. (2) C-myc antisense oligomers reduced the formation of neointima in denuded coronary arteries, implying a therapeutic potential of this approach for the prevention of coronary restenosis. (3) It is postulated that the c-myc proto-oncogene is involved in the process of vascular remodeling, regulating smooth muscle cell proliferation and extracellular matrix synthesis.

Decorin, versican, and biglycan gene expression by keloid and normal dermal fibroblasts: differential regulation by basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is a multipotential heparin-binding factor that belongs to the fibroblast growth factor (FGF) family. The FGFs demonstrate a wide spectrum of biologic activities in vivo and in vitro. In this study, we investigated the potential of bFGF to regulate the expression of various dermal extracellular matrix proteoglycans and type I collagen mRNAs in cultured human fibroblasts from keloid, which is a prototype of dermal fibrosis, and normal skin tissue. We report that bFGF upregulates the expression of the decorin gene in normal and keloid fibroblasts. In contrast, the expression of biglycan is downregulated by bFGF. The mRNA steady-state level of versican, a large proteoglycan, is not altered by bFGF. Type I collagen gene expression is downregulated substantially in keloid and normal fibroblasts by bFGF. The results suggest that the expression of the proteoglycan genes are uncoordinately regulated and that the gene expression of type I collagen and biglycan is coordinately downregulated. The results also demonstrate that keloid fibroblasts respond similarly as do normal fibroblasts to bFGF in the regulation of proteoglycan and collagen expression.