Immunolocalization of collagen types I and III, tenascin, and fibronectin in intramembranous bone.

Structural components of the organic bone matrix were located by immunohistochemical techniques in fresh-frozen sections of normal and dysplastic bone. Fine and coarse birefringent fibers were identified as separate and distinctive features in the extracellular matrix by antibodies raised against human collagen Type III. The glycoprotein tenascin was located on a proportion of the fibers in a characteristic beaded pattern, which was absent in dysplastic bone. The fibers originated in the periosteum or in the fibrous stroma of the marrow cavity and were oriented with regard to both the spatial and the lamellar organization of the bone. The disposition and composition of the fibers suggests that they form a preliminary framework on which intramembranous bone modeling proceeds, and that the specific location of tenascin on the fibers in normal developing membrane bone may be important in determining the alignment of the bone tissue. Epitopes recognized by the collagen Type I and fibronectin antibodies were demonstrated throughout the mineralized matrix, but their incorporation into the collagen "Type III" fibers was evident only outside the mineralized matrix.     

Immunolocalization of collagen types II and III in single fibrils of human articular cartilage.

Type II and III fibrillar collagens were localized by immunogold electron microscopy in resin sections of human femoral articular cartilage taken from the upper radial zone in specimens from patients with osteoarthritis. Tissue samples stabilized by high-pressure cryofixation were processed by freeze-substitution, either in acetone containing osmium or in methanol without chemical fixatives, before embedding in epoxy or Lowicryl resin, respectively. Ultrastructural preservation was superior with osmium-acetone, although it was not possible to localize collagens by this method. In contrast, in tissue prepared by low-temperature methods without chemical fixation, collagens were successfully localized with mono- or polyclonal antibodies to the helical (Types II and III) and amino-propeptide (Type III procollagen) domains of the molecule. Dual localization using secondary antibodies labeled with 5- or 10-nm gold particles demonstrated the presence of Types II and III collagen associated within single periodic banded fibrils. Collagen fibrils in articular cartilage are understood to be heteropolymers mainly of Types II, IX, and XI collagen. Our observations provide further evidence for the complexity of these assemblies, with the potential for interactions between at least 11 distinct collagen types as well as several noncollagenous components of the extracellular matrix.     

Quantification in immunohistochemistry: the measurement of the ratios of collagen types I and III

Summary Quantitative techniques in immunohistochemistry are needed, but they are rarely applied because of doubtful reproducibility. We have developed a method for the detection of collagen types I and III in situ. The method applied was a two-step immuno-alkaline phosphatase technique with visualization of the end-product with Fast Red. The staining intensity was measured with a microdensitometer and the results expressed as ratios. The method yielded results that were unaffected by variations in tissue section thickness but which were proportionally related to time and antigen concentrations. Leiomyoma tissue, with a ratio of collagen types I and III of approximately 1.0, was used to establish the appropriate dilutions of the antibodies, thus assuring identical optical densities. By having the leiomyoma tissue sections incubated together with the heart tissue specimens, leiomyoma tissue was also helpful in correcting deviations from the 1.0 ratio. Accurate measurements of collagen type I/III ratios in normal human heart specimens were obtained with the present quantitative immunohistochemical technique.     

Activity of matrix metalloproteinase-9 against native collagen types I and III

Interstitial collagen types I, II and III are highly resistant to proteolytic attack, due to their triple helical structure, but can be cleaved by matrix metalloproteinase (MMP) collagenases at a specific site, approximately three-quarters of the length from the N-terminus of each chain. MMP-2 and -9 are closely related at the structural level, but MMP-2, and not MMP-9, has been previously described as a collagenase. This report investigates the ability of purified recombinant human MMP-9 produced in insect cells to degrade native collagen types I and III. Purified MMP-9 was able to cleave the soluble, monomeric forms of native collagen types I and III at 37 degrees C and 25 degrees C, respectively. Activity against collagens I and III was abolished by metalloproteinase inhibitors and was not present in the concentrated crude medium of mock-transfected cells, demonstrating that it was MMP-9-derived. Mutated, collagenase-resistant type I collagen was not digested by MMP-9, indicating that the three-quarters/one-quarter locus was the site of initial attack. Digestion of type III collagen generated a three-quarter fragment, as shown by comparison with MMP-1-mediated cleavage. These data demonstrate that MMP-9, like MMP-2, is able to cleave collagens I and III in their native form and in a manner that is characteristic of the unique collagenolytic activity of MMP collagenases.     

Quantitative assay of types I and III collagen synthesized by keloid biopsies and fibroblasts

Molecular sieve column chromatography was used to determine the amount of type I and III collagen synthesized by normal dermis and keloid biopsies and fibroblasts derived from these tissues. After incubation with radioactive proline, the collagen was extracted and separated into types I and III and then quantitated. There was no significant difference in the percent type III collagen synthesized by fresh keloid biopsies compared to normal dermis. Likewise, there was no significant difference in the percent type III collagen synthesized by keloid fibroblasts compared to normal dermal fibroblasts, However, fibroblasts from both keloid and normal dermis synthesized a lower percentage of type III collagen in cell culture compared to the original biopsies. These findings demonstrate that keloid collagen has the same type distribution as normal dermis and suggest that increased collagen synthesis in these lesions is not related to altered collagen types.     

The modulation of contraction of fibroblast populated collagen lattices by types I, II, and III collagen

Human dermal fibroblasts incorporated in a polymerized collagen lattice reduce the size of that matrix. When cell number, collagen concentration, and medium are identical, lattices made with type III collagen contract faster and to a greater degree than those made with type I collagen. The latter contract faster and to a greater degree than those made with type II collagen.     

Microquantitation of insoluble tissue collagen (types I and III) by radiodilution assay

The individual collagen types of the extracellular matrix of small tissue samples have been difficult to quantitate accurately both due to their marked insolubility and their relatively low immunogenicity. Thus no microassay with the sensitivity of a radioimmunoassay is currently available for quantitation of insoluble collagen types I and III in extremely small tissue samples. A radiochemical assay has been developed which allows direct processing of small tissue samples containing as little as 1-3 micrograms of a given collagen alpha chain. Unprocessed lyophilized tissues were digested with cyanogen bromide (CNBr) in the presence of a tritiated probe containing a soluble mixture of 3H-alpha 1(I) and 3H-alpha 1(III) collagen previously extracted and purified from tissue minces incubated with [3H]leucine. The resulting mix of radiolabeled peptides was separated on sodium dodecyl sulfate-polyacrylamide gradient gels. Reduction of the specific radioactivity of free leucine in acid hydrolysates of each individual CNBr peptide can be used to quantitate the amount of collagen types I or III in the original sample. Similar radiodilution analysis using a 3H-alpha 2(I) probe indicated a normal 2:1 ratio of alpha chains of type I collagen in the tissues tested. This method is also applicable to cell culture, easily measuring the collagen associated with fibroblast cell layers or medium in individual microtiter wells. When applied to various tissues of known collagen-type composition, it provides reproducible results which compare well with values published in the literature.     

Correlation of specific sulfated glycosaminoglycans with collagen types I,II, and III

Summary A qualitative and quantitative biochemical study of the glycosaminoglycans was performed in tissues constituted predominantly by one type of collagen, or in tissues containing mixtures of different types of collagen. The results obtained show the presence of dermatan sulfate, chondroitin sulfate, and heparitin sulfate in tissues containing collagen types I, II, or III, respectively, suggesting a specific correlation of different glycosaminoglycans with these three types of collagen.This work was aided by grant N° 79/306 from the Fundação de Amparo à Pesquisa do Estado de São PauloSupported by CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnológico)     

Immunohistochemical expression of types I and III collagen antibodies in the temporomandibular joint disc of human foetuses

The objective was to study the morphology of the articular disc and analyse the immunohistochemical expression of types I and III collagen markers in the temporomandibular joint (TMJ) disc of human foetuses of different gestational ages. Twenty TMJ from human foetuses supplied by Universidade Federal de Uberaba with gestational ages from 17 to 24 weeks were studied. The gestational age of the foetuses was determined by measuring the crown-rump (CR) length. Macroscopically, the foetuses were fixed in 10% formalin solution and dissected by removing the skin and subcutaneous tissue and exposing the deep structures. Immunohistochemical markers of type I and III were used to characterize the existence of collagen fibres. Analysis of the immunohistochemical markers of types I and III collagen revealed the presence of heterotypical fibril networks.     

Immunofluorescent localization of collagen types I, II, and III in the embryonic chick eye.

The appearance and distribution of type I, II, and III collagens in the developing chick eye were studied by specific antibodies and indirect immunofluorescence. At stage 19, only type I collagen was detected in the primary corneal stroma, in the vitreous body, and along the lens surface. At later stages, type I collagen was located in the primary and secondary corneal stroma and in the fibrous sclera, but not around the lens. Type II collagen was first observed at stage 20 in the primary corneal stroma, neural retina, and vitreous body. It was particularly prominent at the interface of the neural retina and vitreous body and, from stage 30 on, in the cartilaginous sclera. The primary corneal stroma consisted of a mixture of type I and II collagens between stages 20 and 27. Invasion of the primary corneal stroma by mesenchyme and subsequent deposition of fibroblast-derived collagen corresponded with a pronounced increase of type I collagen, throughout the entire stroma, and of type II collagen, in the subepithelial region. Type II collagen was also found in Bowman's and Descemet's membranes. A transient appearance of type III collagen was observed in the corneal epithelial cells, but not in the stroma (stages 20–30). The fully developed cornea contained both type I and II collagens, but no type III collagen. Type III collagen was prominent in the fibrous sclera, iris, nictitating membrane, and eyelids.     

Immunocytochemical localization of collagen types I,III, IV,and fibronectin in the human dermis

Summary The distribution of collagen types I, III, IV, and of fibronectin has been studied in the human dermis by light and electron-microscopic immunocytochemistry, using affinity purified primary antibodies and tetramethylrhodamine isothiocyanate-conjugated secondary antibodies. Type I collagen was present in all collagen fibers of both papillary and reticular dermis, but collagen fibrils, which could be resolved as discrete entities, were labeled with different intensity. Type III collagen codistributed with type I in the collagen fibers, besides being concentrated around blood vessels and skin appendages. Coexistence of type I and type III collagens in the collagen fibrils of the whole dermis was confirmed by ultrastructural double-labelling experiments using colloidal immunogold as a probe. Type IV collagen was detected in all basement membranes. Fibronectin was distributed in patches among collagen fibers and was associated with all basement membranes, while a weaker positive reaction was observed in collagen fibers. Ageing caused the thinning of collagen fibers, chiefly in the recticular dermis. The labeling pattern of both type I and III collagens did not change in skin samples from patients of up to 79 years of age, but immunoreactivity for type III collagen increased in comparison to younger skins. A loss of fibronectin, likely related to the decreased morphogenetic activity of tissues, was observed with age.     

Quantitative assay of types I and III collagen synthesized by keloid biopsyes and fibroblasts.

Molecular sieve column chromatography was used to determine the amount of type I and III collagen synthesized by normal dermis and keloid biopsies and fibroblasts derived from these tissues. After incubation with radioactive proline, the collagen was extracted and separated into types I and III and then quantitated. There was no significant difference in the percent type III collagen synthesized by fresh keloid biopsies compared to normal dermis. Likewise, there was no significant difference in the percent type III collagen synthesized by keloid fibroblasts compared to normal dermal fibroblasts. However, fibroblasts from both keloid and normal dermis synthesized a lower percentage of type III collagen in cell culture compared to the original biopsies. These findings demonstrate that keloid collagen has the same type distribution as normal dermis and suggest that increased collagen synthesis in these lesions is not related to altered collagen types.     

The effect of vitamin C on the ratio of collagen types I and III in the periprosthesis area in mice

The aim of this study was to examine the collagen-stimulating effect of ascorbic acid on the growth of the Parieten ProGrip implanted synthetic hernia prosthesis. The characteristics of collagenogenesis in the anterior abdominal wall were assayed with polarized microscopy. We suggest a new way to stimulate a reparation process with ascorbic acid that will help to optimize forming functionally matured structures of connective tissue of the anterior abdominal wall surrounding endoprosthesis. The results of our investigation show that vitamin C has a positive effect on the collagen synthesis in the periprosthetic capsule for any type of endoprosthesis. This was proven by the statistically significant increase in the ratio of collagen I and III types with vitamin C to the animal diet.     

A computational model for collagen fibre remodelling in the arterial wall

As the interaction between tissue adaptation and the mechanical condition within tissues is complex, mathematical models are desired to study this interrelation. In this study, a mathematical model is presented to investigate the interplay between collagen architecture and mechanical loading conditions in the arterial wall. It is assumed that the collagen fibres align along preferred directions, situated in between the principal stretch directions. The predicted fibre directions represent symmetrically arranged helices and agree qualitatively with morphometric data from literature. At the luminal side of the arterial wall, the fibres are oriented more circumferentially than at the outer side. The discrete transition of the fibre orientation at the media-adventitia interface can be explained by accounting for the different reference configurations of both layers. The predicted pressure-radius relations resemble experimentally measured sigma-shaped curves. As there is a strong coupling between the collagen architecture and the mechanical loading condition within the tissue, we expect that the presented model for collagen remodelling is useful to gain further insight into the processes involved in vascular adaptation, such as growth and smooth muscle tone adaptation.     

Distribution of collagen types III and IV in human placental villi

Immunofluorescent examination showed more significant accumulation of interstitial collagen type III in the stroma of mature placenta compared with immature one. Localization of membrane collagen type IV was found neither in basal membranes of epithelium and villous vessels of mature term placenta, nor in their stroma. The described patterns of distribution of collagen types III and IV in human placenta villi were proved by immunoelectronmicroscopic method.     

Synthesis of types I and III procollagen and collagen by monkey aortic smooth muscle cells in vitro.

Analysis of pepsin-resistant proteins produced in culture by monkey aortic smooth muscle cells (SMC) indicates the synthesis of types I and III collagen. As determined by carboxymethylcellulose chromatography and disc gel electrophoresis, SMC cultures synthesize more type III collagen than monkey skin fibroblast cultures; aortic adventitial cell cultures (a mixture of SMC and fibroblasts) synthesize an intermediate amount of type III collagen. Both types I and III procollagens can also be isolated from the culture medium of SMC and skin fibroblasts. The procollagens were separated by diethylaminoethylcellulose (DEAE-cellulose) chromatography in identified by electrophoresis and after cleavage with pepsin and cyanogen bromide. Quantitation of the procollagen by DEAE-cellulose chromatography suggests that 68% of the SMC procollagens and less than 10% of the skin fibroblast procollagens are type III. On the other hand, estimation of the proportions of collagen types secreted by cells, employing pepsin digestion of cell culture medium at 15 degrees C, leads to an underestimation of the amount of type III collagen relative to type I. SMC and fibroblasts may differ in their ability to convert type I procollagen to collagen ad indicated by the observation that skin fibroblast culture medium contains both pN and pC collagen intermediates after 24 h, while cultures of SMC essentially lack the pC collagen intermediates.     

Fibrillogenesis of collagen types I, II, and III with small leucine-rich proteoglycans decorin and biglycan

Collagen has found use as a scaffold material for tissue engineering as well as a coating material for implants with a view to enhancing osseointegration through mimicry of the bone extracellular matrix in vivo. The aim of this study was to compare the collagen types I, II, and III with regard to their ability to bind the small leucine-rich proteoglycans (SLRPs) decorin and biglycan during fibrillogenesis in vitro in phosphate buffer. In addition, the influence of SLRPs on the proportion of collagen molecules incorporated into fibrils during fibrillogenesis in vitro at high and low ionic strength was investigated, as were their effects on the morphology of collagen fibrils and the speed of fibrillogenesis. Considerably more biglycan than decorin was bound by all three collagen types. Collagen II bound significantly more SLRPs in fibrils than collagen I and III. Decorin and biglycan decreased the proportion of collagen molecules of all three collagen types incorporated into fibrils in similar fashion. Biglycan affected neither fibril diameter nor the speed of fibrillogenesis. Decorin reduced the fibril diameter of all three collagen types. The differences in SLRP-binding ability between collagen types could be of significance when selecting collagen type and/or SLRPs as scaffold materials for tissue engineering or implant coatings.