Biomedical subjects
Publications and source records attributed to D Herbage.
Collagen heterogeneity is now well recognised. 11 different types are known, corresponding to 25 polypeptidic chains. In joints, almost all types of collagen are found. Type II collagen is however predominant, due to its fundamental role in cartilage physiology.
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A comparative analysis, by low-angle X-ray diffraction and electron microscopy, of bovine articular cartilage either submitted or not to chemical (0.5-2 M CaCl2, 4 M guanidinium chloride) or enzymatic (hyaluronidase, trypsin) treatments is reported. An analysis of the micrographs using a filtering program on the Fourier transform patterns reveals the absence of modification or alteration of the fibrils after treatment, whereas the X-ray diffraction patterns change. The ratio of the first/third orders intensities increases when the tissue proteoglycans content decreases. These results indicate that proteoglycans are regularly ordered on the type II collagen fibrils in articular cartilage.
Minor disulfide-bonded collagen (previously termed X1-X7 and now called type IX collagen) was isolated from foetal calf cartilage after pepsin treatment. At least three native fractions, containing, respectively, the X1X2X3, X4, and X5X6X7 chains, were separated; and from further biochemical and physicochemical experiments (differential scanning calorimetry, electrical birefringence, rotary shadowing), we propose a tentative model for their organization within a parent molecule. X1 and X2 are molecules composed of three chains of apparent Mr 62,000 and 50,000 linked by interchain disulfide bonds and containing pepsin-sensitive regions. The cleavage of at least three of these sites, present within X2, gives rise to the X3 and X5X6X7 fractions composed of molecules 80-100 nm and 40-55 nm in length, respectively. The X5X6X7 fraction is not digested by pepsin at 30 degrees C owing to its high thermal stability (certainly explained by its high hydroxyproline + proline content). This organization is in good accordance with that proposed for chicken cartilage type IX collagen; differences could only exist in the number and (or) the location of the pepsin-sensitive sites.
Differential scanning calorimetry (DSC) has been applied to the study of connective tissue to evaluate the denaturation process of collagen. We have applied this technique to the study of the ageing of rat skin. We have tried to correlate the variations of the parameters measured by DSC and the modifications of collagen crosslinks with ageing. The thermograms obtained are composed of one main peak located between two shoulders. The relative size of each peak varies with time: the first peak diminishes regularly from 2 to 20 months whilst, at the same time, the third peak increases; the recovery temperature increases with age (+ 16 degrees C between 2 and 20 months); the total denaturation enthalpy does not vary: the main value obtained is 5.9 X 10(-2) J/mg collagen. On the other hand, the assay of reducible collagen crosslinks in rat skin, over the same age range, shows a decrease of heat-labile aldimine crosslink (essentially hydroxylysinonorleucine). These results and the study of thermograms obtained with altered rat skin (animals treated with beta-aminopropionitrile or skin reduced with NaBH4) allow us to conclude that heat-labile and heat-stable crosslinks account for a collagen thermal stabilization which can explain the delay of denaturation characterized by the third peak of DSC thermograms.
The effect of hydrocortisone on the development of dorsal skin was analyzed in the chick embryo by (1) transmission electron microscopy, (2) indirect immunofluorescence histology of extracellular matrix components (collagen types I, III, and IV; fibronectin; and laminin), and (3) quantitative determination of collagen content and proline incorporation, between administration of the drug at 6 or 6.5 days and final retrieval of skin pieces at 11 days of incubation. Treatment caused the formation of featherless skin areas which exhibited an early maturation of the epidermis, a uniform distribution of interstitial collagen and rarefaction of fibronectin in the dermal extracellular matrix, and a significant increase of collagen content and proline incorporation in collagen noncollagen proteins, characterized by an increased hydroxyproline-to-proline ratio. The distribution of type IV collagen and of laminin was unchanged. The absence of feather formation in hydrocortisone-induced apteria is interpreted as resulting primarily from an early extinction of epidermal morphogenetic competence, and secondarily from modifications in the amount and distribution of extracellular matrix components in the dermis.
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Experimental periodontal disease was induced in golden hamsters by feeding the animals with a Keyes 2 000 diet. The aim of the present study was to investigate the effect of 3-methoxy 5,7,3',4'-tetrahydroxyflavan (ME), a flavanoid derivative, on periodontally induced changes by this diet which was fed ad libitum to half of the animals for 2 months prior to the start of drug therapy. The animals were then divided into 4 groups: A) control animals on normal diet; B) ME-treated (200 mg/kg 3 times a week i.p.) animals on normal diet; C) animals on Keyes 2 000 diet; D) ME-treated (200 mg/kg 3 times a week i.p.) animals on Keyes 2 000 diet. ME inhibited the diminution of bone volume as measured morphometrically. The growth of dental plaque as evaluated by a sensitive index (PII) appeared to be retarded by ME. The average PII in the ME-treated animals reached after 2 months of diet remained unchanged during the drug therapy period (4 months); whereas it was significantly increased in the group C hamsters. The biochemical data further suggest that the resorption process of the alveolar bone was decreased by ME.
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Neutral soluble collagen was extracted from lathyritic rat skin under proteolysis-inhibited conditions. Purified solutions were characterized by electric birefringence and heterodyne beat quasi-elastic light-scattering techniques under conditions where the monomeric form was stable (at 4 degrees C in 0.032 M phosphate buffer at pH 7.04). Solutions were then heated and the birefringence and light scattering followed during the fibrillogenesis reaction. The monomer presents a translational diffusion coefficient of 0.85 X 10(-7) cm2/s and a rotary diffusion coefficient of 1150 +/- 50 s-1; these values are consistent with a rodlike molecular model of 220 +/- 10 nm length and 4 +/- 1 nm diameter, substantially different from electron microscopic values of 290 and 1.5 nm, respectively. We propose that at pH 7.04 and relatively high ionic strength, the collagen monomer unit must exhibit substantial deviation from a completely rigid and extended rodlike structure. During the entire lag phase in a thermally induced fibrillogenesis reaction, the relaxation times for both translational and rotational motion remain virtually unchanged. The monomer polarity is also unchanged, as shown by reverse pulse birefringence data. No intermediate size soluble aggregates, such as dimers or trimers, have been detected between monomer and very large aggregates or fibrils during the process, although early multistep assembly products (dimers, trimers) could have been seen if present. These data suggest a model for fibrillogenesis emphasizing a monomer-related nucleation event, such as internal stiffening or conformational transition, followed by a rapid continuous growth up to large fibrils.
New minor disulfide-bonded collagens were recently described in cartilaginous tissues. The localization of these molecules in epiphyseal proper and growth plate cartilage of fetal calf has been studied using light and electron immunoperoxidase microscopy. The labeling was restricted to the pericellular region of the chondrocytes with an increasing intensity form the superficial to the inner zone of the epiphysis. At the ultrastructural level, the fine-non striated fibrils of the pericellular matrix were stained, demonstrating their collagenous nature. These minor collagenous chains are thus new components of the chondrocyte "exoskeleton" in which other molecules (proteoglycans, chondronectin, fibronectin and type V collagen) have been previously demonstrated.
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A combined biochemical, X-ray and electron microscopic study is presented on the molecular structure of bovine and human intervertebral disc. Positively stained electron micrographs of Type I and Type II collagen are shown to have slightly but significantly different banding patterns. New low-angle meridional patterns from different anatomical parts of the intervertebral disc are reported from native and trypsin-extracted discs. Qualitative measurements of these intensities lead to the conclusion that in the nucleus pulposus of the disc, another molecule, probably proteoglycan, is ordered on the collagen fibrils at the axial level of the gap region.
The curves obtained for skin samples of different ages and species by hydrothermal isometric tension ("HIT") measurement are compared to those obtained by differential scanning calorimetry (DSC) under the same thermal conditions (for a rise in temperature at a rate of 1.0 degrees C/min). Collagen denaturation, observed by DSC, directly affects the kinetics of the tension variations in the first part of the "HIT" curves, including the early peak due to the presence and destruction of the heat-labile cross-links in the collagen network. The presence of cross-links is in term shown to delay collagen denaturation to an extent which depends in part on their heat-stability. The final part of the "HIT" curves reflecting the effects of heat in the stable polymeric collagen network is no longer affected by collagen denaturation. Thus, both "HIT" and DSC are useful methods to evaluate collagen reticulation in connective tissues.
In the dermis of inter-appendage and glabrous skin, interstitial collagen types I, III, and V are abundant, while fibronectin is scarce. Conversely, in the morphogenetically active foci of cutaneous appendages, interstitial collagen is scarce or absent, whereas fibronectin is abundant. Type IV collagen and laminin are localized at the dermal-epidermal junction and distributed evenly.
The distribution of collagen type III throughout the pulp tissue from human developing tooth was studied using specific antibodies, immuno-fluorescence as well as immuno-peroxidase labelling for electron microscopy. Our results indicate that type III and type I collagen are present in the pulp. The staining intensity seems to correlate with the relatively high proportions of type III collagen biochemically found in pulp. In addition, type III collagen and reticulin fibres are similarly distributed, except that the Von Korff fibres were never detected with anti-type III collagen antibodies. Correspondingly, at the ultrastructural level, type III collagen appears as fine, branched filaments or electron dense material distributed throughout the tissue and particularly in close association with the plasma membrane of pulp fibroblasts. In contrast, type I collagen appears as typical coarse cross banded fibres.