A Quantitative Study of the Relationship between the Distribution of Different Types of Collagen and the Mechanical Behavior of Rabbit Medial Collateral Ligaments

The mechanical properties of ligaments are key contributors to the stability and function of musculoskeletal joints. Ligaments are generally composed of ground substance, collagen (mainly type I and III collagen), and minimal elastin fibers. However, no consensus has been reached about whether the distribution of different types of collagen correlates with the mechanical behaviors of ligaments. The main objective of this study was to determine whether the collagen type distribution is correlated with the mechanical properties of ligaments. Using axial tensile tests and picrosirius red staining-polarization observations, the mechanical behaviors and the ratios of the various types of collagen were investigated for twenty-four rabbit medial collateral ligaments from twenty-four rabbits of different ages, respectively. One-way analysis of variance was used in the comparison of the Young''s modulus in the linear region of the stress-strain curves and the ratios of type I and III collagen for the specimens (the mid-substance specimens of the ligaments) with different ages. A multiple linear regression was performed using the collagen contents (the ratios of type I and III collagen) and the Young''s modulus of the specimens. During the maturation of the ligaments, the type I collagen content increased, and the type III collagen content decreased. A significant and strong correlation () was identified by multiple linear regression between the collagen contents (i.e., the ratios of type I and type III collagen) and the mechanical properties of the specimens. The collagen content of ligaments might provide a new perspective for evaluating the linear modulus of global stress-strain curves for ligaments and open a new door for studying the mechanical behaviors and functions of connective tissues.     

A comparative analysis of structure and spatial distribution of decorin in human leiomyoma and normal myometrium

Leiomyoma is a benign smooth muscle tumor of the uterus that affects many women in active reproductive life. It is composed by bundles of smooth muscle cells surrounded by extracellular matrix. We have recently shown that the glycosylation of extracellular matrix proteoglycans is modified in leiomyoma: increased amounts of galactosaminoglycans with structural modifications are present. The data here presented show that decorin is present in both normal myometrium and leiomyoma but tumoral decorin is glycosylated with longer galactosaminoglycan side chains. Furthermore, these chains contain a higher ratio D-glucuronate/L-iduronate, as compared to normal tissue. To determine if these changes in proteoglycan glycosylation correlates with modifications in the extracellular matrix organization, we compared the general structural architecture of leiomyoma to normal myometrium. By histochemical and immunofluorescence methods, we found a reorganization of muscle fibers and extracellular matrix, with changes in the distribution of glycoproteins, proteoglycans, and collagen. Thin reticular fibers, possibly composed by types I and III collagen, were replaced by thick fibers, possibly richer in type I collagen. Type I collagen colocalized with decorin both in leiomyoma and normal myometrium, in contrast to type IV collagen that did not. The relative amount of decorin was increased and the distribution of decorin and collagen was totally modified in the tumor, as compared to the normal myometrium. These findings reveal that not only decorin structure is modified in leiomyoma but also the tissue architecture changed, especially concerning extracellular matrix.     

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.     

Type V collagen. Quantitation in normal lungs and in lungs of rats with bleomycin-induced pulmonary fibrosis

Type V collagen was first isolated in 1976; there is still controversy as to how many molecular species of type V collagen exist. Although its structural and functional roles remain unclear, reports of changes in the relative amount of type V collagen from that present in normal tissue have been reported in such diverse pathologic conditions as atherosclerotic aortas, prolapsed mitral valves, and fibrotic lungs. Methods for quantitating type V collagen relative to other collagens have consisted of solubilizing the collagen with pepsin and then analyzing the ratios of the intact chains by gel electrophoresis or by column chromatography. In tissues in which only a small percentage of the total collagen can be solubilized by pepsin, such analyses may not accurately reflect changes in the total collagen present. In this report, a method for quantitating type V collagen relative to types I and III collagens based on CNBr peptide mapping is presented. CNBr solubilizes virtually all the collagen present in any tissue. The method is applied to a model of bleomycin-induced pulmonary fibrosis in rats. It was found that type I collagen increased relative to types III and V collagens, which seemed to remain at values comparable to those observed in lungs from control (normal) rats, both in terms of newly synthesized collagen (collagen synthesized by lung minces during 4 h in culture) and total unlabeled lung collagen (collagen synthesized during the life of the animal).     

Quantitation of Type I to type III collagen ratios in small samples of human tendon,blood vessels,and atherosclerotic plaque

A method is described whereby the ratio of the major interstitial collagens (Types I and III) can be measured in biopsy specimens of human tissue weighing as little as 25 mg. Marker peptides are solubilized from the tissue by digestion with cyanogen bromide. These peptides which are not known to be involved in collagen crosslinking are isolated and quantified by a combination of carboxymethyl-cellulose chromatography and polyacrylamide gel electrophoresis. The peptides used are α1(I)-CB7 and α1(III)-CB5. The use of the method is illustrated by analyzing the collagen type ratio in small specimens of tendon, aorta, and vena cava.     

Quantitation of type I and III collagens using electrophoresis of alpha chains and cyanogen bromide peptides

The methods of quantitating the relative amounts of type I and III collagens in samples containing crosslinked collagen chains were evaluated using electrophoresis of alpha chains and cyanogen bromide peptides. The densitometry areas of the alpha I(I) chains from type I collagen and the alpha I(III) chains from type III collagen were reduced because of the failure of the crosslinked chains to dissociate. However, the ratios of the unit densitometry areas of these chains (area of chain/micrograms type I or III collagen loaded) were constant for type I and III collagens prepared from the same samples of tissue. A calibration factor, which was the same for dermis and mitral valve, was derived to convert the densitometry area ratios to the weight ratios of type I to III collagens. In contrast, the densitometry areas of the alpha I(I) CB8 (type I collagen marker) and the alpha I(III) CB5 (type III collagen marker) were not reduced by crosslinked collagen chains. A calibration factor was also derived to convert the ratios of the densitometry areas of the marker peptides to weight ratios of type I to type III collagens. Almost identical results were obtained when electrophoresis of alpha chains and of cyanogen bromide peptides was used with these calibration factors to quantitate the relative amounts of type I and III collagens in tissue extracts which contained different amounts of crosslinked chains.     

Analyzing the feasibility of discriminating between collagen types I and II using polarization‐resolved second harmonic generation

According to previous studies, the nonlinear susceptibility tensor ratio χ₃₃/χ₃₁ obtained from polarization‐resolved second harmonic generation (P‐SHG) under the assumption of cylindrical symmetry can be used to distinguish between fibrillar collagen types. Discriminating between collagen fibrils of types I and II is important in tissue engineering of cartilage. However, cartilage has a random organization of collagen fibrils, and the assumption of cylindrical symmetry may be incorrect. In this study, we simulated the P‐SHG response from different collagen organizations and demonstrated a possible method to exclude areas where cylindrical symmetry is not fulfilled and where fibrils are located in the imaging plane. The χ₃₃/χ₃₁‐ratio for collagen type I in tendon and collagen type II in cartilage was estimated to be 1.33 and 1.36, respectively, using this method. These ratios are now much closer than what has been reported previously in the literature, and the larger reported differences between collagen types can be explained by variation in the structural organization.      

Characterization of the collagen synthesized by cultured human smooth muscle cells from fetal and adult aorta.

Smooth muscle cells were grown from explants of the tunica media of fetal and adult human aorta. Collagen was isolated after incubation with [14C]glycine and was characterized by ion-exchange chromatography. All cells investigated synthesized two types of collagen: Type I (chain composition [alpha1(I)]2alpha2) and type III (chain composition [alpha1(III)]3). The collagen made by cells from adult donors contained approximately 70% type I and 30% type III collagen. This corresponds to the collagen composition in teh original tissue. No age-relate change in the type I/type III ratio was found with cells from donors between 9 and 67 years of age. On the other hand, the type III portion of the collagen made by fetal cells was markedly less (about 15-20% of total collagen).     

Pulmonary macrophages alter the collagen phenotype of lung fibroblasts

Cultured lung fibroblasts produced and secreted interstitial collagen types I and III. The relative proportion of type III collagen increased as a linear function of cell density, with confluent cultures producing 8.6% type III collagen. When human lung fibroblasts were cultured in the presence of newly harvested lung macrophages, the proportion of type III collagen secreted rose to 15.5%. This high level of type III collagen synthesis was greater than could be induced by withdrawal of serum, a perturbation known to alter the proportion of types I and III collagen synthesized by fibroblasts. This effect on fibroblast phenotype was independent of cell density, as both low and high density cultures of fibroblasts responded similarly when cultured with macrophages. There was no evidence that fibroblasts synthesize new or different collagen types (such as type I trimer) in response to macrophages. Optimal conditions for eliciting an effect on fibroblast connective tissue metabolism required interaction of the two cell types for 5–8 days. These in vitro changes are analogous to the sequence of interactions and changes in connective tissue metabolism seen during recovery from tissue injury.     

Immunohistochemical studies on the tissue localization of collagen types I, III, IV, V and VI in schwannomas. Correlation with ultrastructural features of the extracellular matrix

The distinctive tissue localization of collagen types in typical schwannomas with Antoni type A and B areas was demonstrated immunohistochemically using affinity-purified antibodies against types I, III, IV, V and VI collagen and comparative ultrastructural studies were made on the extracellular matrix components. Antoni type A tissue, which was composed of tightly packed spindle cells with long cytoplasmic processes surrounded by a continuous basement membrane and a few fibrillar components of the extracellular matrix, was almost exclusively immunoreactive for type IV collagen, presumably representing the basement membrane. Verocay bodies, which are organoid structures of Antoni type A tissue, had a variety of more abundant extracellular fibrous components, such as banded collagen fibrils, fibrous long-spacing fibrils and microfibrils. These were positive for type I and III, as well as type IV collagen. In Antoni type B areas, where two types to tumor cells designated Schwann cell-like and fibroblast-like were scattered in large amounts of amorphous extracellular matrix containing microfibrils and thick banded collagen fibrils, type VI collagen as well as types I, III and IV collagen were consistently detected. Type V collagen was localized in dense fibrous tissue areas and around blood vessels. These findings indicate that the differently organized cellular patterns of schwannomas, identified as Antoni types A and B, are characterized not only by the ultrastructural features of the extracellular matrix, but also by the distinctive collagen types produced by neoplastic Schwann cells.     

A simplified method for quantitation of the relative amounts of type I and type III collagen in small tissue samples

A method is described for the quantitation of the relative amounts of types I and III collagens in rabbit lung tissue. This involved (i) repeated homogenization in the presence of 2% sodium dodecyl sulfate and the production of an acetone dried powder, (ii) reaction with cyanogen bromide, (iii) polyacrylamide gel electrophoresis, and (iv) densitometric scanning of proteins stained by Coomassie blue R-250. Several features of this procedure were shown to offer advantages over methods previously employed. First, the sodium dodecyl sulfate solution was shown to remove the bulk of noncollagen proteins leaving an insoluble residue which could then be reacted with cyanogen bromide without further purification. Second, cyanogen bromide was shown to solubilize essentially all of the collagen in the residue leaving an insoluble pellet with an amino acid analysis similar to elastin. Finally, to facilitate accurate quantitation, types I and III collagen standards were included with each gel so that a standard curve of protein versus staining density could be constructed. This method is assessed to be simpler and more accurate than those employed previously for the quantitation of collagens and can be applied to small tissue samples (<100 mg) such as would be obtained by lung biopsy.     

Collagen types I, III, and V in human embryonic and fetal skin

The dermis of human skin develops embryonically from lateral plate mesoderm and is established in an adult-like pattern by the end of the first trimester of gestation. In this study the structure, biochemistry, and immunocytochemistry of collagenous matrix in embryonic and fetal dermis during the period of 5 to 26 weeks of gestation was investigated. The dermis at five weeks contains fine, individual collagen fibrils draped over the surfaces of mesenchymal cells. With increasing age, collagen matrix increases in abundance in the extracellular space. The size of fibril diameters increases, and greater numbers of fibrils associate into fiber bundles. By 15 weeks, papillary and reticular regions are recognized. Larger-diameter fibrils, larger fibers, denser accumulations of collagen, and fewer cells distinguish the deeper reticular region from the finer, more cellular papillary region located beneath the epidermis. The distribution of collagen types I, III, and V were studied at the light microscope level by immunoperoxidase staining and at the ultrastructural level by transmission (TEM) and scanning electron microscopy (SEM) with immunogold labeling. By immunoperoxidase, types I and III were found to be evenly distributed, regardless of fetal age, throughout the dermal and subdermal connective tissue with an intensification of staining at the dermal-epidermal junction (DEJ). Staining for types III and V collagen was concentrated around blood vessels. Type V collagen was also localized in basal and periderm cells of the epidermis. By immuno-SEM, types I and III were found associated with collagen fibrils, and type V was localized to dermal cell surfaces and to a more limited extent with fibrils. The results of biochemical analyses for relative amounts of types I, III, and V collagen in fetal skin extracts were consistent with immunoperoxidase data. Type I collagen was 70-75%, type III collagen was 18-21%, and type V was 6-8% of the total of these collagens at all gestational ages tested, compared to 85-90% type I, 8-11% type III, and 2-4% type V in adult skin. The enrichment of both types III and V collagen in fetal skin may reflect in part the proportion of vessel- and nerve-associated collagen versus dermal fibrillar collagen. The accumulation of dermal fibrillar collagen with increasing age would enhance the estimated proportion of type I collagen, even though the ratios of type III to I in dermal collagen fibrils may be similar at all ages.     

The effects of mechanical stability on the macromolecules of the connective tissue matrices produced during fracture healing. I. The collagens

Summary The distribution of types I, II, III, V and IX collagens in healing fractures of the rabbit tibia has been demonstrated by immunofluorescent techniques. It has also been shown that the mechanical stability of the healing fracture affects both the distribution and types of the collagens present.The initial fibrous matrix contains types III and V collagens; type I collagen was only located in this matrix if unfixed tissue was used. In mechanically stable fractures, cancellous bone forms over the entire periosteal surface by 5–7 days; type I collagen is laid down within the previous fibrous matrix. The trabeculae are heterogeneous in their collagen content. The cavities contain a matrix of types III and V collagens. Small nodules of cartilage may be present between 7 and 14 days; these contain types II and IX collagens.In mechanically unstable fractures, cancellous bone is initially formed away from the fracture gap. The fibrous tissue over the gap is replaced by cartilage; types II and IX collagens are laid down on the pre-existing fibrous matrix. The cartilage is replaced by endochondral ossification. At the ossification front, type I collagen is found around the chondrocyte lacunae of the spicules of cartilage. The new trabeculae contain a core of cartilage which is surrounded by a bone matrix of types I and V collagens.The fracture gaps are invaded by fibrous tissue, which contain types III and V collagens. This is later replaced by cancellous bone.     

Immunohistochemical Video-microdensitometry of Myocardial Collagen Type I and Type III

Collagen is an essential part of the cardiac interstitium. Collagen subtypes, their location, total amount and the architecture of the fibrillar network are of functional importance. Architecture in terms of density of the fibrillar network is assumed to be reflected by the intensity of immunohistochemical staining of collagen. The aim of this study was to evaluate a video-based microdensitometric method for quantifying density expressed as absorbance of collagen subtypes I and III stained with an indirect immunoperoxidase method in myectomy specimens of patients with hypertrophic obstructive cardiomyopathy. Various factors influencing the immunohistochemical staining product and the technical properties of the image analysis system were investigated. Linearity between collagen concentration and the absorbance of the immunohistochemical staining product was demonstrated for collagen I using a dot-blot technique. Immunohistochemical collagen staining and density measure ment were easily reproducible. The cardiac disability of the patients was assessed according to the New York Heart Association (NYHA) criteria. There was a significant increase in collagen type I density with higher NYHA class, whereas no significant association was found for total collagen area fraction. Thus, video-based microdensitometry gives further insight into the structural remodelling of myocardial collagens and reveals their significance in the process of heart failure in hypertrophic cardiomyopathy.     

Collagen metabolism in the murine colon following X irradiation.

Female CBA mice, aged 16 weeks, were irradiated to the total pelvic region with either single doses (5-20 Gy) or two equal fractions (10- to 30-Gy total dose, 24-h interval) of 240 kV X rays. Total protein and collagen synthesis rates, collagen breakdown, and net collagen content of the colon were measured at various times postirradiation using a radioisotope incorporation method and HPLC analysis. Immunohistochemical staining and computerized image analysis were used to assess the relative amounts of collagen types I and III at various times postirradiation, in various regions of the colon. Total protein and collagen synthesis rates were elevated above control levels at 4 and 8 weeks postirradiation, as was collagen degradation. Values had returned to control levels by 16 weeks postirradiation, and there were no further changes up to 71 weeks postirradiation. The net amount of collagen in the colon did not change relative to controls at any time during the investigation. There was, however, increased immunohistochemical staining for collagen type I 52 weeks postirradiation in all regions of the colon and decreased staining of type III in the circular muscle layer and villi. Altered ratios of these two collagen isotypes are consistent with changes in mechanical properties of the tissue.     

Morphological alterations and NO-synthase expression in the heart after continuous light exposure of rats

Although exposure to continuous light is associated with hypertension and modulates the outcome of ischemia-reperfusion injury, less attention has been paid to its effects on cardiac morphology. We investigated whether 4-week exposure of experimental rats to continuous 24 h/day light can modify cardiac morphology, with focus on heart weight, fibrosis and collagen I/III ratio in correlation with NO-synthase expression. Two groups of male adult Wistar rats were studied: controls exposed to normal light/dark cycle (12 h/day light, 12 h/day dark) and rats exposed to continuous light. After 4 weeks of treatment the absolute and the relative heart weights were determined and myocardial fibrosis and collagen type I/III ratio were evaluated using picrosirius red staining. Endothelial and inducible NO-synthase expression was detected immunohistochemically. The exposure of rats to continuous light resulted in an increase of body weight with proportionally increased heart weight. Myocardial fibrosis remained unaffected but collagen I/III ratio increased. Neither endothelial nor inducible NO-synthase expression was altered in light-exposed rats. We conclude that the loss of structural homogeneity of the myocardium in favor of collagen type I might increase myocardial stiffness and contribute to functional alterations after continuous light exposure.     

The influence of tissue section thickness on the study of collagen by the Picrosirius-polarization method

Summary The influence of tissue section thickness on the color and intensity of birefringence displayed by collagen in tissue sections studied by means of the Picrosirius-polarization method, is reported in this paper. When dermal collagen sections of different thicknesses (ranging from 0.25 to 11 m) were studied by this method, it became evident that not only did the intensity of birefringence increase proportionally to tissue section thickness, as was to be expected, but also a gradual shift in color from green through yellow to red could be observed as tissue section thickness increased. The limitations of the Picrosirius-polarization method for the localization of collagen types I, II, and III in routinely used histological slides is discussed, showing that this method is useful for the study of the distribution of the different types of interstitial collagen in normal adult vertebrate organs.     

Quantitation of type I, III, and V collagens in human tissue samples by high-performance liquid chromatography of selected cyanogen bromide peptides

A method to determine the proportions of the major fiber-forming collagens (types I, III, and V) in noncartilaginous human tissues is presented. The procedure relies on direct solubilization of tissue collagen as cyanogen bromide peptides. The peptides are subjected to cation exchange chromatography followed by gel permeation chromatography in a manner consistent with the rapid resolution and quantitation of relatively low-molecular-weight marker peptides for each collagen. The marker peptides utilized for type I, III, and V collagens are alpha 1 (I)-CB2, alpha 1 (III)-CB2, and alpha 1 (V)-CB1, respectively. Quantitation of the peptides is attained as a function of ultraviolet absorbance during gel permeation chromatography. The nature of the marker peptides, the use of high-performance liquid chromatography techniques, and quantitation of the peptides by ultraviolet absorbance renders the method suitably rapid, sensitive, and accurate for routine evaluations of collagen composition. The utility of the method is illustrated in the presentation of analyses on specimens of placental membranes and blood vessel walls.