Inhibition of lysyl oxidase activity can delay phenotypic modulation of chondrocytes in two-dimensional culture.
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OBJECTIVE: Chondrocytes frequently de-differentiate in two-dimensional (2D) culture, especially in the presence of serum. To examine the role of lysyl oxidase (LOX) induced cross-linking in this phenomenon, the effect of the specific LOX inhibitor beta-aminopropionitrile (BAPN) was studied in 2D chondrocyte culture. DESIGN: Chick embryo sternal chondrocytes (both proliferative and hypertrophic, from caudal and cranial zones, respectively) were cultured in the presence and absence of BAPN. The production and activities of LOX and LOX-like (LOXL) were assessed by enzyme assay and the use of specific antibodies. Seventeen batches of serum of different origin were compared. Chondrocyte phenotype was assessed both morphologically and biochemically, the latter by quantitative analysis of production of radiolabeled cartilage collagens II, IX, X and XI, and the de-differentiation marker collagen I, for up to 4 weeks in culture. RESULTS: LOX and LOXL were identified, by Western blotting and immunofluorescence, and LO activity was measured in the medium, with both proliferative and hypertrophic chondrocytes. Inhibition of LO activity prevented or delayed chondrocyte de-differentiation, as characterized by changes in cell shape and synthesis of the five different collagen types, from the first days of culture for up to 4 weeks, depending on the origin of the serum added to the culture medium. CONCLUSION: LO activity may be involved in the control of chondrocyte phenotype, in addition to serum factors. Inhibition of LO activity by BAPN may be useful for the maintenance of the chondrocyte phenotype in 2D culture. Specific variations in the relative proportions of collagens II, IX and XI could be involved in the mechanism underlying these observations. (+info)
Adjacent tissues (cartilage, bone) affect the functional integration of engineered calf cartilage in vitro.
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OBJECTIVE: An in vitro model was used to test the hypothesis that culture time and adjacent tissue structure and composition affected chondrogenesis and integrative repair in engineered cartilage. METHOD: Engineered constructs made of bovine calf chondrocytes and hyaluronan benzyl ester non-woven mesh were press-fitted into adjacent tissue rings made of articular cartilage (AC), devitalized bone (DB), or vital bone (VB) and cultured in rotating bioreactors for up to 8 weeks. Structure (light and electron microscopy), biomechanical properties (interfacial adhesive strength, construct compressive modulus), biochemical composition (construct glycosaminoglycans (GAG), collagen, and cells), and adjacent tissue diffusivity were assessed. RESULTS: Engineered constructs were comprised predominately of hyaline cartilage, and appeared either closely apposed to adjacent cartilage or functionally interdigitated with adjacent bone due to interfacial deposition of extracellular matrix. An increase in culture time significantly improved construct adhesive strength (P<0.001), modulus (P=0.02), GAG (P=0.04) and cellularity (P<0.001). The type of adjacent tissue significantly affected construct adhesion (P<0.001), modulus (P<0.001), GAG (P<0.001) and collagen (P<0.001). For constructs cultured in rings of cartilage, negative correlations were observed between ring GAG content (log transformed) and construct adhesion (R2=0.66, P<0.005), modulus (R2=0.49, P<0.05) and GAG (R2=0.44, P<0.05). Integrative repair was better for constructs cultured adjacent to bone than cartilage, in association with its solid architectural structure and high GAG content, and best for constructs cultured adjacent to DB, in association with its high diffusivity. CONCLUSIONS: Chondrogenesis and integrative repair in engineered cartilage improved with time and depended on adjacent tissue architecture, composition, and transport properties. (+info)
Relationships among microstructural properties of bone at the human midshaft femur.
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Mineralization density and collagen fibre orientation are two aspects of a bone's microstructural organization that influence its mechanical properties. Previous studies by our group have demonstrated a distinctly non-random, though highly variable, spatial distribution of these two variables in the human femoral cortex. In this study of 37 specimens, these variables are examined relative to one another in order to determine whether regions of bone demonstrating higher or lower mineralization density also demonstrate a prevalence of either transversely or longitudinally oriented collagen fibres. An analysis of rank-transformed collagen fibre orientation (as determined by circularly polarized light) and mineralization density (as determined by backscattered electron microscopy) data sets demonstrated that areas of low mineralization density (predominantly in the anterior-lateral cortex) tended to correspond to regions of higher proportions of longitudinally oriented collagen fibres. Conversely, areas of higher mineralization density (postero-medially) tended to correspond to regions of higher proportions of transversely oriented collagen fibres. High variability in the sample led to generally low correlations between the two data sets, however. A second analysis focused only on the orientation of collagen fibres within poorly mineralized bone (representing bone that was newly formed). This analysis demonstrated a lower proportion of transverse collagen fibres in newly formed bone with age, along with some significant regional differences in the prevalence of collagen fibres of either orientation. Again high variability characterized the sample. These results are discussed relative to the hypothesized forces experienced at the midshaft femur. (+info)
Age-related changes in the structure of myocardial collagen network of auricle of the right atrium in healthy persons and ischemic heart disease patients.
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The objective of the study was to examine and evaluate morphometrically age-related changes in the structure of myocardium collagen network of auricle of the right atrium in control group persons, who were not diagnosed with cardiac pathology leading to heart lesion or overload, and in ischemic heart disease patients. Material of 56 persons of both genders aged 20-94 years was used for study purposes. Biopsy material of 17 healthy persons (control group, average age 60.53+/-9.89 years) and autopsy material of 39 ischemic heart disease patients (average age 63.83+/-15.67 years) taken from the basis of auricle of the right atrium (specimen size--2 mm x 2 mm) were examined. Morphometric analysis of collagen network was performed using histologic and video morphometric methods. After this investigation we evaluated quantitative parameters of the bundles of collagen net--namely area, number, perimeter. The percentile occupied area of bundles in control group was 17.6+/-2.5%; ischemic heart disease patients group--26.8+/-2.9%; number of bundles was 4179+/-1073 and 2523+/-867; perimeter--24163+/-3308 mm and 23426+/-409 mm, respectively. After investigation of age-related changes of collagen network in control group and ischemic heart disease patients' group, which did not statistically significantly differed by age, we determined that collagen network area in auricle of the right atrium increased with age in both groups, however, spatial distribution of collagen network was different. Collagen network area enlarged with lengthening of its fibers along cardiomyocytes in control group. In ischemic heart disease group, it enlarged both in parallel to cardiomyocytes and by separate collagen fibers merging into bigger bundles. Fibrillar collagen network area and its total perimeter of healthy persons increased with age, and number of fibers did not change. Consequently, collagen fiber area of one location increased with age; its shape, judging by in parallel increasing total perimeter, became branchier, i.e. proliferated in endomysium in parallel with cardiomyocytes. In ischemic heart disease group fibrillar collagen network percentage area increased with age, however, total perimeter and number of separate fibers in visual field decreased. Consequently, in ischemic heart disease group separate collagen fibers merged, their locations enlarged, taking an integral structure, which allowed assuming development of interstitial fibrosis. (+info)
Procollagen trafficking, processing and fibrillogenesis.
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Collagen fibrils in the extracellular matrix allow connective tissues such as tendon, skin and bone to withstand tensile forces. The fibrils are indeterminate in length, insoluble and form elaborate three-dimensional arrays that extend over numerous cell lengths. Studies of the molecular basis of collagen fibrillogenesis have provided insight into the trafficking of procollagen (the precursor of collagen) through the cellular secretory pathway, the conversion of procollagen to collagen by the procollagen metalloproteinases, and the directional deposition of fibrils involving the plasma membrane and late secretory pathway. Fibril-associated molecules are targeted to the surface of collagen fibrils, and these molecules play an important role in regulating the diameter and interactions between the fibrils. (+info)
The new collagen gene COL27A1 contains SOX9-responsive enhancer elements.
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The most recently discovered collagen gene, COL27A1, codes for type XXVII collagen. The COL27A1 gene is strongly expressed in developing cartilage and weakly expressed in many other tissue types. The present study was undertaken to identify transcriptional regulatory mechanisms that govern the expression of COL27A1 in cartilage, and in particular to determine whether SOX9, a key regulator of chondrogenesis, could activate COL27A1. The first intron of COL27A1 was examined to identify sites with homology to the Sox consensus sequence (A)/(T)(A)/(T)CAA(A)/(T)G. Three 50-bp regions that contained paired Sox sites arranged in opposite orientation to each other and separated by 3 or 4 bp were targeted for further analysis. The elements were tested by transient transfection of reporter plasmids, and two of the three elements showed enhancer activity in chondrocytic cells. The same two elements bound SOX9 in electrophoretic mobility shift assays (EMSA). They were not transcriptionally active in fibroblasts, but cotransfection with a SOX9 expression plasmid resulted in activation. The independent mutation of either Sox site in a pair prevented SOX9 binding to the enhancers in EMSA experiments, indicating that SOX9 binds these enhancers only as a dimer. Mutation of either site in a pair also abolished enhancer activity in chondrocytes, indicating that dimeric binding of SOX9 is required for transcriptional activation of the two new enhancers. In summary, these results suggest that SOX9 may play an important role in the transcriptional activation of the newest collagen gene, COL27A1. (+info)
No effect of menstrual cycle on myofibrillar and connective tissue protein synthesis in contracting skeletal muscle.
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We tested the hypothesis that acute exercise would stimulate synthesis of myofibrillar protein and intramuscular collagen in women and that the phase of the menstrual cycle at which the exercise took place would influence the extent of the change. Fifteen young, healthy female subjects were studied in the follicular (FP, n=8) or the luteal phase (LP, n=7, n=1 out of phase) 24 h after an acute bout of one-legged exercise (60 min of kicking at 67% W(max)), samples being taken from the vastus lateralis in both the exercised and resting legs. Rates of synthesis of myofibrillar and muscle collagen proteins were measured by incorporation of [(13)C]leucine. Myofibrillar protein synthesis (means+/-SD; rest FP: 0.053+/-0.009%/h, LP: 0.055+/-0.013%/h) was increased at 24-h postexercise (FP: 0.131+/-0.018%/h, P<0.05, LP: 0.134+/-0.018%/h, P< 0.05) with no differences between phases. Similarly, muscle collagen synthesis (rest FP: 0.024+/- 0.017%/h, LP: 0.021+/- 0.006%/h) was elevated at 24-h postexercise (FP: 0.073+/- 0.016%/h, P<0.05, LP: 0.072+/- 0.015%/h, P<0.05), but the responses did not differ between menstrual phases. Therefore, there is no effect of menstrual cycle phase, at rest or in response to an acute bout of exercise, on myofibrillar protein synthesis and muscle collagen synthesis in women. (+info)
Targeted ablation of the abcc6 gene results in ectopic mineralization of connective tissues.
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Pseudoxanthoma elasticum (PXE), characterized by connective tissue mineralization of the skin, eyes, and cardiovascular system, is caused by mutations in the ABCC6 gene. ABCC6 encodes multidrug resistance-associated protein 6 (MRP6), which is expressed primarily in the liver and kidneys. Mechanisms producing ectopic mineralization as a result of these mutations remain unclear. To elucidate this complex disease, a transgenic mouse was generated by targeted ablation of the mouse Abcc6 gene. Abcc6 null mice were negative for Mrp6 expression in the liver, and complete necropsies revealed profound mineralization of several tissues, including skin, arterial blood vessels, and retina, while heterozygous animals were indistinguishable from the wild-type mice. Particularly striking was the mineralization of vibrissae, as confirmed by von Kossa and alizarin red stains. Electron microscopy revealed mineralization affecting both elastic structures and collagen fibers. Mineralization of vibrissae was noted as early as 5 weeks of age and was progressive with age in Abcc6(-/-) mice but was not observed in Abcc6(+/-) or Abcc6(+/+) mice up to 2 years of age. A total body computerized tomography scan of Abcc6(-/-) mice revealed mineralization in skin and subcutaneous tissue as well as in the kidneys. These data demonstrate aberrant mineralization of soft tissues in PXE-affected organs, and, consequently, these mice recapitulate features of this complex disease. (+info)