Pepsin-solubilized bovine corium collagen was purified, reconstituted, and treated with various levels of glutaraldehyde. Treatment of suspensions of fibrillar collagen with low concentrations of glutaraldehyde appeared to have little effect on the gross morphology of fibrils, as judged by electron microscopy, but did have a significant impact on their physicochemical stability. Fibrillar collagen treated with glutaraldehyde at a concentration equal to or greater than 0.0075% demonstrated significant decreases in neutral solubility at elevated temperatures as compared to noncross-linked controls. Differential scanning calorimetry provided a convenient and quantitative means to correlate increases in melting temperature with increases in glutaraldehyde treatment concentration. Fibrillar collagen cross-linked with glutaraldehyde concentrations as low as 0.0075% demonstrated a significantly greater resistance to proteolytic degradation than did noncross-linked fibrillar collagen samples. The residual, extractable aldehyde content of such preparations was between 1 and 3 ppm. Rheological measurements on such cross-linked suspensions demonstrated that they were non-Newtonian, shear-thinning fluids, and that they were two- to threefold more viscous than corresponding preparations of noncross-linked collagen.
SynopsisMeasurements of translational diffusion coefficients by quasielastic laser light scattering, sedimentation coefficients, and intrinsic viscosities a t zero shear of proteoglycan subunit fraction A1-D1-D1 isolated from bovine nasal septa are reported. Molecular weights and hydrodynamic dimensions are compared with those expected on the basis of structural models previously proposed. Comparison of the concentration dependence of the diffusion coefficient in the presence of NaCl and GdnHCl leads to the conclusion that significant self-association behavior of subunit occurs in the absence of GdnHCl. In the absence of added salt, anomalous nonlinear concentration dependence of Dt estimated from wide-angle light-scattering experiments is observed. In addition, Dt apparently becomes angle dependent. These results are interpreted in terms of the perturbation of normal translational diffusion of the monomer by strong repulsive intermolecular interactions due to the combined effects of long-range electrostatic forces and macromolecular congestion a t higher concentrations. By carrying out experiments a t small scattering angles, it is possible to determine Dp for proteoglycan subunit in the absence of supporting electrolyte. Titration of a dilute solution of subunit with hyaluronic acid results in a sigmoidal behavior of the Stokes radius, indicating the formation of complexes of higher molecular weight results from the noncovalent association of proteoglycan subunits with hyaluronate. Observation of Dt appears to provide a useful method for studying the proteoglycan subunit-hyaluronate interactions.
Aqueous suspensions of glutaraldehyde cross-linked fibrillar collagen and non-cross-linked fibrillar collagen were examined by rheometry, particle size analysis, and microscopic techniques. Although cross-linked collagen suspensions were similar to non-cross-linked suspensions by microscopic and size analyses, they differed in rheometric properties. Concentric cylinder Couette flow, shear creep, uniaxial creep, and porous bed flow all revealed that cross-linked collagen was more resistant to deformation and flow than non-cross-linked collagen. These results were in agreement with in vivo dermal implantation studies, both in pig and human; i.e., compared to non-cross-linked collagen, the cross-linked formulation was more difficult to inject into tissue and did not spread uniformly, sometimes giving rise to palpable lumps or large masses evident in histological sections. When hyaluronic acid was blended with cross-linked collagen to achieve a final hyaluronate concentration of 5 mg/mL, there was a significant improvement in ease of injection into tissue. Rheometry on blends of hyaluronate and cross-linked collagen demonstrated that the blend required lower forces to achieve deformation and flow, compared to cross-linked collagen alone. Particle size analysis on the blend showed a reduction in fiber aggregate dimensions, compared to cross-linked collagen alone.
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