In vitro Synthesis of Native,Fibrous Long Spacing and Segmental Long Spacing Collagen

Collagen fibrils are present in the extracellular matrix of animal tissue to provide structural scaffolding and mechanical strength. These native collagen fibrils have a characteristic banding periodicity of ~67 nm and are formed in vivo through the hierarchical assembly of Type I collagen monomers, which are 300 nm in length and 1.4 nm in diameter. In vitro, by varying the conditions to which the monomer building blocks are exposed, unique structures ranging in length scales up to 50 microns can be constructed, including not only native type fibrils, but also fibrous long spacing and segmental long spacing collagen. Herein, we present procedures for forming the three different collagen structures from a common commercially available collagen monomer. Using the protocols that we and others have published in the past to make these three types typically lead to mixtures of structures. In particular, unbanded fibrils were commonly found when making native collagen, and native fibrils were often present when making fibrous long spacing collagen. These new procedures have the advantage of producing the desired collagen fibril type almost exclusively. The formation of the desired structures is verified by imaging using an atomic force microscope.     

Reduction of type V collagen using a dominant-negative strategy alters the regulation of fibrillogenesis and results in the loss of corneal- specific fibril morphology

A number of factors have been implicated in the regulation of tissue- specific collagen fibril diameter. Previous data suggest that assembly of heterotypic fibrils composed of two different fibrillar collagens represents a general mechanism regulating fibril diameter. Specifically, we hypothesize that type V collagen is required for the assembly of the small diameter fibrils observed in the cornea. To test this, we used a dominant-negative retroviral strategy to decrease the levels of type V collagen secreted by chicken corneal fibroblasts. The chicken alpha 1(V) collagen gene was cloned, and retroviral vectors that expressed a polycistronic mRNA encoding a truncated alpha 1(V) minigene and the reporter gene LacZ were constructed. The efficiency of viral infection was 30-40%, as determined by assaying beta- galactosidase activity. To assess the expression from the recombinant provirus, Northern analysis was performed and indicated that infected fibroblasts expressed high steady-state levels of retroviral mRNA. Infected cells synthesized the truncated alpha 1(V) protein, and this was detectable only intracellularly, in a distribution that colocalized with lysosomes. To assess endogenous alpha 1(V) protein levels, infected cell cultures were assayed, and these consistently demonstrated reductions relative to control virus-infected or uninfected cultures. Analyses of corneal fibril morphology demonstrated that the reduction in type V collagen resulted in the assembly of large- diameter fibrils with a broad size distribution, characteristics similar to fibrils produced in connective tissues with low type V concentrations. Immunoelectron microscopy demonstrated the amino- terminal domain of type V collagen was associated with the small- diameter fibrils, but not the large fibrils. These data indicate that type V collagen levels regulate corneal fibril diameter and that the reduction of type V collagen is sufficient to alter fibril assembly so that abnormally large-diameter fibrils are deposited into the matrix.     

Probing the effect of glycosaminoglycan depletion on integrin interactions with collagen I fibrils in the native extracellular matrix environment

Fibrillar collagen–integrin interactions in the extracellular matrix (ECM) regulate a multitude of cellular processes and cell signalling. Collagen I fibrils serve as the molecular scaffolding for connective tissues throughout the human body and are the most abundant protein building blocks in the ECM. The ECM environment is diverse, made up of several ECM proteins, enzymes, and proteoglycans. In particular, glycosaminoglycans (GAGs), anionic polysaccharides that decorate proteoglycans, become depleted in the ECM with natural aging and their mis-regulation has been linked to cancers and other diseases. The impact of GAG depletion in the ECM environment on collagen I protein interactions and on mechanical properties is not well understood. Here, we integrate ELISA protein binding assays with liquid high-resolution atomic force microscopy (AFM) to assess the effects of GAG depletion on the interaction of collagen I fibrils with the integrin α2I domain using separate rat tails. ELISA binding assays demonstrate that α2I preferentially binds to GAG-depleted collagen I fibrils in comparison to native fibrils. By amplitude modulated AFM in air and in solution, we find that GAG-depleted collagen I fibrils retain structural features of the native fibrils, including their characteristic D-banding pattern, a key structural motif. AFM fast force mapping in solution shows that GAG depletion reduces the stiffness of individual fibrils, lowering the indentation modulus by half compared to native fibrils. Together these results shed new light on how GAGs influence collagen I fibril–integrin interactions and may aid in strategies to treat diseases that result from GAG mis-regulation.     

Thermal Memory in Self-Assembled Collagen Fibril Networks

Collagen fibrils form extracellular networks that regulate cell functions and provide mechanical strength to tissues. Collagen fibrillogenesis is an entropy-driven process promoted by warming and reversed by cooling. Here, we investigate the influence of noncovalent interactions mediated by the collagen triple helix on fibril stability. We measure the kinetics of cold-induced disassembly of fibrils formed from purified collagen I using turbimetry, probe the fibril morphology by atomic force microscopy, and measure the network connectivity by confocal microscopy and rheometry. We demonstrate that collagen fibrils disassemble by subunit release from their sides as well as their ends, with complex kinetics involving an initial fast release followed by a slow release. Surprisingly, the fibrils are gradually stabilized over time, leading to thermal memory. This dynamic stabilization may reflect structural plasticity of the collagen fibrils arising from their complex structure. In addition, we propose that the polymeric nature of collagen monomers may lead to slow kinetics of subunit desorption from the fibril surface. Dynamic stabilization of fibrils may be relevant in the initial stages of collagen assembly during embryogenesis, fibrosis, and wound healing. Moreover, our results are relevant for tissue repair and drug delivery applications, where it is crucial to control fibril stability.     

Live imaging of collagen remodeling during angiogenesis

To better understand interstitial matrix remodeling during angiogenesis, we probed endogenous optical signatures of collagen fibrils and cells with multiphoton microscopy to noninvasively visualize, in real-time, changes to fibril organization around angiogenic sprouts and growing neovessels. From analyses of the second-harmonic generation signal from fibrillar collagen and two-photon excited fluorescence, as well as coherent transmitted light from vascular cells, we found that microvessel fragments interacting with the collagen matrix exhibited two key features: a strong association of fibrillar collagen around the parent vessel fragment during vessel construct reconstitution and a substantial collagen fibril reorganization by sprout and neovessel tips. Results indicate that angiogenic sprouts and growing neovessels actively and differentially remodel existing collagen fibrils. This imaging approach to assess local changes in matrix organization may have a broader impact on tissue biology and mechanics during angiogenesis and allow for new insights in cardiovascular, diabetes, and cancer research.     

Structural investigations on native collagen type I fibrils using AFM

This study was carried out to determine the elastic properties of single collagen type I fibrils with the use of atomic force microscopy (AFM). Native collagen fibrils were formed by self-assembly in vitro characterized with the AFM. To confirm the inner assembly of the collagen fibrils, the AFM was used as a microdissection tool. Native collagen type I fibrils were dissected and the inner core uncovered. To determine the elastic properties of collagen fibrils the tip of the AFM was used as a nanoindentor by recording force-displacement curves. Measurements were done on the outer shell and in the core of the fibril. The structural investigations revealed the banding of the shell also in the core of native collagen fibrils. Nanoindentation experiments showed the same Young's modulus on the shell as well as in the core of the investigated native collagen fibrils. In addition, the measurements indicate a higher adhesion in the core of the collagen fibrils compared to the shell.     

Tensile mechanical properties of three-dimensional type I collagen extracellular matrices with varied microstructure

The importance and priority of specific micro-structural and mechanical design parameters must be established to effectively engineer scaffolds (biomaterials) that mimic the extracellular matrix (ECM) environment of cells and have clinical applications as tissue substitutes. In this study, three-dimensional (3-D) matrices were prepared from type I collagen, the predominant compositional and structural component of connective tissue ECMs, and structural-mechanical relationships were studied. Polymerization conditions, including collagen concentration (0.3-3 mg/mL) and pH (6-9), were varied to obtain matrices of collagen fibrils with different microstructures. Confocal reflection microscopy was used to assess specific micro-structural features (e.g., diameter and length) and organization of component fibrils in 3-D. Microstructural analyses revealed that changes in collagen concentration affected fibril density while maintaining a relatively constant fibril diameter. On the other hand, both fibril length and diameter were affected by the pH of the polymerization reaction. Mechanically, all matrices exhibited a similar stress-strain curve with identifiable "toe," "linear," and "failure" regions. However the linear modulus and failure stress increased with collagen concentration and were correlated with an increase in fibril density. Additionally, both the linear modulus and failure stress showed an increase with pH, which was related to an increasedfibril length and a decreasedfibril diameter. The tensile mechanical properties of the collagen matrices also showed strain rate dependence. Such fundamental information regarding the 3-D microstructural-mechanical properties of the ECM and its component molecules are important to our overall understanding of cell-ECM interactions (e.g., mechanotransduction) and the development of novel strategies for tissue repair and replacement.     

An axial periodic fibrillar arrangement of antigenic determinants for fibronectin and procollagen on ascorbate treated human fibroblasts

Fibronectin and collagens are major constituents of the cell matrix of fibroblasts. Fibronectin is a 220,000 dalton glycoprotein that mediates a variety of adhesive functions of cells examined in vitro. Fibronectin is secreted in a soluble form and interacts with collagen to form extracellular filaments. Fibronectin and procollage type I were localized using the peroxidase anti-peroxidase method. Under standard culture conditions, fibronectin and procollagen were localized to non-periodic 10 nm extracellular fibrils, the cell membrane and plasma membrane vesicles. Ascorbate treatment of cells leads to a new larger fibril with a diameter of approximately 40 nm. Antibodies to fibronectin and procollagen I react to these native collagen fibrils with an axial periodicity of approximately 70 nm. Fibronectin is clearly associated with native collagen fibrils produced by ascorbate treated cells and there is an asymetric distribution or segregation of fibronectin on these collagen fibrils with a 70 nm axial repeat.     

In vitro reconstitution of fibrillar collagen type I assemblies at reactive polymer surfaces

The reconstitution of fibrillar collagen and its assemblies with heparin and hyaluronic acid was studied in vitro. Fibril formation kinetics were analyzed by turbidity and depletion measurements in solutions containing varied concentrations of collagen and glycosaminoglycans. Fibril-forming collagen solutions were further applied for the coating of planar substrates which had been modified with alternating maleic anhydride copolymer films before. The immobilized collagen assemblies were characterized with respect to the deposited amount of protein using ellipsometry and acidic hydrolysis/HPLC-based amino acid analysis, respectively. AFM, SEM, and cLSM were utilized to gain information on structural features and patterns formed by surface-attached fibrils depending on the initial solution concentrations of collagen. The results revealed that the addition of heparin and hyaluronic acid affected both the fibril dimensions and the meshwork characteristics of the surface-bound fibrils.     

Type V collagen controls the initiation of collagen fibril assembly

Vertebrate collagen fibrils are heterotypically composed of a quantitatively major and minor fibril collagen. In non-cartilaginous tissues, type I collagen accounts for the majority of the collagen mass, and collagen type V, the functions of which are poorly understood, is a minor component. Type V collagen has been implicated in the regulation of fibril diameter, and we reported recently preliminary evidence that type V collagen is required for collagen fibril nucleation (Wenstrup, R. J., Florer, J. B., Cole, W. G., Willing, M. C., and Birk, D. E. (2004) J. Cell. Biochem. 92, 113-124). The purpose of this study was to define the roles of type V collagen in the regulation of collagen fibrillogenesis and matrix assembly. Mouse embryos completely deficient in pro-alpha1(V) chains were created by homologous recombination. The col5a1-/- animals die in early embryogenesis, at approximately embryonic day 10. The type V collagen-deficient mice demonstrate a virtual lack of collagen fibril formation. In contrast, the col5a1+/- animals are viable. The reduced type V collagen content is associated with a 50% reduction in fibril number and dermal collagen content. In addition, relatively normal, cylindrical fibrils are assembled with a second population of large, structurally abnormal collagen fibrils. The structural properties of the abnormal matrix are decreased relative to the wild type control animals. These data indicate a central role for the evolutionary, ancient type V collagen in the regulation of fibrillogenesis. The complete dependence of fibril formation on type V collagen is indicative of the critical role of the latter in early fibril initiation. In addition, this fibril collagen is important in the determination of fibril structure and matrix organization.     

Organisation of collagen fibrils in tendon: changes induced by an anabolic steroid

Morphometric and stereologic analysis of the organisation of collagen fibrils in tendon tissue after a treatment with an anabolic steroid hormone allowed the following observations: In a short-term study stereological data revealed a potent accumulation of collagen fibrils in the extra-cellular matrix after the administration of an anabolic steroid. Compared with controls, the anabolic steroid significantly increased the number of dysplastic collagen fibrils dependent on duration of treatment. Inter- and intrafibrillary dysplastic collagen fibrils possess characteristic diameter distributions which differ considerably from those of normal collagen fibrils. The functional significance of the changes in mean diameter, diameter distribution, numerical density and volume fraction of collagen fibrils in tendons following hormone treatment may be relevant to the use of these drugs in clinical practice and in competitive sports.     

Fibrous long spacing collagen ultrastructure elucidated by atomic force microscopy.

Fibrous long spacing collagen (FLS) fibrils are collagen fibrils in which the periodicity is clearly greater than the 67-nm periodicity of native collagen. FLS fibrils were formed in vitro by the addition of alpha1-acid glycoprotein to an acidified solution of monomeric collagen and were imaged with atomic force microscopy. The fibrils formed were typically approximately 150 nm in diameter and had a distinct banding pattern with a 250-nm periodicity. At higher resolution, the mature FLS fibrils showed ultrastructure, both on the bands and in the interband region, which appears as protofibrils aligned along the main fibril axis. The alignment of protofibrils produced grooves along the main fibril, which were 2 nm deep and 20 nm in width. Examination of the tips of FLS fibrils suggests that they grow via the merging of protofibrils to the tip, followed by the entanglement and, ultimately, the tight packing of protofibrils. A comparison is made with native collagen in terms of structure and mechanism of assembly.     

Tip-mediated fusion involving unipolar collagen fibrils accounts for rapid fibril elongation, the occurrence of fibrillar branched networks in skin and the paucity of collagen fibril ends in vertebrates.

Collagen fibrils are the principal source of mechanical strength of connective tissues such as tendon, skin, cornea, cartilage and bone. The ability of these tissues to withstand tensile forces is directly attributable to the length and diameter of the fibrils, and to interactions between individual fibrils. Although electron microscopy studies have provided information on fibril diameters, little is known about the length of fibrils in tissue and how fibrils interact with each other. The question of fibril length has been difficult to address because fibril ends are rarely observed in cross-sections of tissue. The paucity of fibril ends, or tips, has led to controversy about how long individual fibrils might be and how the fibrils grow in length and diameter. This review describes recent discoveries that are relevant to these questions. We now know that vertebrate collagen fibrils are synthesised as short (1-3 microm) early fibrils that fuse end-to-end in young tissues to generate very long fibrils. The diameter of the final fibril is determined by the diameter of the collagen early fibrils. During a late stage of tissue assembly fibril tips fuse to fibril shafts to generate branched networks. Of direct relevance to fibril fusion is the fact that collagen fibrils can be unipolar or bipolar, depending on the orientation of collagen molecules in the fibril. Fusion relies on: (1) specific molecular interactions at the carboxyl terminal ends of unipolar collagen fibrils; and (2) the insulator function of small proteoglycans to shield the surfaces of fibrils from inappropriate fusion reactions. The fusion of tips to shafts to produce branched networks of collagen fibrils is an elegant mechanism to increase the mechanical strength of tissues and provides an explanation for the paucity of fibril tips in older tissue.     

Candidate cell and matrix interaction domains on the collagen fibril, the predominant protein of vertebrates

Type I collagen, the predominant protein of vertebrates, polymerizes with type III and V collagens and non-collagenous molecules into large cable-like fibrils, yet how the fibril interacts with cells and other binding partners remains poorly understood. To help reveal insights into the collagen structure-function relationship, a data base was assembled including hundreds of type I collagen ligand binding sites and mutations on a two-dimensional model of the fibril. Visual examination of the distribution of functional sites, and statistical analysis of mutation distributions on the fibril suggest it is organized into two domains. The "cell interaction domain" is proposed to regulate dynamic aspects of collagen biology, including integrin-mediated cell interactions and fibril remodeling. The "matrix interaction domain" may assume a structural role, mediating collagen cross-linking, proteoglycan interactions, and tissue mineralization. Molecular modeling was used to superimpose the positions of functional sites and mutations from the two-dimensional fibril map onto a three-dimensional x-ray diffraction structure of the collagen microfibril in situ, indicating the existence of domains in the native fibril. Sequence searches revealed that major fibril domain elements are conserved in type I collagens through evolution and in the type II/XI collagen fibril predominant in cartilage. Moreover, the fibril domain model provides potential insights into the genotype-phenotype relationship for several classes of human connective tissue diseases, mechanisms of integrin clustering by fibrils, the polarity of fibril assembly, heterotypic fibril function, and connective tissue pathology in diabetes and aging.     

Cartilage Fibrils of Mammals are Biochemically Heterogeneous: Differential Distribution of Decorin and Collagen IX

Cartilage fibrils contain collagen II as the major constituent, but the presence of additional components, minor collagens, and noncollagenous glycoproteins is thought to be crucial for modulating several fibril properties. We have examined the distribution of two fibril constituents—decorin and collagen IX—in samples of fibril fragments obtained after bovine cartilage homogenization. Decorin was preferentially associated with a population of thicker fibril fragments from adult articular cartilage, but was not present on the thinnest fibrils. The binding was specific for the gap regions of the fibrils, and depended on the decorin core protein. Collagen IX, by contrast, predominated in the population with the thinnest fibrils, and was scarce on wider fibrils. Double-labeling experiments demonstrated the coexistence of decorin and collagen IX in some fibrils of intermediate diameter, although most fibril fragments from adult cartilage were strongly positive for one component and lacked the other. Fibril fragments from fetal epiphyseal cartilage showed a different pattern, with decorin and collagen IX frequently colocalized on fragments of intermediate and large diameters. Hence, the presence of collagen IX was not exclusive for fibrils of small diameter. These results establish that articular cartilage fibrils are biochemically heterogeneous. Different populations of fibrils share collagen II, but have distinct compositions with respect to macromolecules defining their surface properties.     

Amianthoid (asbestoid) transformation: electron microscopical studies on aging human costal cartilage

The present study reports on the fine structure of human costal cartilage at different ages in order to obtain information on the morphogenesis of amianthoid fibers. Our results reveal an overall increase of collagen fibril diameter with increasing age, even in areas with no signs of amianthoid transformation. Ultrastructural evidence is presented that this increase in diameter is due to a gathering of the preexisting collagen fibrils. The age-related change in collagen fibril diameter is paralleled by changes in the composition and ultrastructural appearance of cartilage proteoglycans (as revealed by acridine orange staining). Acridine-orange-positive filaments indicative for proteoglycans are markedly reduced in size with advancing age in centrally located regions of costal cartilage. Treatment with testicular hyaluronidase previous to acridine-orange staining leaves these small proteoglycan filaments unaffected. By contrast, the filaments visible after acridine-orange staining in the extracellular matrix near to the perichondrium are susceptible to hyaluronidase treatment. Infrequently, a sharp increase in collagen fibril diameter can be observed in territorial matrix areas of degenerating chondrocytes. This observation is conspicuous at ages of 10 and 20 years. Amianthoid transformation is characterized by the appearance of collagen fibrils strictly arranged in parallel. These amianthoid fibers are embedded in a matrix rich in small acridine-orange-positive filaments similar to the proteoglycan filaments observed in centrally located matrix regions. It can be concluded that extensive remodelling not only of the collagen fibrils but also of the cartilage proteoglycans is involved in the development of amianthoid transformation.     

Growth of Collagen Fibril Seeds from Embryonic Tendon: Fractured Fibril Ends Nucleate New Tip Growth

Collagen fibrils are the principal tensile element of vertebrate tissues where they occur in the extracellular matrix as spatially organised arrays. A major challenge is to understand how the mechanisms of nucleation, growth and remodelling yield fibrils of tissue-specific diameter and length. Here we have developed a seeding system whereby collagen fibrils were isolated from avian embryonic tendon and added to purified collagen solution, in order to characterise fibril surface nucleation and growth mechanisms. Fragmentation of tendon in liquid nitrogen followed by Dounce homogenisation generated fibril length fragments. Most (> 94%) of the fractured ends of fibrils, which show an abrupt square profile, were found to act as nucleation sites for further growth by molecular accretion. The mechanism of this nucleation and growth process was investigated by transmission electron microscopy, atomic force microscopy and scanning transmission electron microscopy mass mapping. Typically, a single growth spur occurred on the N-terminal end of seed fibrils whilst twin spurs frequently formed on the C-terminal end before merging into a single tip projection. The surface nucleation and growth process generated a smoothly tapered tip that achieved maximum diameter when the axial extension reached ∼ 13 μm. Lateral growth also occurred along the entire length of all seed fibrils that contained tip projections. The data support a model of collagen fibril growth in which the broken ends of fibrils are nucleation sites for propagation in opposite axial directions. The observed fibril growth behaviour has direct relevance to tendon matrix remodelling and repair processes that might involve rupture of collagen fibrils.     

Monomer and oligomer of type I collagen: molecular properties and fibril assembly

Type I collagen purified from calf skin was further separated into monomeric and oligomeric fractions and characterized with gel electrophoresis and measurement of solution viscosity. The thermal stabilities of the triple-helical structure of the collagen molecules of these preparations and the fibrils assembled therefrom were determined with differential UV spectroscopy and scanning microcalorimetry. The monomeric collagen was reduced with NaBH4-, and the kinetics and equilibrium of the reversible fibril assembly-disassembly were examined in detail. Fibril assembly and disassembly of the collagen induced by slow scans of temperature showed hysteresis. The assembly curve was very sharp whereas the disassembly curve was gradual. Equilibrium centrifugation showed the collagen disassembled from the fibrils to be predominantly monomers. However, unlike the unassembled collagen, the collagen disassembled from fibrils by cooling showed no lag phase in subsequent cycles of fibril assembly. The thermodynamic parameters of fibril growth were derived from a fibril disassembly curve. Fibril growth was weaker for the NaBH4-reduced monomeric collagen than the native crude collagen, perhaps due to the removal of oligomers and the changes in the molecular structure brought by the reduction. The results corroborated the strongly cooperative mechanism for the fibril assembly proposed in the preceding paper.     

The in situ supermolecular structure of type I collagen.

BACKGROUND: The proteins belonging to the collagen family are ubiquitous throughout the animal kingdom. The most abundant collagen, type I, readily forms fibrils that convey the principal mechanical support and structural organization in the extracellular matrix of connective tissues such as bone, skin, tendon, and vasculature. An understanding of the molecular arrangement of collagen in fibrils is essential since it relates molecular interactions to the mechanical strength of fibrous tissues and may reveal the underlying molecular pathology of numerous connective tissue diseases. RESULTS: Using synchrotron radiation, we have conducted a study of the native fibril structure at anisotropic resolution (5.4 A axial and 10 A lateral). The intensities of the tendon X-ray diffraction pattern that arise from the lateral packing (three-dimensional arrangement) of collagen molecules were measured by using a method analogous to Rietveld methods in powder crystallography and to the separation of closely spaced peaks in Laue diffraction patterns. These were then used to determine the packing structure of collagen by MIR. CONCLUSIONS: Our electron density map is the first obtained from a natural fiber using these techniques (more commonly applied to single crystal crystallography). It reveals the three-dimensional molecular packing arrangement of type I collagen and conclusively proves that the molecules are arranged on a quasihexagonal lattice. The molecular segments that contain the telopeptides (central to the function of collagen fibrils in health and disease) have been identified, revealing that they form a corrugated arrangement of crosslinked molecules that strengthen and stabilize the native fibril.     

Time-lapse confocal reflection microscopy of collagen fibrillogenesis and extracellular matrix assembly in vitro

The development of the next generation of biomaterials for restoration of tissues and organs (i.e., tissue engineering) requires a better understanding of the extracellular matrix (ECM) and its interaction with cells. Extracellular matrix is a macromolecular assembly of natural biopolymers including collagens, glycosaminoglycans (GAGs), proteoglycans (PGs), and glycoproteins. Interestingly, several ECM components have the ability to form three-dimensional (3D), supramolecular matrices (scaffolds) in vitro by a process of self-directed polymerization, "self-assembly". It has been shown previously that 3D matrices with distinct architectural and biological properties can be formed from either purified type I collagen or a complex mixture of interstitial ECM components derived from intestinal submucosa. Unfortunately, many of the imaging and analysis techniques available to study these matrices either are unable to provide insight into 3D preparations or demand efforts that are often prohibitory to observations of living, dynamic systems. This is the first report on the use of reflection imaging at rapid time intervals combined with laser-scanning confocal microscopy for analysis of structural properties and kinetics of collagen and ECM assembly in 3D. We compared time-lapse confocal reflection microscopy (TL-CRM) with a well-established spectrophotometric method for determining the self-assembly properties of both purified type I collagen and soluble interstitial ECM. While both TL-CRM and spectrophotometric techniques provided insight into the kinetics of the polymerization process, only TL-CRM allowed qualitative and quantitative evaluation of the structural parameters (e.g., fibril diameter) and 3D organization (e.g., fibril density) of component fibrils over time. Matrices formed from the complex mixture of soluble interstitial ECM components showed an increased rate of assembly, decreased opacity, decreased fibril diameter, and increased fibril density compared to that of purified type I collagen. These results suggested that the PG/GAG components of soluble interstitial ECM were affecting the polymerization of the component collagens. Therefore, the effects of purified and complex mixtures of PG/GAG components on the assembly properties of type I collagen and interstitial ECM were evaluated. The data confirmed that the presence of PG/GAG components altered the kinetics and the 3D fibril morphology of assembled matrices. In summary, TL-CRM was demonstrated to be a new and useful technique for analysis of the 3D assembly properties of collagen and other natural biopolymers which requires no specimen fixation and/or staining.Copyright 2000 John Wiley & Sons, Inc.