Single amino acid substitutions in the C-terminus of collagen II alter its affinity for collagen IX

The structural integrity of cartilage depends on the presence of extracellular matrices (ECM) formed by heterotypic fibrils composed of collagen II, collagen IX, and collagen XI. The formation of these fibrils depends on the site-specific binding between relatively small regions of interacting collagen molecules. Single amino acid substitutions in collagen II change the physicochemical and structural characteristics of those sites, thereby leading to an alteration of intermolecular collagen II/collagen IX interaction. Employing a biosensor to study interactions between R75C, R789C or G853E collagen II mutants and collagen IX, we demonstrated significant changes in the binding affinities. Moreover, analyses of computer models representing mutation sites defined exact changes in physicochemical characteristics of collagen II mutants. Our study shows that changes in collagen II/collagen IX affinity could represent one of the steps in a cascade of changes occurring in the ECM of cartilage as a result of single amino acid substitutions in collagen II.     

Guilty by association: some collagen II mutants alter the formation of ECM as a result of atypical interaction with fibronectin

Among the structural components of extracellular matrices (ECM) fibrillar collagens play a critical role, and single amino acid substitutions in these proteins lead to pathological changes in tissues in which they are expressed. Employing a biologically relevant experimental model consisting of cells expressing R75C, R519C, R789C, and G853E procollagen II mutants, we found that the R789C mutation causing a decrease in the thermostability of collagen not only alters individual collagen molecules and collagen fibrils, but also has a negative impact on fibronectin. We propose that thermolabile collagen molecules are able to bind to fibronectin, thereby altering intracellular and extracellular processes in which fibronectin takes part, and we postulate that such an atypical interaction could change the architecture of the ECM of affected tissues in patients harboring mutations in genes encoding fibrillar collagens.     

COMP acts as a catalyst in collagen fibrillogenesis

We have previously reported that COMP (cartilage oligomeric matrix protein) is prominent in cartilage but is also present in tendon and binds to collagens I and II with high affinity. Here we show that COMP influences the fibril formation of these collagens. Fibril formation in the presence of pentameric COMP was much faster, and the amount of collagen in fibrillar form was markedly increased. Monomeric COMP, lacking the N-terminal coiled-coil linker domain, decelerated fibrillogenesis. The data show that stimulation of collagen fibrillogenesis depends on the pentameric nature of COMP and not only on collagen binding. COMP interacts primarily with free collagen I and II molecules, bringing several molecules to close proximity, apparently promoting further assembly. These assemblies further join in discrete steps to a narrow distribution of completed fibril diameters of 149 +/- 16 nm with a banding pattern of 67 nm. COMP is not found associated with the mature fibril and dissociates from the collagen molecules or their early assemblies. However, a few COMP molecules are found bound to more loosely associated molecules at the tip/end of the growing fibril. Thus, COMP appears to catalyze the fibril formation by promoting early association of collagen molecules leading to increased rate of fibrillogenesis and more distinct organization of the fibrils.     

Cartilage contains mixed fibrils of collagen types II, IX, and XI

The distribution of collagen XI in fibril fragments from 17-d chick embryo sternal cartilage was determined by immunoelectron microscopy using specific polyclonal antibodies. The protein was distributed throughout the fibril fragments but was antigenically masked due to the tight packing of collagen molecules and could be identified only at sites where the fibril structure was partially disrupted. Collagens II and IX were also distributed uniformly along fibrils but, in contrast to collagen XI, were accessible to the antibodies in intact fibrils. Therefore, cartilage fibrils are heterotypically assembled from collagens II, IX, and XI. This implies that collagen XI is an integral component of the cartilage fibrillar network and homogeneously distributed throughout the tissue. This was confirmed by immunofluorescence.     

Covalent cross-linking of the NC1 domain of collagen type IX to collagen type II in cartilage

From a study to understand the mechanism of covalent interaction between collagen types II and IX, we present experimental evidence for a previously unrecognized molecular site of cross-linking. The location relative to previously defined cross-linking sites predicts a specific manner of interaction and folding of collagen IX on the surface of nascent collagen II fibrils. The initial evidence came from Western blot analysis of type IX collagen extracted by pepsin from fetal human cartilage, which showed a molecular species that had properties indicating an adduct between the alpha1(II) chain and the C-terminal domain (COL1) of type IX collagen. A similar component was isolated from bovine cartilage in sufficient quantity to confirm this identity by N-terminal sequence analysis. Using an antibody that recognized the putative cross-linking sequence at the C terminus of the alpha1(IX) chain, cross-linked peptides were isolated by immunoaffinity chromatography from proteolytic digests of human cartilage collagen. They were characterized by immunochemistry, N-terminal sequence analysis, and mass spectrometry. The results establish a link between a lysine near the C terminus (in the NC1 domain) of alpha1(IX) and the known cross-linking lysine at residue 930 of the alpha1(II) triple helix. This cross-link is speculated to form early in the process of interaction between collagen IX molecules and collagen II polymers. A model of molecular folding and further cross-linking is predicted that can spatially accommodate the formation of all six known cross-linking interactions to the collagen IX molecule on a fibril surface. Of particular biological significance, this model can accommodate potential interfibrillar as well as intrafibrillar links between the collagen IX molecules themselves, so providing a mechanism whereby collagen IX could stabilize a collagen fibril network.     

Interaction of collagen molecules from the aspect of fibril formation: acid-soluble, alkali-treated, and MMP1-digested fragments of type I collagen.

Collagen type I extracted with acid or digested with pepsin forms fibrils under physiological conditions, but this ability is lost when the collagen is treated with alkaline solution or digested with matrix metalloproteinase 1 (MMP1). When acid-soluble collagen was incubated with alkali-treated collagen, the fibril formation of acid-soluble collagen was inhibited. At 37 degrees C, at which alkali-treated collagen is denatured, the lag time was prolonged but the growth rate of fibrils was not affected. At 30 degrees C, at which the triple helical conformation of alkali-treated collagen is retained, the lag time was prolonged and the growth rate reduced. Heat-denatured alkali-treated collagen and MMP1-digested fragments have no inhibitory effect on the fibril formation of acid-soluble collagen. This means that the triple helical conformation and the molecular length are important factors in the interaction of collagen molecules and that alkali-treated collagen acts as a competitive inhibitor for fibril formation of collagen. We found that alkali-treated collagen and MMP1-digested fragments form fibrils that lack the D periodic banding pattern and twisted morphology under acidic conditions at the appropriate ionic strength. We also calculated the relative strengths of hydrophobic and electrostatic interactions between collagen molecules. When the hydrophobic interaction between linear collagen molecules was considered, we found a pattern of periodic maximization of the interactive force including the D period. On the other hand, the electrostatic interaction did not show the periodic pattern, but the overall interaction score affected fibril formation.     

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.     

NC4 Domain of cartilage-specific collagen IX inhibits complement directly due to attenuation of membrane attack formation and indirectly through binding and enhancing activity of complement inhibitors C4B-binding protein and factor H

Collagen IX containing the N-terminal noncollagenous domain 4 (NC4) is unique to cartilage and a member of the family of fibril-associated collagens with both collagenous and noncollagenous domains. Collagen IX is located at the surface of fibrils formed by collagen II and a minor proportion of collagen XI, playing roles in tissue stability and integrity. The NC4 domain projects out from the fibril surface and provides sites for interaction with other matrix components such as cartilage oligomeric matrix protein, matrilins, fibromodulin, and osteoadherin. Fragmentation of collagen IX and loss of the NC4 domain are early events in cartilage degradation in joint diseases that precedes major damage of collagen II fibrils. Our results demonstrate that NC4 can function as a novel inhibitor of the complement system able to bind C4, C3, and C9 and to directly inhibit C9 polymerization and assembly of the lytic membrane attack complex. NC4 also binds the complement inhibitors C4b-binding protein and factor H and enhances their cofactor activity in degradation of activated complement components C4b and C3b. NC4 interactions with fibromodulin and osteoadherin inhibited binding to C1q and complement activation by these proteins. Taken together, our results suggest that collagen IX and its interactions with matrix components are important parts of a machinery that protects the cartilage from complement activation and chronic inflammation seen in diseases like rheumatoid arthritis.     

Dysfunctional tendon collagen fibrillogenesis in collagen VI null mice

Tendons are composed of fibroblasts and collagen fibrils. The fibrils are organized uniaxially and grouped together into fibers. Collagen VI is a non-fibrillar collagen expressed in developing and adult tendons. Human collagen VI mutations result in muscular dystrophy, joint hyperlaxity and contractures. The purpose of this study is to determine the functional roles of collagen VI in tendon matrix assembly. During tendon development, collagen VI was expressed throughout the extracellular matrix, but enriched around fibroblasts and their processes. To analyze the functional roles of collagen VI a mouse model with a targeted inactivation of Col6a1 gene was utilized. Ultrastructural analysis of Col6a1−/− versus wild type tendons demonstrated disorganized extracellular micro-domains and associated collagen fibers in the Col6a1−/− tendon. In Col6a1−/− tendons, fibril structure and diameter distribution were abnormal compared to wild type controls. The diameter distributions were shifted significantly toward the smaller diameters in Col6a1−/− tendons compared to controls. An analysis of fibril density (number/μm²) demonstrated a ~ 2.5 fold increase in the Col6a1−/− versus wild type tendons. In addition, the fibril arrangement and structure were aberrant in the peri-cellular regions of Col6a1−/− tendons with frequent very large fibrils and twisted fibrils observed restricted to this region. The biomechanical properties were analyzed in mature tendons. A significant decrease in cross-sectional area was observed. The percent relaxation, maximum load, maximum stress, stiffness and modulus were analyzed and Col6a1−/− tendons demonstrated a significant reduction in maximum load and stiffness compared to wild type tendons. An increase in matrix metalloproteinase activity was suggested in the absence of collagen VI. This suggests alterations in tenocyte expression due to disruption of cell-matrix interactions. The changes in expression may result in alterations in the peri-cellular environment. In addition, the absence of collagen VI may alter the sequestering of regulatory molecules such as leucine rich proteoglycans. These changes would result in dysfunctional regulation of tendon fibrillogenesis indirectly mediated by collagen VI.     

Axial structure of the heterotypic collagen fibrils of vitreous humour and cartilage

We have compared the axial structures of negatively stained heterotypic, type II collagen-containing fibrils with computer-generated staining patterns. Theoretical negative-staining patterns were created based upon the "bulkiness" of the individual amino acid side-chains in the primary sequence and the D-staggered arrangement of the triple-helices. The theoretical staining pattern of type II collagen was compared and cross-correlated with the experimental staining pattern of both reconstituted type II collagen fibrils, and fibrils isolated from adult and foetal cartilage and vitreous humour. The isolated fibrils differ markedly in both diameter and composition. Correlations were significantly improved when a degree of theoretical hydroxylysine glycosylation was applied, showing for the first time that this type of glycosylation influences the negative-staining pattern of collagen fibrils. Increased correlations were obtained when contributions from types V/XI and IX collagen were included in the simulation model. The N-propeptide of collagen type V/XI and the NC2 domain of type IX collagen both contribute to prominent stain-excluding peaks in the gap region. With decreasing fibril diameter, an increase of these two peaks was observed. Simulations of the fibril-derived staining patterns with theoretical patterns composed of proportions of types II, V/XI and IX collagen confirmed that the thinnest fibrils (i.e. vitreous humour collagen fibrils) have the highest minor collagen content. Comparison of the staining patterns showed that the organisation of collagen molecules within vitreous humour and cartilage fibrils is identical. The simulation model for vitreous humour, however, did not account for all stain-excluding mass observed in the staining pattern; this additional mass may be accounted for by collagen-associated macromolecules.     

D-periodic distribution of collagen type IX along cartilage fibrils

It has recently become apparent that collagen fibrils may be composed of more than one kind of macromolecule. To explore this possibility, we developed a procedure to purify fibril fragments from 17-d embryonic chicken sternal cartilage. The fibril population obtained shows, after negative staining, a uniformity in the banding pattern and diameter similar to the fibrils in situ. Pepsin digestion of this fibril preparation releases collagen types II, IX, and XI in the proportion of 8:1:1. Rotary shadowing of the fibrils reveals a d-periodic distribution of 35-40-nm long projections, each capped with a globular domain, which resemble in form and dimensions the aminoterminal globular and collagenous domains, NC4 and COL3, of type IX collagen. The monoclonal antibody (4D6) specific for an epitope close to the amino terminal of the COL3 domain of type IX collagen bound to these projections, thus confirming their identity. Type IX collagen is therefore distributed in a regular d-periodic arrangement along cartilage fibrils, with the chondroitin sulfate chain of type IX collagen in intimate contact with the fibril.     

Relationship between collagen fibril diameters and body size

Summary Suspensions of collagen fibrils obtained from derma of Elasmobranchia and Actinopterygia of different body sizes and developmental stages were examined by transmission electron microscopy. Fibril diameters were measured and classified into groups comprising a 20 nm diameter interval. Diagrams showing fibril populations of each fish were made. The measurements were averaged and their confidence intervals and standard errors determined. For each species other diagrams were plotted in which the mean diameters were correlated to the body length of each sample. The results show that: 1) a correlation exists between an increase in diameter of collagen fibrils and somatic growth until sexual maturity is reached; 2) fibril populations are subsequently spread over a wider range due to the presence in the derma of classes of newly formed and therefore thinner fibrils. The deposition of new fibrils is possibly influenced by individual factors; 3) no relationship exists between mean fibril diameter and body size; 4) no relationship exists between phylogenetic position and pattern of diameter distribution.Research supported by a grant from C.N.R. Roma (69.02087.0115.1150)     

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.     

Time-dependent increase in the stability of collagen fibrils formed in vitro. I. Effects of pH and salt concentration on the dissolution of the fibrils.

The time-dependent increase in stability, as measured in terms of the rate of dissolution, of collagen fibrils formed in vitro from pepsin-treated collagen was significantly affected only by temperature, and not by either ionic strength or pH. This is in contrast with collagen fibril formation, a process which is greatly affected by ionic strength and pH. Within the range of temperature 29-37 degrees C, lower temperature caused slower fibril formation and faster fibril stabilization. These results suggest that the intermolecular interactions involved in stabilizing collagen fibrils are entirely different from those involved in fibril formation. Based on kinetic analysis of the dissolution and stabilization of the fibrils, it is proposed that collagen molecules first form unstable fibrils which become gradually stabilized on prolonged incubation, without necessarily introducing covalent cross-links.     

In situ atomic force microscopy of partially demineralized human dentin collagen fibrils

Dentin collagen fibrils were studied in situ by atomic force microscopy (AFM). New data on size distribution and the axial repeat distance of hydrated and dehydrated collagen type I fibrils are presented. Polished dentin disks from third molars were partially demineralized with citric acid, leaving proteins and the collagen matrix. At this stage collagen fibrils were not resolved by AFM, but after exposure to NaOCl(aq) for 100-240 s, and presumably due to the removal of noncollagenous proteins, individual collagen fibrils and the fibril network of dentin connected to the mineralized substrate were revealed. High-aspect-ratio silicon tips in tapping mode were used to image the soft fibril network. Hydrated fibrils showed three distinct groups of diameters: 100, 91, and 83 nm and a narrow distribution of the axial repeat distance at 67 nm. Dehydration resulted in a broad distribution of the fibril diameters between 75 and 105 nm and a division of the axial repeat distance into three groups at 67, 62, and 57 nm. Subfibrillar features (4 nm) were observed on hydrated and dehydrated fibrils. The gap depth between the thick and thin repeating segments of the fibrils varied from 3 to 7 nm. Phase mode revealed mineral particles on the transition from the gap to the overlap zone of the fibrils. This method appears to be a powerful tool for the analysis of fibrillar collagen structures in calcified tissues and may aid in understanding the differences in collagen affected by chemical treatments or by diseases.     

Observing growth steps of collagen self-assembly by time-lapse high-resolution atomic force microscopy

Insights into molecular mechanisms of collagen assembly are important for understanding countless biological processes and at the same time a prerequisite for many biotechnological and medical applications. In this work, the self-assembly of collagen type I molecules into fibrils could be directly observed using time-lapse atomic force microscopy (AFM). The smallest isolated fibrillar structures initiating fibril growth showed a thickness of approximately 1.5 nm corresponding to that of a single collagen molecule. Fibrils assembled in vitro established an axial D-periodicity of approximately 67 nm such as typically observed for in vivo assembled collagen fibrils from tendon. At given collagen concentrations of the buffer solution the fibrils showed constant lateral and longitudinal growth rates. Single fibrils continuously grew and fused with each other until the supporting surface was completely covered by a nanoscopically well-defined collagen matrix. Their thickness of approximately 3 nm suggests that the fibrils were build from laterally assembled collagen microfibrils. Laterally the fibrils grew in steps of approximately 4 nm, indicating microfibril formation and incorporation. Thus, we suggest collagen fibrils assembling in a two-step process. In a first step, collagen molecules assemble with each other. In the second step, these molecules then rearrange into microfibrils which form the building blocks of collagen fibrils. High-resolution AFM topographs revealed substructural details of the D-band architecture of the fibrils forming the collagen matrix. These substructures correlated well with those revealed from positively stained collagen fibers imaged by transmission electron microscopy.     

Modulation of collagen fibrillogenesis by tenascin-X and type VI collagen

Tenascin-X (TNX) is an extracellular matrix glycoprotein. We previously demonstrated that TNX regulates the expression of type VI collagen. In this study, we investigated the binding of TNX to type I collagen as well as to type VI collagen and the effects of these proteins on fibrillogenesis of type I collagen. Full-length recombinant TNX, which is expressed in and purified from mammalian cell cultures, and type VI collagen purified from bovine placenta were used. Solid-phase assays revealed that TNX or type VI collagen bound to type I collagen, although TNX did not bind to type VI collagen, fibronectin, or laminin. The rate of collagen fibril formation and its quantity, measured as increased turbidity, was markedly increased by the presence of TNX, whereas type VI collagen did not increase the quantity but accelerated the rate of collagen fibril formation. Combined treatment of both had an additive effect on the rate of collagen fibril formation. Furthermore, deletion of the epidermal growth factor-like (EGF) domain or fibrinogen-like domain of TNX attenuated the initial rate of collagen fibril formation. Finally, we observed abnormally large collagen fibrils by electron microscopy in the skin from TNX-deficient (TNX-/-) mice during development. These findings demonstrate a fundamental role for TNX and type VI collagen in regulation of collagen fibrillogenesis in vivo and in vitro.     

Crystalline fibril structure of type II collagen in lamprey notochord sheath

We report here the existence of a crystalline molecular packing of type II collagen in the fibrils of the lamprey notochord sheath. This is the first finding of a crystalline structure in any collagen other than type I.The lamprey notochord sheath has a composition similar to that of cartilage, with type II collagen, a minor collagen component with 1α, 2α and 3α chains, and cartilage-like proteoglycan. The high degree of orientation of fibrils in the notochord makes it possible to use X-ray diffraction to determine collagen fibril organization in this type II-containing tissue. The low angle equatorial scattering shows the fibrils are all about 17 nm in diameter and have an average center-to-center separation of 31 nm. These results are supported by electron microscope observations. A set of broad equatorial diffraction maxima at higher angles represents the sampling of the collagen molecular transform by a limited crystalline lattice, extending over a lateral dimension close to the diameter of one fibril. This indicates that each 17 nm fibril contains a crystalline array of molecules and, although a unit cell is difficult to determine because of the broad overlapping reflections, it is clear that the quasi-hexagonal triclinic unit cell of type I collagen in rat tail tendon is not consistent with the data. The meridional diffraction pattern showed 26 orders with the characteristic 67 nm periodicity found for tendon. However, the intensities of these reflections differ markedly from those found for tendon and cannot be explained by an unmodified gap/ overlap model within each 67 nm period. Both X-ray diffraction and electron microscope data indicate a low degree of contrast along the fibril axis and are consistent with a periodic binding of a non-collagenous component in such a way as to obscure the gap region.     

Chondroitin sulfate perlecan enhances collagen fibril formation. Implications for perlecan chondrodysplasias

Inactivation of the perlecan gene leads to perinatal lethal chondrodysplasia. The similarity to the phenotypes of the Col2A1 knock-out and the disproportionate micromelia mutation suggests perlecan involvement in cartilage collagen matrix assembly. We now present a mechanism for the defect in collagen type II fibril assembly by perlecan-null chondrocytes. Cartilage perlecan is a heparin sulfate or a mixed heparan sulfate/chondroitin sulfate proteoglycan. The latter form binds collagen and accelerates fibril formation in vitro, with more defined fibril morphology and increased fibril diameters produced in the presence of perlecan. Interestingly, the enhancement of collagen fibril formation is independent on the core protein and is mimicked by chondroitin sulfate E but neither by chondroitin sulfate D nor dextran sulfate. Furthermore, perlecan chondroitin sulfate contains the 4,6-disulfated disaccharides typical for chondroitin sulfate E. Indeed, purified glycosaminoglycans from perlecan-enriched fractions of cartilage extracts contain elevated levels of 4,6-disulfated chondroitin sulfate disaccharides and enhance collagen fibril formation. The effect on collagen assembly is proportional to the content of the 4,6-disulfated disaccharide in the different cartilage extracts, with growth plate cartilage glycosaminoglycan being the most efficient enhancer. These findings demonstrate a role for perlecan chondroitin sulfate side chains in cartilage extracellular matrix assembly and provide an explanation for the perlecan-null chondrodysplasia.     

Interactions of collagen types I and II with chondroitin sulfates A-C and their effect on osteoblast adhesion

Collagen has found use as a scaffold material for tissue engineering as well as a coating material for implants. The main aim of this study was to compare the ability of the collagen types I and II to bind preparations of the chondroitin sulfate types A-C (CS A, CS B, CS C). In addition, the effect of the three CS preparations on the extent of collagen incorporated into fibrils and the morphology of collagen fibrils was investigated, as was the influence of collagen fibril coatings containing CS A-C on titanium surfaces on the adhesion of primary rat osteoblasts. Fibrils of both collagen types bound a higher mass of CS C than CS B and a greater mass of CS B than CS A per milligram of fibrils formed. Fibrils of collagen type II bound a higher mass of CS B and C than collagen I fibrils. The proportion of collagen incorporated into fibrils decreased with increasing CS A and CS C concentration but not with increasing CS B concentration. All three CS preparations caused collagen I and II fibrils to become thinner. CS A and CS B but not CS C appeared to stimulate the formation of focal adhesions by osteoblasts after incubation for 2 hours. These results could be of importance when selecting collagen type or CS type as materials for implant coatings or tissue engineering scaffolds.