Injectable cross-linked collagen with improved flow properties.

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.

Proteoglycans from bovine fetal epiphyseal cartilage. Sedimentation velocity and light scattering studies of the effect of link protein on proteoglycan aggregate size and stability.

Proteoglycan monomer and link proteins were isolated from bovine fetal epiphyseal cartilage and characterized. The physical characteristics of proteoglycan monomer were: s0(20) = 21.3 S, D0t,z = 4.25 x 10(-8)cm2/sec, Mw = 3 x 10(6) and Rg,z = 980A. Link protein preparations contained link proteins 1 and 2, but little or none of the fragment, link protein 3. Link protein-stabilized and link protein-free proteoglycan aggregates were reassembled from proteoglycan monomer, link protein and hyaluronate. The effect of epiphyseal cartilage link protein on aggregate size and stability was examined in sedimentation velocity studies. Compared with link protein from mature bovine nasal and articular cartilages, which contain appreciable amounts of link protein 3, epiphyseal cartilage link protein dramatically stabilized aggregates at pH 5. In the presence of link protein, 92% of the proteoglycan monomers were bound as aggregate at pH 7, and 81% were bound at pH 5. In the absence of link protein, 51% of monomers were bound at pH 7, and only 32% were bound at pH 5. The progressive dissociation of link protein-free aggregates as a function of decreasing pH, and of increasing temperature, was also examined in dynamic light scattering studies. The results of the light scattering studies were in perfect accord with the results of the sedimentation velocity studies. However, compared with the sedimentation velocity studies, the dynamic light scattering studies provided a more detailed and informative description of the dissociation of the link-free aggregate as a function of pH, as a function of temperature, and of the capacity of link protein to stabilize aggregate against dissociation at decreased pH or elevated temperature.

Thermal and solvent stability of proteoglycan aggregates by quasielastic laser light-scattering.

The dissociation behavior of several species of proteoglycan aggregates (PGA) has been studied quantitatively by monitoring the changes in particle size by dynamic laser light-scattering. Firstly, studies of the thermal dissociation of reconstituted PGA from bovine nasal septum and bovine fetal epiphysus are described. The effect of link protein in stabilizing reconstituted PGA against changes in temperature has been demonstrated. It was also confirmed that, upon prolonged heating at 70 degrees, the dissociation of link-containing, reconstituted PGA is effectively irreversible. Secondly, the dissociation characteristics of native PGA isolated from chick chondrocyte and rat chondrosarcoma cell-cultures were investigated in aqueous solvents containing increasing concentrations of guanidine hydrochloride. It was found that the more densely packed, native, aggregate structure has a higher susceptibility to thermal disruption than the reconstituted, link-containing material. Considerable loss of subunits from the native aggregates occurs at temperature where the link protein retains its activity. The dissociation of native PGA is irreversible, in the sense that reconstituted PGA always exhibits smaller sizes than the native PGA, reflecting a smaller degree of aggregation of the subunits.

The preparation and physicochemical characterization of an injectable form of reconstituted, glutaraldehyde cross-linked, bovine corium collagen.

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.