Modulation of collagen proteolysis by chemical modification of amino acid side-chains in acellularized arteries.

In this study, we have examined the effects of specific chemical modifications of amino acid side-chains on the in vitro degradation of "native" collagen obtained from acellular matrix (ACM)-processed arteries. Two monofunctional epoxides of different size and chemistry were used to modify lysine, with or without methylglyoxal modification of arginine. Biochemical, thermomechanical, tensile mechanical, and multi-enzymatic (collagenase, cathepsin B, acetyltrypsin, and trypsin) degradation analyses were used to determine the effects of modifications.Collagen solubilization by enzymes was found to depend upon the size and chemistry of epoxides used to modify lysine residues. In general, the solubilization of native ACM collagen by collagenase, cathepsin B, trypsin, and acetyltrypsin was either unaltered or decreased after modification with glycidol. In contrast, n-butylglycidylether (n-B) treatment increased solubilization by all enzymes. Subsequent arginine modification significantly reduced collagen solubilization by acetyltrypsin for glycidol-treated ACM arteries, whereas increased collagen solubilization was observed for n-B-treated ACM arteries with all enzymes. Gel chromatographic analyses of collagen fragments solubilized by trypsin revealed that both the amount and sites of cleavage were altered after lysine and arginine modification. The ability to modulate the enzymatic degradation of tissue-derived materials as demonstrated in this study may facilitate the design of novel engineering scaffolds for tissue regeneration or collagen-based drug delivery systems.

Control of pH alters the type of cross-linking produced by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) treatment of acellular matrix vascular grafts.

Carbodiimide cross-linking of bioprosthetic materials has been shown to provide tissue stabilization equivalent to that of glutaraldehyde cross-linking, but without the risk of the release of unreacted or depolymerized cytotoxic reagent after implantation. In this study, the effects of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) treatment on acellularized ovine carotid arteries were studied under two different pH conditions: (i) pH controlled at an optimal value of 5.5; and (ii) a simpler, but industrially significant, uncontrolled pH system. A multimode approach was employed involving biochemical assays, thermomechanical, tensile, and shear mechanical testing, and in vitro enzyme degradation analyses. EDC treatment decreased the hoop tangent modulus of acellular matrix (ACM) arterial grafts measured at 20 kPa of stress regardless of pH control. Extensibility of ACM arterial grafts measured at 20 kPa of stress was reduced after EDC treatment with pH control only. In contrast, shear stiffness of ACM arterial grafts increased to a greater degree under cross-linking without pH control (21 x compared to 14 x with pH control). Thermomechanical analyses revealed that EDC cross-linking with pH control also increased the collagen denaturation temperature of ACM arteries to a greater degree (a rise of 24.3 +/- 0.6 degrees C vs. 21.7 +/- 0.7 degrees C for no pH control), whereas cross-linking without pH control consumed a larger amount of lysine residues after 3 h of treatment. Most interestingly, both EDC treatments were equally effective in stabilizing ACM arteries against multiple degradative enzymes in vitro. The observed differences between EDC treatments under different pH conditions are attributed to differences in the location and types of the exogenous cross-links formed. The absence of pH control may have favored the formation of interfibrillar or intermolecular cross-links in collagen as well as involvement of other extracellular matrix components (proteoglycans and glycosaminoglycans). Furthermore, it may be emphasized that the location or type of cross-links differentially affected the mechanical behavior of treated materials without affecting the increase in resistance to enzymatic degradation.

Altered mechanical properties in aortic elastic tissue using glutaraldehyde/solvent solutions of various dielectric constant.

The extent to which elastic tissue can be crosslinked in aldehydes and the mechanism of such action is unresolved in the literature. We have used glutaraldehyde/solvent solutions of decreasing dielectric constant (phosphate buffer, methanol, 95% ethanol, n-propanol, n-butanol) to alter the mechanical properties of aortic elastic tissue obtained from autoclaved and CNBr-purified bovine aortae. Treated and untreated hoop samples were examined for stress-strain and stress relaxation behavior and for residual stress using opening angle experiments as per Fung. The extent of exogenous crosslinking was analyzed through amino acid analysis. Mechanical properties of autoclaved elastic tissue varied with dielectric constant in glutaraldehyde/solvent treatments; however, solvent treatment alone produced no effect. Extensibility decreased with decreasing dielectric constant while tensile modulus changed over a range from -2.4% (-0.86 kPa) for glutaraldehyde/buffer to +35.3% (+14.3 kPa) for glutaraldehyde/n-propanol (untreated-treated). Residual stress experiments similarly showed a systematic decrease in opening angle with decreasing dielectric constant. Differences ranged from 10.5 degrees for glutaraldehyde/buffer to 22.2 degrees for glutaraldehyde/n-butanol. Interestingly, purification of aortae with CNBr reduced the effects of glutaraldehyde/n-butanol treatment. We hypothesize that CNBr differentially degraded the elastin-associated microfibrillar proteins in aortic elastic tissue, thus producing the observed differences in mechanical behavior. The observed phenomena in this study may be attributed to the composite structure of elastic tissue: elastin and microfibrillar protein. During treatment, conformational changes in elastin facilitated by polar/nonpolar interactions occurred which then were "locked" in by glutaraldehyde crosslinking of the microfibrillar proteins. By this mechanism the increases in both stiffness and time-dependent behavior observed after treatment may be explained.

Effect of applied uniaxial stress on rate and mechanical effects of cross-linking in tissue-derived biomaterials.

Conformational changes in collagen fibrils, and indeed the triple helix, can be produced by application of mechanical stress or strain. We have demonstrated that the rate of cross-linking in glutaraldehyde and epoxide homobifunctional reagents can be modulated by uniaxial stress (strain). Two poly(glycidyl ether) epoxides were used: Denacol EX-810 (a small bifunctional reagent), and Denacol EX-512 (a large polyfunctional reagent). To prevent any possible effect from being masked by saturation of cross-linking sites, bovine pericardium was cross-linked to such an extent that the increase in collagen denaturation temperature, Td, was one-half of the maximal rise achievable with each reagent. Uniaxial tensile stress of 0, 15, 124 or 233 kPa was applied during cross-linking. Cross-linking rate (as observed by increase in Td) increased with increasing stress to a maximum at 124 kPa in glutaraldehyde at pH 7 but decreased in EX-810 at pH 7. In each case, the effect was small but statistically significant. No effect was observed with the larger EX-512. Cross-linking under increasing stress also showed systematic effects on mechanical properties: decreasing extensibility and plastic strain while increasing tensile strength. In each case, the effects of the epoxides were slightly different from those of glutaraldehyde. In preparation for the above experiments, studies of the effect of pH, temperature, and exposure time were carried out for each epoxide and (to a lesser extent) for glutaraldehyde. Again, systematic changes in mechanical properties were observed with increasing Td. Conformational changes in collagen produced by mechanical stress (strain) modulate the rate of cross-linking and the resulting mechanical properties; however, the effects are sensitive to the reagent employed.

Solvent environment modulates effects of glutaraldehyde crosslinking on tissue-derived biomaterials.

Bioprosthetic materials utilized in the construction of heart valves and vascular grafts possess limited performance and viability in vivo. This is due (in part) to the failure of these materials to mimic the mechanical properties of the host tissue they replace. If bioprosthetic materials could be engineered to meet the mechanical performance required in vivo, the functional lifetime of implants would be increased. In this study, glutaraldehyde/solvent solutions of decreasing dielectric constant (polarity) were utilized to modify the properties of crosslinked collagen in whole bovine pericardial tissue. Solvents included phosphate buffer, methanol, 95% (w/w) ethanol, n-propanol, and n-butanol. Exogenous crosslinking was verified in collagen by thermal denaturation tests and amino acid analyses. Tensile mechanical behavior of collagenous pericardial samples was found to depend upon the dielectric constant (polarity) of the glutaraldehyde/solvent solutions employed; however, treatment in the solvents alone had little, if any, effect. As the dielectric constant of the solvents decreased, three mechanical properties were systematically altered: plastic strain fell from a mean of 8.9 +/- 1.5% (buffer) to 1.6 +/- 0.4% (n-butanol); strain at fracture increased from 32.2 +/- 2.6% (buffer) to 55.6 +/- 4.6% (n-butanol); and percent stress remaining after 1000-s stress relaxation from an 80-g initial load fell from 86.3 +/- 1.1% (buffer) to 76.9 +/- 1.0% (n-butanol). Crosslinking using a glutaraldehyde/n-butanol solution produced materials with tensile mechanical behavior that was very close to that of fresh tissue; however, the flexural properties of the treated tissue were different from those of fresh tissue. This decoupling of the flexural and tensile mechanical behaviors of crosslinked bioprosthetic materials is unique to this form of treatment. The observed phenomena may be the results of conformational changes in collagen facilitated by polar/nonpolar interactions with the solvent that are "locked in" by the action of glutaraldehyde. This technique may aid in the "customized" design of mechanical properties in tissue-derived biomaterials.