Contacts with fibrils containing collagen I, but not collagens II, IX, and XI, can destabilize the cartilage phenotype of chondrocytes. (1/125)

OBJECTIVE: Cell-matrix interactions are important regulators of cellular functions, including matrix synthesis, proliferation and differentiation. This is well exemplified by the characteristically labile phenotype of chondrocytes that is lost in monolayer culture but is stabilized in suspension under appropriate conditions. We were interested in the role of collagen suprastructures in maintaining or destabilizing the cartilage phenotype of chondrocytes. DESIGN: Primary sternal chondrocytes from 17-day-old chick embryos were cultured in gels of fibrils reconstituted from soluble collagen I from various sources. The culture media either contained or lacked FBS. Cells were cultured for up to 28 days and the evolution of the phenotype of the cells was assessed by their collagen expression (collagens II and X for differentiated chondrocytes and hypertrophic chodrocytes, repectively; collagen I for phenotypically modulated cells), or by their secretion of alkaline phosphatase (hypertrophic cartilage phenotype). RESULTS: The cells often retained their differentiated phenotype only if cultured with serum. Under serum-free conditions, cartilage characteristics were lost. The cells acquired a fibroblast-like shape and, later, synthesized collagen I instead of cartilage collagens. Shape changes were influenced by beta1-integrin-activity, whereas other matrix receptors were important for alterations of collagen patterns. Heterotypic fibrils reconstituted from collagens II, IX, and XI did not provoke this phenotypic instability. CONCLUSIONS: Chondrocytes sensitively recognize the suprastructures of collagen fibrils in their environment. Cellular interactions with fibrils with appropriate molecular organizations, such as that in cartilage fibrils, result in the maintenance of the differentiated cartilage phenotype. However, other suprastructures, e.g. in reconstituted fibrils mainly containing collagen I, lead to cell-matrix interactions incompatible with the cartilage phenotype. The maintenance of the differentiated traits of chondrocytes is pivotal for the normal function of, e.g., articular cartilage. If pathologically altered matrix suprastructures lead to a dysregulation of collagen production also in vivo compromised cartilage functions inevitably will be propagated further.  (+info)

Articular cartilage repair using a tissue-engineered cartilage-like implant: an animal study. (2/125)

OBJECTIVE: Because articular cartilage has limited ability to repair itself, treatment of (osteo)chondral lesions remains a clinical challenge. We aimed to evaluate how well a tissue-engineered cartilage-like implant, derived from chondrocytes cultured in a novel patented, scaffold-free bioreactor system, would perform in minipig knees with chondral, superficial osteochondral, and full-thickness articular defects. DESIGN: For in vitro implant preparation, we used full-thickness porcine articular cartilage and digested chondrocytes. Bioreactors were seeded with 20x10(6) cells and incubated for 3 weeks. Subsequent to culture, tissue cartilage-like implants were divided for assessment of viability, formaldehyde-fixed and processed by standard histological methods. Some samples were also prepared for electron microscopy (TEM). Proteoglycans and collagens were identified and quantified by SDS-PAGE gels. For in vivo studies in adult minipigs, medial parapatellar arthrotomy was performed unilaterally. Three types of defects were created mechanically in the patellar groove of the femoral condyle. Tissue-engineered cartilage-like implants were placed using press-fit fixation, without supplementary fixation devices. Control defects were not grafted. Animals could bear full weight with an unlimited range of motion. At 4 and 24 weeks postsurgery, explanted knees were assessed using the modified ICRS classification for cartilage repair. RESULTS: After 3-4 weeks of bioreactor incubation, cultured chondrocytes developed a 700-microm- to 1-mm-thick cartilage-like tissue. Cell density was similar to that of fetal cartilage, and cells stained strongly for Alcian blue and safranin O. The percentage of viable cells remained nearly constant (approximately 90%). Collagen content was similar to that of articular cartilage, as shown by SDS-PAGE. At explantation, the gross morphological appearance of grafted defects appeared like normal cartilage, whereas controls showed irregular fibrous tissue covering the defect. Improved histologic appearance was maintained for 6 months postoperatively. Although defects were not always perfectly level upon implantation at explanation the implant level matched native cartilage levels with no tissue hypertrophy. Once in place, implants remodelled to tissues with decreased cell density and a columnar organization. CONCLUSIONS: Repair of cartilage defects with a tissue-engineered implant yielded a consistent gross cartilage repair with a matrix predominantly composed of type II collagen up to 6 months after implantation. This initial result holds promise for the use of this unique bioreactor/tissue-engineered implant in humans.  (+info)

COL11A1 in FAP polyps and in sporadic colorectal tumors. (3/125)

BACKGROUND: We previously reported that the alpha-1 chain of type 11 collagen (COL11A1), not normally expressed in the colon, was up-regulated in stromal fibroblasts in most sporadic colorectal carcinomas. Patients with germline mutations in the APC gene show, besides colonic polyposis, symptoms of stromal fibroblast involvement, which could be related to COL11A1 expression. Most colorectal carcinomas are suggested to be a result of an activated Wnt- pathway, most often involving an inactivation of the APC gene or activation of beta-catenin. METHODS: We used normal and polyp tissue samples from one FAP patient and a set of 37 sporadic colorectal carcinomas to find out if the up-regulation of COL11A1 was associated with an active APC/beta-catenin pathway. RESULTS: In this study we found a statistically significant difference in COL11A1 expression between normal tissue and adenomas from one FAP patient, and all adenomas gave evidence for an active APC/beta-catenin pathway. An active Wnt pathway has been suggested to involve stromal expression of WISP-1. We found a strong correlation between WISP-1 and COL11A1 expression in sporadic carcinomas. CONCLUSIONS: Our results suggest that expression of COL11A1 in colorectal tumors could be associated with the APC/beta-catenin pathway in FAP and sporadic colorectal cancer.  (+info)

Expression of collagen and aggrecan genes in normal and osteoarthritic murine knee joints. (4/125)

OBJECTIVE: The STR/ort mouse strain develops osteoarthritis (OA) of the medial tibial cartilage whilst CBA mice do not develop this disease. We investigated whether changes occur in the expression of genes encoding major extracellular matrix proteins in the connective tissue of the murine knee joint in OA. DESIGN: Expression of the genes encoding collagens II (Col2alpha1), X (Col10alpha1), alpha2(XI) (Col11alpha2) and aggrecan (Agc) was detected in skeletally mature and immature male mice of the CBA and STR/ort strains by in situ hybridization. RESULTS: Col2alpha1 was expressed by chondrocytes of the tibial and patella-femoral cartilage and by the meniscal cartilage in all young mice (4-9 weeks) but only in the patella-femoral cartilage in older mice of both strains (36-45 weeks). In contrast Col2alpha1 was expressed by growth plate chondrocytes of both species at all ages. Similarly, Col2alpha1 transcripts were detected in cruciate ligament cells in both strains at all ages. Col10alpha1 transcripts were detected in cruciate ligament cells in both strains at all ages. Col10alpha1 expression was evident in the hypertrophic chondrocytes in the growth plate of young CBA and STR mice, but was not active in these cells in mature animals. However, Col10alpha1 was transcribed in articular chondrocytes of the tibia, meniscal and patella-femoral cartilages of all ages, in normal and osteoarthritic mice. Transcripts were also present in ligament of some mature animals. Col11alpha2 followed a similar pattern of expression in CBA cartilages to Col2alpha1, being active in adult growth plate but generally inactive in adult articular cartilages. Young CBA and STR/ort mice expressed Col11alpha2 in articular cartilage and very strongly throughout the growth plate. Agc expression was detected in all articular cartilages at all ages in both strains. Interestingly, transcripts for all four genes were absent in tibial articular chondrocytes located close to osteoarthritic lesions in STR/ort mice, indicating that these cells are unable to synthesize matrix proteins. Adult STR/ort mice also showed evidence of tissue remodeling around the periphery of the knee joint. Cells in remodeling areas actively transcribed Col2alpha1, Col10alpha1, Col11alpha2 and Agc. CONCLUSION: It is unlikely that OA develops in STR/ort mice because of failure to express major proteins in joint tissue. However, once lesions develop in articular cartilage neighbouring chondrocytes fail to express genes encoding several matrix proteins.  (+info)

A Kruppel-associated box-zinc finger protein, NT2, represses cell-type-specific promoter activity of the alpha 2(XI) collagen gene. (5/125)

Type XI collagen is composed of three chains, alpha 1(XI), alpha 2(XI), and alpha 3(XI), and plays a critical role in the formation of cartilage collagen fibrils and in skeletal morphogenesis. It was previously reported that the -530-bp promoter segment of the alpha 2(XI) collagen gene (Col11a2) was sufficient for cartilage-specific expression and that a 24-bp sequence from this segment was able to switch promoter activity from neural tissues to cartilage in transgenic mice when this sequence was placed in the heterologous neurofilament light gene (NFL) promoter. To identify a protein factor that bound to the 24-bp sequence of the Col11a2 promoter, we screened a mouse limb bud cDNA expression library in the yeast one-hybrid screening system and obtained the cDNA clone NT2. Sequence analysis revealed that NT2 is a zinc finger protein consisting of a Kruppel-associated box (KRAB) and is a homologue of human FPM315, which was previously isolated by random cloning and sequencing. The KRAB domain has been found in a number of zinc finger proteins and implicated as a transcriptional repression domain, although few target genes for KRAB-containing zinc finger proteins has been identified. Here, we demonstrate that NT2 functions as a negative regulator of Col11a2. In situ hybridization analysis of developing mouse cartilage showed that NT2 mRNA is highly expressed by hypertrophic chondrocytes but is minimally expressed by resting and proliferating chondrocytes, in an inverse correlation with the expression patterns of Col11a2. Gel shift assays showed that NT2 bound a specific sequence within the 24-bp site of the Col11a2 promoter. We found that Col11a2 promoter activity was inhibited by transfection of the NT2 expression vector in RSC cells, a chondrosarcoma cell line. The expression vector for mutant NT2 lacking the KRAB domain failed to inhibit Col11a2 promoter activity. These results demonstrate that KRAB-zinc finger protein NT2 inhibits transcription of its physiological target gene, suggesting a novel regulatory mechanism of cartilage-specific expression of Col11a2.  (+info)

Adjacent DNA sequences modulate Sox9 transcriptional activation at paired Sox sites in three chondrocyte-specific enhancer elements. (6/125)

Expression of the type XI collagen gene Col11a2 is directed to cartilage by at least three chondrocyte-specific enhancer elements, two in the 5' region and one in the first intron of the gene. The three enhancers each contain two heptameric sites with homology to the Sox protein-binding consensus sequence. The two sites are separated by 3 or 4 bp and arranged in opposite orientation to each other. Targeted mutational analyses of these three enhancers showed that in the intronic enhancer, as in the other two enhancers, both Sox sites in a pair are essential for enhancer activity. The transcription factor Sox9 binds as a dimer at the paired sites, and the introduction of insertion mutations between the sites demonstrated that physical interactions between the adjacently bound proteins are essential for enhancer activity. Additional mutational analyses demonstrated that although Sox9 binding at the paired Sox sites is necessary for enhancer activity, it alone is not sufficient. Adjacent DNA sequences in each enhancer are also required, and mutation of those sequences can eliminate enhancer activity without preventing Sox9 binding. The data suggest a new model in which adjacently bound proteins affect the DNA bend angle produced by Sox9, which in turn determines whether an active transcriptional enhancer complex is assembled.  (+info)

Collagen XI sequence variations in nonsyndromic cleft palate, Robin sequence and micrognathia. (7/125)

Cleft palate is a common birth defect, but its etiopathogenesis is mostly unknown. Several studies have shown that cleft palate has a strong genetic component. Robin sequence consists of three of the following four findings: micrognathia, glossoptosis, obstructive apnea, and cleft palate. While cleft palate is mainly nonsyndromic, about 80 percent of Robin sequence cases are associated with syndromes. Mutations in genes coding for cartilage collagens II and XI, COL2A1, COL11A1 and COL11A2, have been shown to cause chondrodysplasias that are commonly associated with Robin sequence, micrognathia or cleft palate. We therefore analyzed a cohort of 24 patients with nonsyndromic Robin sequence, 17 with nonsyndromic cleft palate and 21 with nonsyndromic micrognathia for mutations in COL11A2. A total of 23 Robin sequence patients were also analyzed for mutations in COL2A1 and COL11A1. We detected two disease-associated mutations in patients with Robin sequence, an Arg to stop codon mutation in COL11A2 and a splicing mutation in COL11A1. Two putatively disease-associated sequence variations were found in COL11A1 in Robin sequence patients, one in COL11A2 in a patient with micrognathia and one in COL2A1 in two patients with Robin sequence. The results showed that sequence variations in these genes can play a role in the etiology of Robin sequence, cleft palate and micrognathia but are not common causes of these phenotypes.  (+info)

Loss of DNA-dependent dimerization of the transcription factor SOX9 as a cause for campomelic dysplasia. (8/125)

Campomelic dysplasia (CD) is a semilethal osteochondrodysplasia, characterized by skeletal anomalies that include bending of the long bones, and by XY sex reversal. CD results from haploinsufficiency for the transcription factor SOX9, a key regulator at various steps of cartilage differentiation and of early testis development. Two functional domains are so far recognized for SOX9, a high-mobility group (HMG) DNA-binding domain and a C-terminal transactivation domain. We present two CD patients with de novo mutations in a conserved region preceding the HMG domain. A long-term survivor with the acampomelic form of CD has an A76E amino acid substitution, while a severely affected CD patient had an in-frame deletion of amino acid residues 66-75. The conserved domain has been shown to function in the related transcription factor SOX10 as a DNA-dependent dimerization domain. We show that, like SOX10, SOX9 also binds cooperatively as a dimer to response elements in regulatory regions of some target genes such as the cartilage genes Col11a2 and CD-Rap. Dimerization and the resulting capacity to activate promoters via dimeric binding sites is lost in both mutant SOX9 proteins while other features involved in SOX9 function remained unaltered. These findings establish the dimerization domain as the third domain essential for SOX9 function during chondrogenesis.  (+info)