Edward J. Miller

[2] Preparation and characterization of the different types of collagen

Publisher Summary This chapter presents methods generally applicable to the preparation of native collagen from a variety of sources. The preparation of a collagen sample generally involves several different steps. These include acquisition and preliminary processing of an appropriate tissue or organ, extraction of the collagen, and its purification. The latter process requires not only elimination of the noncollagenous components that are present in the extract, but may also require selective removal of alternative collagen types. This can usually be accomplished by a judicious use of selective precipitation techniques accompanied ultimately by one or two chromatographic steps. Given the extreme diversity of the tissues or organs in which collagen occurs as well as the multiplicity of collagen types that may be present in a given source, there understandably exists no single standard or preferred method for the preparation of collagen. Unless otherwise indicated, all procedures are conducted at relatively low temperatures, in the range of 4-8 °. This minimizes bacterial growth, enhances the solubility of native collagens, and ensures the retention of native conformation on the part of the solubilized collagens.

[1] The collagens: An overview and update

Publisher Summary This chapter emphasizes on new developments concerning the relatively well-characterized collagens as well as on studies describing the more recently discovered collagens. It is abundantly clear that although collagen molecules function primarily as components of supporting aggregates, they constitute a large family of proteins with a wide spectrum of chemical and structural features. The nature of the aggregates derived from various types of molecules reflects this diversity. The molecular diversity of the collagen family of proteins is, of course, specified ultimately by the various genes utilized in synthesizing different collagen chains. The genetic heterogeneity is, however, considerably amplified through intracellular posttranslational modifications of the nascent chains as well as through extracellular processing of secreted molecules. A number of factors have greatly facilitated the acquisition of data on most of the collagens described above. Use of limited proteolysis with pepsin to facilitate the release of fragments of the molecules is one of these factors. Fragmentation of the molecules constitutes a serious limitation when this approach is utilized. However, it appears likely that many of the collagens for which extensive data are now available would have remained undetected were it not possible to isolate their fragments by means of limited proteolysis.

Effect of physical crosslinking methods on collagen-fiber durability in proteolytic solutions

We previously demonstrated that ultraviolet (UV) or dehydrothermal (DHT) crosslinking partially denatured fibers extruded from an insoluble type I collagen dispersion. In this study denaturation effects were evaluated by measuring collagen-fiber sensitivity to trypsin. Shrinkage-temperature measurements and sensitivity to collagenase served as indices of crosslinking. UV or DHT crosslinking increased the collagen-fiber shrinkage temperature, resistance to degradation in collagenase, and durability under load in collagenase. However, in trypsin solutions, solubility was significantly increased for UV (approximately 11%) or DHT (approximately 15%) crosslinked fibers compared with uncrosslinked fibers (approximately 4%). Size-exclusion chromatography indicated that no intact collagen alpha-chains were present in the soluble fraction of fibers exposed to trypsin (MW < 1 kD). Interestingly, UV-crosslinked collagen fibers remained intact an order of magnitude longer (4840 +/- 739 min) than DHT-crosslinked (473 +/- 39 min) or uncrosslinked (108 +/- 53 min) fibers when placed under load in trypsin solutions. These data indicate that mechanical loading during incubation in a trypsin solution measures denaturation effects not detected by the trypsin-solubility assay. Our results suggest that DHT-crosslinked collagen fibers should not be used as load-bearing implants. UV-crosslinked fibers may retain more native structure and should exhibit greater resistance to nonspecific proteases in vivo.

The Structure of Fibril‐forming Collagens

Although collagen molecules are designed primarily to serve as constituents of supporting aggregates in various tissues, they are present as a relatively large family of proteins that exhibit a wide diversity in structural and chemical features. Molecular diversity is, of course, specified primarily by the different genes for synthesis of the various collagen chains. However, intracellular post-translational modifications of the nascent chains as well as extracellular processing of newly assembled molecules contribute to, and considerably amplify, the diversity specified by the genome. Moreover, the nature of the aggregates derived from various molecular species of collagen reflects this diversity. In this fashion, a great deal of chemical and biological variation is created in otherwise highly similar molecules such as those classified here as belonging to group 1. It is anticipated that further developments regarding these and other molecular species of collagen will considerably refine our understanding of the spectrum of structure and function associated with this unique family of proteins.

Collagen molecules comprised of α1(V)-chains (B-chains): an apparent localization in the exocytoskeleton

Antibodies specific for alpha 1(V) chains and native collagen molecules containing the alpha 1(V) chain have been used to study the localization of alpha 1(V)-containing molecules in differentiating hyaline cartilage. Immunofluorescence data show that the undifferentiated mesenchyme contains significant quantities of these molecules throughout the cell-rich tissue matrix. Examination of fully differentiated hyaline cartilage reveals a unique staining pattern wherein the immunofluorescent material is restricted to the pericellular matrix within the chondrocyte lacunae. We conclude from these data in conjunction with other evidence that the Type V collagens function as components of an exocytoskeleton for connective tissue cells.

The collagenous exocytoskeleton of smooth muscle cells.

Abstract Using specific antibodies in immunoelectron microscope studies with vascular tissues, type V collagen has been localized in basement membrane structures associated with discrete regions of the smooth muscle cell surface as well as membranous structures adjacent to endothelial cell membranes. These results confirm and extend earlier light microscope observations suggesting that type V collagen is largely associated with the pericellular environment in such tissues.

The effect of embryo extract on the types of collagen synthesized by cultured chick chondrocytes

Abstract Growth of embryonic chick chondrocytes in dialyzed embryo extract results in both a change in morphology of the cells toward that of a fibroblast and a change in the type of collagen synthesized from the cartilage-specific Type II collagen (chain composition [α1(II)] 3 ) to a mixture of Type I collagen (chain composition [α1(I)] 2 α2) and the Type I trimer (chain composition [α1(I)] 3 ). Analyses after 6 days of growth in embryo extract show that the synthesis of only Type I collagen and the Type I trimer can be detected. However, on subculturing the cells to a low density and allowing a period of growth without embryo extract, colonies of chondrocytes reappear and the synthesis of Type II collagen apparently resumes. It is suggested that the observed changes represent a “modulation” in cell behavior, this being expressed not only by the morphological changes but also by changes in cell-specific protein synthesis as demonstrated by the changes in the type of collagen synthesized.

Evidence for the Existence of an α1(V)α2(V)α3(V) Collagen Molecule in Human Placental Tissue

Abstract Chromatography of native type V collagen from human placenta on phosphocellulose using nondenaturing conditions results in the partial resolution of two fractions. The first fraction contains each of the three type V α chains in approximately equal proportions and upon thermal denaturation exhibits a melting temperature of 33°C. Fraction two contains the α1 (V) and α2 (V) chains in approximately a 2:1 ratio, respectively, and has a melting temperature of 35°C. These data indicate the presence of two molecular species of type V collagen in placenta, namely an α1 (V) α2 (V) α3 (V) molecule and the previously described [α1 (V)] 2 -α2 (V) molecule.

Antibodies to human native and denatured collagens in synovial fluids of patients with rheumatoid arthritis

Abstract Specimens of joint fluids and sera from 24 patients with rheumatoid arthritis (RA) and 31 patients with other rheumatic conditions were tested by a passive hemagglutination assay for the presence of antibodies to human native collagens and to their respective chains. A large percentage of the RA patients displayed a high occurrence of anticollagen antibodies in the serum, and the titers and occurrence of these antibodies were even higher in the respective synovial fluids. Synovial fluids, but not sera, of three osteoarthritic patients possessed antibodies to collagen chains. Patients with gouty arthritis, pseudogout, and/or traumatic effusions had no anticollagen antibodies.

Packaging and delivery of bone induction factors in a collagenous implant

Extracts of demineralized rat bones which contained factors stimulating bone induction were reconstituted with highly purified human type I collagen to provide a suitable and easily manipulated delivery system for surgical implantation. When implanted subcutaneously in rats, the implants governed and delineated the dimensions of the resulting bony tissue. It is proposed that this implant system has clinical application in the filling of osseous defects within the scope of orthopaedic and oral and maxillofacial surgery. It is presented here as a potential improvement over conventional implant materials without osteoinductive properties.