Collagen and related research最新文献

Cervical tissue from ewes at various stages of pregnancy was examined for evidence that collagenase is involved in the process of cervical softening. Collagenase activity was detected in medium after 2–3 days culture of ovine cervical explants, but there was no significant difference in total enzymic activity produced by explants from non-pregnant, early pregnant or late pregnant animals when expressed as units/mg wet weight of tissue over five days in culture. Oestradiol infusion into ewes prior to parturition did not alter the enzyme activity subsequently produced in explant culture. However, the DNA concentration, and hence the number of cells per unit volume, decreased significantly with length of pregnancy, this effect being due to expansion of cervical tissue which occurs late in pregnancy. Thus, if collagenase activity is expressed relative to DNA and hence cell number, there is evidence for increased production per cell in order to keep the tissue concentration constant. However, as the concentration of collagen in cervix remains constant during pregnancy, the ratio of collagenase activity to collagen is also constant. It is therefore concluded that there is no evidence of a role for increased collagenase activity in cervical softening in the ewe.

A method for quantitating nicked or shortened molecules (fragments) in pepsinizedbovine type I collagen preparations using polarimetry thermal denaturation curves is described.^The shortened molcules denature about 4°C lower than intact collagen molecules. The analog output of a polarimeter was digitized and stored on a microcomputer disk. A BASIC program was written which retrieves the specific rotation data from the disk, smooths the data with a boxcar average, and plots the derivative of the denaturation curve. The derivative curve was deconvoluted by fitting three Gaussian curves to the derivative curve using published algorithms. The area of the Gaussian centered at 37°C was proportional to the amount of collagen fragments. A good correlation between the amount of fragments determined by polarimetry and by a trypsin sensitivity assay was observed. The overall precision of the method was about 10% RSD, and the method was repeatable by multiple analysts. Application of the method to reconstituted fibrillar collagen samples showed that more fragments are generated when pepsin digestion time is lengthened. By fitting a fourth Gaussian component to the derivative curve, the method can also be used to determine relative amounts of denatured collagen (helix partially unwound but a chains not nicked). The detection limit for denatured collagen is about 20%.

The effect of interleukin 1 (IL1) on the synthesis and degradation of collagen was examined in explants of cultured cartilage. IL1 induced a reversible, dose-dependent (10-100pM) inhibition of type II collagen synthesis. The proportion of collagen produced decreased selectively from 7% to 1.2% of total protein after 72 h exposure to IL1 (100 pM). There was no change in the rate of degradation of newly synthesized collagen. Analysis of newly synthesized material showed that the type II collagen synthesized in the presence of IL1 had the same characteristics as that extracted from unstimulated cartilage. The relative amounts of type II procollagen mRNA were estimated by Northern blot hybridization. The levels were decreased in cartilage cultured with IL1 to a similar extent as that seen for the type II collagen protein. Exposure to IL1 (10-350 pM) for 3 days did not induce increased resorption of extracellular collagen in the cultured explants. These data demonstrate that decreased collagen production in cartilage exposed to IL1 is due primarily to decreased amounts of type II procollagen mRNA.

Implantation of rat demineralized bone matrix into intramuscular pouches has beenshown to cause a complex cellular transition of mesenchymal-type cells into well developed mature bone. Demineralized bone matrix was surgically implanted into rat muscle pouches and removed at various intervals between 7 and 28 days. Histological sections of the implants revealed bone formation by endochondral ossification and appositional bone growth. Biochemical analysis of collagen synthesis demonstrated the following: (1) synthesis of type X collagen, a collagen produced by hypertrophic chondrocytes in the growth plate and in fracture callus. (2) Synthesis of a collagenase-sensitive 17k protein which seems to increase in the early stages of bone induction. Pulse chase analysis indicates that 17k is not a degradation product of another protein and appears to be synthesized without a large M_r precursor. The 17k component contains one or more collagenous domains that are partially resistant to proteolysis with pepsin. Our results confirm the appearance of a cartilage intermediate during demineralized bone matrix induced ossification and implicate the existence of proteins which may be useful markers in future studies on matrix mineralization and ossification.

Subcutaneous implantation of demineralized collagenous bone matrix results in localinduction of new bone formation. The dose dependence of bone induction was investigated using different amounts of rat demineralized bone matrix (DBM) and also osteogenin-enriched fraction with and without inactive collagenous bone matrix (ICBM). There is a threshold for bone induction; at least lOmg of DBM is required. There is a dose dependent increase in bone induction between 10 to 25 mg. Exogenous type I collagen was found to be stimulatory to bone induction when suboptimal doses of DBM are employed. The activity of osteogenin-enriched dissociative extract was enhanced by addition of ICBM. These results imply that optimal bone induction requires the combined action of soluble osteogenin-enriched fraction and insoluble collagenous substratum.