The stabilization of in vivo assembled collagen fibrils by proteoglycans/glycosaminoglycans

The effects of proteoglycans/glycosaminoglycans on the thermal stability of in vivo assembled collagen fibrils have been examined. The shrinkage temperature of tendon collagen was found to be linearly dependent on the concentration of chondroitin sulphate in the surrounding fluid. Enzymic pretreatment of articular cartilage, to reduce its glycosaminoglycan content, resulted in decreased stability of the collagen present. The stability of the collagen in hyaluronidase-treated cartilage was found to be higher when measured in a solution of chondroitin sulphate (30 g/dl) than in buffer alone. The results of this study demonstrate that the proteoglycans stabilize collagen fibrils in tissues such as articular cartilage.     

A role for glycosaminoglycans in the development of collagen fibrils

Extensive data on the glycosaminoglycan (GAG) composition and the collagen fibril diameter distribution have been collected for a diverse range of connective tissues. It is shown that tissues with the smallest diameter collagen fibrils (mass-average diameter less than 60 nm) have high concentrations of hyaluronic acid and that tissues with the largest diameter collagen fibrils (mass-average diameter approximately 200 nm) have high concentrations of dermatan sulphate. It is suggested that the lateral growth of fibrils beyond a diameter of about 60 nm is inhibited by the presence of an excess of hyaluronic acid but that this inhibitory effect may be removed by an increasing concentration of chondroitin sulphate and/or dermatan sulphate. It is also postulated that high concentrations of chondroitin sulphate will inhibit fibril growth beyond a mass-average diameter of approximately 150 nm. Such an inhibition may in turn be removed by an increasing concentration of dermatan sulphate such that it becomes the dominant GAG present in the tissue.     

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.     

Effects of glycosaminoglycans and proteoglycan on the in vitro assembly and thermal stability of collagen fibrils

The effects of three glycosaminoglycans (chondroitin 6-sulfate, dermatan sulfate, and hyaluronate) and a proteoglycan on the kinetics of fibril formation and on the thermal stability of the in vitro assembled collagen fibrils, under physiological conditions of ionic strength and pH, have been examined. The glycosaminoglycans were found to influence the kinetics of collagen precipitation but not the thermal stability of the in vitro assembled fibrils. The proteoglycan was found to influence the kinetics of collagen precipitation and to reduce the thermal stability of the in vitro assembled fibrils. Comparison of the interaction occurring between chondroitin 6-sulfate and collagen under acidic conditions (0.05M acetic acid) and that occurring under physiological conditions showed that markedly different interaction products were formed under the different conditions.     

The precipitation of toroidal collagen fibrils

The morphology of aggregates of calf-skin tropocollagen, precipitated by continuous injection into neutral phosphate buffers at 35 degrees , has been studied by electron microscopy. Although most of the collagen is precipitated as normal native fibrils, a small proportion forms closed toroidal structures having the usual native band-interband pattern. Theoretical considerations, based on elastic energies in a general microfibril model, predict that the toroids should have a simple super-helical structure, and this is not inconsistent with the observations. From the theoretical energies it was possible to estimate a crude lower limit of 3kcal./mole for the free energy of association of the tropocollagen macromolecules.     

The intermolecular space of reconstituted collagen fibrils

The extent, geometry and heterogeneity of the intermolecular space of hydrated, purified and reconstituted steer skin collagen fibrils has been characterized. The extent of the space has been assessed experimentally by an X-ray diffraction method and a new physical chemical technique, and found to be 1.14 ml per gram collagen. A theoretical model relating the intermolecular space to X-ray diffraction parameters has been presented, and this suggests that the geometry of the intermolecular space arises from a near-hexagonal packing of the collagen molecules. On the basis of an assumed microfibrillar packing model and a geometric construction of the shape of a collagen molecule, the distribution of the space within reconstituted collagen fibrils has been characterized as follows: 0.13 ml of the intermolecular space/g collagen can be attributed to the helical groove of the collagen molecules per se and 1.01 ml/g is interstitial; 0.66 ml/g is present in the form of “pores” (hexagonally-closed packed spaces), whereas 0.48 ml/g is present in the form of “holes” (hexagonal volume defects); 0.73 ml/g of the intermolecular space is associated with a region of the collagen fibrils where holes are localized and 0.41 ml/g is attributable to the regions of the fibril in which pores only are present.     

Bovine corneal stroma contains a structural glycoprotein located in the gap region of the collagen fibrils

Treatment of bovine corneal stroma using SDS-containing extracting solutions removes a 135,000 MW glycoprotein from the main collagen framework of the tissue. Low-angle synchrotron X-ray diffraction patterns obtained from corneas extracted in this way indicate that the glycoprotein has been removed from the gap regions of the collagen fibrils and is thus an important structural component of the corneal stroma. The glycoprotein (GP 135) shares a number of properties with one of the subunits of type VI collagen, but tests have so far failed to establish their identity.     

Complexing of fibronectin glycosaminoglycans and collagen

Collagen-fibronectin complexes, formed by binding of fibronectin to gelatin or collagen insolubilized on Sepharose, were found to bind 20–40% of radioactivity in [³⁵S]heparin. Fibronectin attached directly to Sepharose also bound [³⁵S]heparin, while gelatin-Sepharose without fibronectin did not. Unlabeled heparin and highly sulfated heparan sulfate efficiently inhibited the binding of [³⁵S]heparin, hyaluronic acid and dermatan sulfate were slightly inhibitory, while chondroitin sulfates and heparan sulfate with a low sulfate content did not inhibit.The interaction of heparin with fibronectin bound to gelatin resulted in complexes which required higher concentrations of urea to dissociate than complexes of fibronectin and gelatin alone. Heparin as well as highly sulfated heparan sulfate and hyaluronic acid brought about agglutination of plastic beads coated with gelatin when fibronectin was present. Neither fibronectin nor glycosaminoglycans alone agglutinated the beads.It is proposed that the multiple interactions of fibronectin, collagen and glycosaminoglycans revealed in these assays could play a role in the deposition of these substances as an insoluble extracellular matrix. Alterations of the quality or quantity of any one of these components could have important effects on cell surface interactions, including the lack of cell surface fibronectin in malignant cells.     

Crystalline regions in collagen fibrils

A new image processing technique, content-dependent anisotropic spatial frequency filtering, has been developed to visualize the location and orientation of crystalline regions in collagen fibril cross-sections. The results show that most crystalline regions are oriented with their approximately 4 nm periodicity directed radially from the fibril centre. This periodicity corresponds to the separation between rows of molecular ends in the quasi-hexagonal molecular packing scheme. The extent of crystallinity increases with radius and frequently the lattice is either continuously distorted or interrupted by sharp discontinuities.     

Mechanical properties of collagen fibrils

The formation of collagen fibers from staggered subfibrils still lacks a universally accepted model. Determining the mechanical properties of single collagen fibrils (diameter 50-200 nm) provides new insights into collagen structure. In this work, the reduced modulus of collagen was measured by nanoindentation using atomic force microscopy. For individual type 1 collagen fibrils from rat tail, the modulus was found to be in the range from 5 GPa to 11.5 GPa (in air and at room temperature). The hypothesis that collagen anisotropy is due to the subfibrils being aligned along the fibril axis is supported by nonuniform surface imprints performed by high load nanoindentation.     

Structure and function of bone collagen fibrils

The intermolecular volume of fully hydrated collagen fibrils from a number of mineralized and non-mineralized tissues of adult rats has been determined both by an exclusion technique and by a method which involves the monitoring of specific X-ray diffraction parameters. The intermolecular volume of either bone or dentinal fibrils is approximately twice that of either tail or achilles tendon, and the most frequent intermolecular distance in bone or dentine fibrils is approximately 3 Å larger than of the tendons.A number of fibrillar structures are most compatible with the intermolecular volume of rat tail tendon. These include hexagonal molecular packing and orthogonal arrays of microfibrils comprising seven parallel molecular strands. The intermolecular volume of bone or dentinal collagen fibrils, on the other hand, appears to arise from structures having a disordered or pseudo-hexagonal molecular packing, in which the most frequent intermolecular distance is about 19 Å.The space associated with collagen fibrils in adult bone is such that 70 to 80% of the mineral is located within the intermolecular space of the fibrils—approximately equal amounts of mineral being in spaces having lateral dimensions of 25 to 75 Å and 6 to 12 Å, respectively. Particles located in the latter kind of intermolecular space probably constitute, to a large extent, the non-crystalline mineral phase of adult bone.The stereo-chemical constraints on the transport of mineral ions into and within collagen fibrils of bone and tendon support the postulate that bone collagen is an in vivo catalyst for mineral deposition and further suggests that its catalytic activity may be partially regulated through its molecular packing.     

The crystalline structure of collagen fibrils in tendon.

New data have been collected on the crystalline structure of collagen fibrils in tendon. The unit cell in decrimped tendon has been determined by measurements of the Bragg reflections in the X-ray diffraction pattern. The results are consistent with a triclinic cell with $\text{b = 75.5}\text{A}\text{?}$, β = 93 °, $\text{a =}\text{b}\text{sin}\text{β}$, a = 90 °, $\text{c = n × 668}\text{A}\text{?}$, where n is probably 4 and γ = 90 °. A selection rule observed for prominent reflections is explicable either in terms of a specific orientation of the microfibrils on the lattice, or by a helical distortion of the microfibril axis. The cell parameter β can be varied by changing the ionic envirionment.     

Low strain nanomechanics of collagen fibrils

The high stiffness of collagenous tissues such as tendon and ligament is derived in large part from the mechanics and geometries of the constituent collagen's hierarchical forms. The primary structural unit in connective tissues is the collagen fibril for which there exists little direct mechanical or deformational study. Therefore, the current understanding of the mechanisms involved is extrapolated from whole tissue data. To address this, the elastic response due to bending of readily extractable adult collagen fibrils was studied, and the results were compared to previously reported radial indentation experiments. A demonstration of a material anisotropy arising without loss of the assumptions of homogeneity is presented.     

Elastic model for crimped collagen fibrils

A physiologic constitutive expression is presented in algorithmic format for the nonlinear elastic response of wavy collagen fibrils found in soft connective tissues. The model is based on the observation that crimped fibrils in a fascicle have a three-dimensional structure at the micron scale that we approximate as a helical spring. The symmetry of this wave form allows the force/displacement relationship derived from Castigliano's theorem to be solved in closed form: all integrals become analytic. Model predictions are in good agreement with experimental observations for mitral-valve chordae tendinece.     

Viscoelastic properties of isolated collagen fibrils

Understanding the viscoelastic behavior of collagenous tissues with complex hierarchical structures requires knowledge of the properties at each structural level. Whole tissues have been studied extensively, but less is known about the mechanical behavior at the submicron, fibrillar level. Using a microelectromechanical systems platform, in vitro coupled creep and stress relaxation tests were performed on collagen fibrils isolated from the sea cucumber dermis. Stress-strain-time data indicate that isolated fibrils exhibit viscoelastic behavior that could be fitted using the Maxwell-Weichert model. The fibrils showed an elastic modulus of 123 ± 46 MPa. The time-dependent behavior was well fit using the two-time-constant Maxwell-Weichert model with a fast time response of 7 ± 2 s and a slow time response of 102 ± 5 s. The fibrillar relaxation time was smaller than literature values for tissue-level relaxation time, suggesting that tissue relaxation is dominated by noncollagenous components (e.g., proteoglycans). Each specimen was tested three times, and the only statistically significant difference found was that the elastic modulus is larger in the first test than in the subsequent two tests, indicating that viscous properties of collagen fibrils are not sensitive to the history of previous tests.     

Liquid crystallinity in collagen solutions and magnetic orientation of collagen fibrils

Studies of the optical birefringence of solutions of acid-soluble collagen from rat-tail tendon at 22°C in the pH range 1.0–6.0 show that collagen exhibits an isotropic to mesophase transition only between pH 2.4 and 3.0 at 10% weight concentration. Such liquid crystalline order is probably essential for the orientation of collagen in a magnetic field. When solutions of neutral salt-soluble collagen were precipitated at pH 7.0 by warming to 37°C (“heat gelling”) in a magnetic field of ca. 20 kG, the resulting fibrils wee oriented perpendicular to the direction of the field. Heat gelling is shown to be a useful technique for maintaining the orientation induced in precursor solutions even after the sample is removed from the magnetic field.     

Supramolecular interactions in the dermo-epidermal junction zone: anchoring fibril-collagen VII tightly binds to banded collagen fibrils

The dermis and the epidermis of normal human skin are functionally separated by a basement membrane but, together, form a stable structural continuum. Anchoring fibrils reinforce this connection by insertion into the basement membrane and by intercalation with banded collagen fibrils of the papillary dermis. Structural abnormalities in collagen VII, the major molecular constituent of anchoring fibrils, lead to a congenital skin fragility condition, dystrophic epidermolysis bullosa, associated with skin blistering. Here, we characterized the molecular basis of the interactions between anchoring fibrils and banded collagen fibrils. Suprastructural fragments of the dermo-epidermal junction zone were generated by mechanical disruption and by separation with magnetic Immunobeads. Anchoring fibrils were tightly attached to banded collagen fibrils. In vitro binding studies demonstrated that a von Willebrand factor A-like motif in collagen VII was essential for binding of anchoring fibrils to reconstituted collagen I fibrils. Since collagen I and VII molecules reportedly undergo only weak interactions, the attachment of anchoring fibrils to collagen fibrils depends on supramolecular organization of their constituents. This complex is stabilized in situ and resists dissociation by strong denaturants.