Low oxygen tension stimulates the redifferentiation of dedifferentiated adult human nasal chondrocytes.
(17/80)
OBJECTIVE: To determine the effect of dissolved oxygen tension (DO) on the redifferentiation of dedifferentiated adult human nasal septum chondrocytes cultured as pellets. DESIGN: After isolation, human nasal chondrocytes were expanded in monolayer culture, which resulted in their dedifferentiation. Dedifferentiated cells were pelleted, transferred to a bioreactor and maintained for up to 21 days at 100% DO (21% oxygen), 25% DO (5.25% oxygen) or 5% DO (1% oxygen), which was controlled in the liquid phase. Redifferentiation was assessed by staining the extracellular matrix with safranin-O and by the immunolocalization of collagen types I, II, IX and of a fibroblast membrane marker (11-fibrau). In addition, glycosaminoglycans (GAG) and DNA content were determined spectrophotometrically. RESULTS: In monolayer culture, cells dedifferentiated and multiplied 90- to 100-fold. Cell pellets cultured in a bioreactor under conditions of low oxygen tension (25% DO or 5% DO) stained intensely for GAGs and for collagen type II, but very weakly for collagen type I. After 14 days of culturing, cell pellets maintained at 5% DO stained more intensely for collagen IX and more weakly for 11-fibrau than did those incubated at 25% DO. After 21 days of culturing the GAG content of cell pellets maintained at 5% DO was significantly greater than that of those incubated at 25% DO. Under air-saturated conditions (100% DO), the DNA and GAG contents of cell pellets decreased with time in culture. After 21 days of culturing, both parameters were substantially lower in cell pellets maintained at 100% DO than in those incubated at low oxygen tensions. The staining signals for collagen types II and IX were much weaker, and those for the markers of dedifferentiation (collagen type I and 11-fibrau) much stronger under air-saturated conditions than at low oxygen tensions. CONCLUSION: These observations demonstrate that using the present set-up, low oxygen tension stimulates the redifferentiation of dedifferentiated adult human nasal chondrocytes in pellet cultures. (+info)
Age-related changes on the surface of vitreous collagen fibrils.
(18/80)
PURPOSE: To determine whether aging vitreous collagen fibrils undergo ultrastructural changes that might underlie vitreous liquefaction and posterior vitreous detachment. METHODS: Vitreous collagen fibrils from 21 human subjects (age range, 3-89 years) and from bovine eyes were isolated on electron microscopy grids. Cupromeronic blue labeling in the presence of 0.3 M MgCl(2) and immunogold labeling for collagen types II and IX were analyzed by transmission electron microscopy. RESULTS: Aging was associated with marked changes on the surface of human vitreous collagen fibrils, including an exponential loss of type IX collagen along with its chondroitin sulfate side-chains (half-life, 11 years) and a fourfold increase in the exposure of type II collagen. CONCLUSIONS: Despite being a minor component of vitreous collagen fibrils, type IX collagen, probably by virtue of its chondroitin sulfate side-chains, shields type II collagen from exposure on the fibril surface. With aging, this shielding diminishes, resulting in the surface exposure of "sticky" type II collagen and thus predisposing the vitreous collagen fibrils to fusion. These changes could underlie vitreous liquefaction and weakening of vitreoretinal adhesion. (+info)
Characterization of recombinant amino-terminal NC4 domain of human collagen IX: interaction with glycosaminoglycans and cartilage oligomeric matrix protein.
(19/80)
The N-terminal NC4 domain of collagen IX is a globular structure projecting away from the surface of the cartilage collagen fibril. Several interactions have been suggested for this domain, reflecting its location and its characteristic high isoelectric point. In an attempt to characterize the NC4 domain in more detail, we set up a prokaryotic expression system to produce the domain. The purified 27.5-kDa product was analyzed for its glycosaminoglycan-binding potential by surface plasmon resonance and solid-state assays. The results show that the NC4 domain of collagen IX specifically binds heparin with a K(d) of 0.6 microm, and the full-length recombinant collagen IX has an even stronger interaction with heparin, with an apparent K(d) of 3.6 nm. The heparin-binding site of the NC4 domain was located in the extreme N terminus, containing a heparin-binding consensus sequence, whereas electron microscopy suggested the presence of at least three additional heparin-binding sites on full-length collagen IX. The NC4 domain was also shown to bind cartilage oligomeric matrix protein. This interaction and the association of cartilage oligomeric matrix protein with other regions of collagen IX were found to be heparin-competitive. Circular dichroism analyses of the NC4 domain indicated the presence of stabilizing disulfide bonds and a thermal denaturation point of about 80 degrees C. The pattern of disulfide bond formation within the NC4 domain was identified by tryptic peptide mass mapping of the NC4 in native and reduced states. A similar pattern was demonstrated for the NC4 domain of full-length recombinant collagen IX. (+info)
Hyaline cartilage engineered by chondrocytes in pellet culture: histological, immunohistochemical and ultrastructural analysis in comparison with cartilage explants.
(20/80)
Cartilage engineering is a strategic experimental goal for the treatment of multiple joint diseases. Based on the process of embryonic chondrogenesis, we hypothesized that cartilage could be engineered by condensing chondrocytes in pellet culture and, in the present study, examined the quality of regenerated cartilage in direct comparison with native cartilage. Chondrocytes isolated from the sterna of chick embryos were cultured in pellets (4 x 10(6) cells per pellet) for 2 weeks. Cartilage explants from the same source were cultured as controls. After 2 weeks, the regenerated cartilage from pellet culture had a disc shape and was on average 9 mm at the longest diameter. The chondrocyte phenotype was stabilized in pellet culture as shown by the synthesis of type II collagen and aggrecan, which was the same intensity as in the explant after 7 days in culture. During culture, chondrocytes also continuously synthesized type IX collagen. Type X collagen was negatively stained in both pellets and explants. Except for fibril orientation, collagen fibril diameter and density in the engineered cartilage were comparable with the native cartilage. In conclusion, hyaline cartilage engineered by chondrocytes in pellet culture, without the transformation of cell phenotypes and scaffold materials, shares similarities with native cartilage in cellular distribution, matrix composition and density, and ultrastructure. (+info)
The fibril-associated collagen IX provides a novel mechanism for cell adhesion to cartilaginous matrix.
(21/80)
Collagen IX is the prototype fibril-associated collagen with interruptions in triple helix. In human cartilage it covers collagen fibrils, but its putative cellular receptors have been unknown. The reverse transcription-PCR analysis of human fetal tissues suggested that based on their distribution all four collagen receptor integrins, namely alpha1beta1, alpha2beta1, alpha10beta1, and alpha11beta1, are possible receptors for collagen IX. Furthermore primary chondrocytes and chondrosarcoma cells express the four integrins simultaneously. Chondrosarcoma cells, as well as Chinese hamster ovary cells transfected to express alpha1beta1, alpha2beta1, or alpha10beta1 integrin as their only collagen receptor, showed fast attachment and spreading on human recombinant collagen IX indicating that it is an effective cell adhesion protein. To further study the recognition of collagen IX we produced recombinant alphaI domains in Escherichia coli. For each of the four alphaI domains, collagen IX was among the best collagenous ligands, making collagen IX exceptional compared with all other collagen subtypes tested so far. Rotary shadowing electron microscopy images of both alpha1I- and alpha2I-collagen IX complexes unveiled only one binding site located in the COL3 domain close to the kink between it and the COL2 domain. The recognition of collagen IX by alpha2I was considered to represent a novel mechanism for two reasons. First, collagen IX has no GFOGER motif, and the identified binding region lacks any similar sequences. Second, the alpha2I domain mutations D219R and H258V, which both decreased binding to collagen I and GFOGER, had very different effects on its binding to collagen IX. D219R had no effect, and H258V prevented type IX binding. Thus, our results indicate that collagen IX has unique cell adhesion properties when compared with other collagens, and it provides a novel mechanism for cell adhesion to cartilaginous matrix. (+info)
The association of lumbar spondylolisthesis with collagen IX tryptophan alleles.
(22/80)
Two collagen type IX gene polymorphisms that introduce a tryptophan residue into the protein's triple-helical domain have been linked to an increased risk of lumbar disc disease. To determine whether a particular subset of symptomatic lumbar disease is specifically associated with these polymorphisms, we performed a prospective case-control study of 107 patients who underwent surgery of the lumbar spine. Patients were assigned to one of five clinical categories (fracture, disc degeneration, disc herniation, spinal stenosis without spondylolisthesis and spinal stenosis with spondylolisthesis) based on history, imaging results, and findings during surgery. Of the 11 tryptophan-positive patients, eight had spinal stenosis with spondylolisthesis and three had disc herniation. The presence of the tryptophan allele was significantly associated with African-American or Asian designation for race (odds ratio 4.61, 95% CI 0.63 to 25.35) and with the diagnosis of spinal stenosis with spondylolisthesis (odds ratio 6.81, 95% CI 1.47 to 41.95). Our findings indicate that tryptophan polymorphisms predispose carriers to the development of symptomatic spinal stenosis associated with spondylolisthesis which requires surgery. (+info)
Mutations in the known genes are not the major cause of MED; distinctive phenotypic entities among patients with no identified mutations.
(23/80)
Multiple epiphyseal dysplasia (MED) is a clinically and genetically heterogeneous chondrodysplasia. Mutations in six genes (COMP, COL9A1, COL9A2, COL9A3, MATN3 and DTDST) have been reported, but the genotype-phenotype correlations and the proportions of cases due to mutations in these genes are still not well characterized. We performed a clinical, radiological and molecular analysis of known MED genes on 29 consecutive MED patients. The mutation analysis resulted in identification of the DTDST mutation in four patients (14%), the COMP mutation in three (10%) and the MATN3 mutation in three (10%). Thus, a disease-causing mutation was identified in 10 patients altogether (34%). The phenotypic features observed in the patients with mutations were in accordance with previously described phenotypes, but two new distinct phenotypic entities were identified in patients in whom no mutation was found. One of them was characterized by severe, early-onset dysplasia of the proximal femurs with almost complete absence of the secondary ossification centres and abnormal development of the femoral necks. The other phenotype was characterized by 'mini-epiphyses', resulting in severe dysplasia of the proximal femoral heads. The findings suggest that mutations in the known genes are not the major cause of MED and are responsible for less than half of the cases. The existence of additional MED loci is supported by the exclusion of known loci by mutation analysis and finding of specific subgroups among these patients. (+info)
Cellular invasion of the chicken corneal stroma during development: regulation by multiple matrix metalloproteases and the lens.
(24/80)
Avian corneal development requires cellular invasion into the acellular matrix of the primary stroma. Previous results show that this invasion is preceded by the removal of the fibril-associated type IX collagen, which possibly stabilizes matrices through interfibrillar cross-bridges secured by covalent crosslinks. In the present study, we provide evidence for the expression of three matrix metalloproteinases (MMPs) in early corneas, two of which act cooperatively to selectively remove type IX collagen in situ. In organ cultures, MMP inhibitors (either TIMP-2 or a synthetic inhibitor) resulted in arrested development, in which collagen IX persisted, and the stroma remained compact and acellular. We also show that blocking covalent crosslinking of collagen allows for cellular invasion to occur, even when the removal of type IX collagen is prevented. Thus, one factor regulating corneal invasion is the physical structure of the matrix, which can be modified by either selective proteolysis or reducing interfibrillar cross-bridges. We also detected another level of regulation of cellular invasion involving inhibition by the underlying lens. This block, which seems to influence invasive behavior independently of matrix modification, is a transient event that is released in ovo just before invasion proceeds. (+info)