Christopher A. Pereira

A multi-sample denaturation temperature tester for collagenous biomaterials.

The temperature at which collagen denatures from a triple helix to a random coil structure is a useful measure of the degree of crosslinking. A new multi-sample denaturation temperature tester (DTT) has been constructed for rapid determination of the collagen denaturation temperature of natural tissues and collagenous biomaterials. To validate the system, the denaturation temperatures measured for the DTT are compared with results from differential scanning calorimetry (DSC). Data are presented for bovine pericardium in three states with denaturation temperatures ranging from 68 to 85 degrees C: fresh, or crosslinked with glutaraldehyde or the epoxide reagent Denacol EX-512 poly (glycidyl ether). Denaturation temperatures measured by DTT were not significantly different from those measured by differential scanning calorimetry (DSC); however, DSC onset systematically occurred at a slightly lower temperature than that measured by DTT. This result, seen only for fresh tissue is in agreement with earlier experiments using hydrothermal isometric tension (HIT) testing. By contrast, DTT and DSC onset were identical for the exogenously crosslinked materials. Since the measured transition temperature was independent of initial load, this variable may be chosen to yield sharper force-temperature transitions with a given sample geometry. This instrument allows accurate assessment of collagen denaturation temperatures for multiple samples in a fraction of the time required by other methods.

Crosslinking of tissue-derived biomaterials in 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)

In contrast to bifunctional reagents such as glutaraldehyde or polyfunctional reagents such as polyepoxides, carbodiimides belong to the class of zero-length crosslinkers which modify amino acid side-groups to permit crosslink formation, but do not remain as part of that linkage. The authors have compared the effects of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and glutaraldehyde (the de facto industrial standard crosslinker) on the hydrothermal, biochemical, and uniaxial mechanical properties of bovine pericardium. EDC crosslinking was optimized for maximum increase in collagen denaturation temperature using variables of pH, concentration, and ratio of EDC to N-hydroxysuccinimide (NHS): a reagent for formation of activated esters. EDC and glutaraldehyde crosslinked materials were subjected to hydrothermal denaturation tests, biochemical degradation by enzymes (collagenase, trypsin) and CNBr, amino acid analysis for unreacted lysine, and to high strain rate mechanical tests including: large deformation stress-strain studies (0.1 to 10 Hz), stress relaxation experiments (loading time 0.1 s) and small deformation forced vibration (1 and 10 Hz). A protocol for EDC crosslinking was developed which used 1.15% EDC (2:1 EDC:NHS) at pH 5.5 for 24 h. The increase in denaturation temperature for EDC (from 69.7±1.2°C to 86.0±0.3°C) was equivalent to that produced by glutaraldehyde (85.3±0.4°C). Both treatments equivalently increased resistance to collagenase and CNBr degradation; however, after denaturation, the EDC-treated tissue was slightly more resistant to collagenase, and markedly more resistant to trypsin. EDC-treated materials were more extensible and more elastic than glutaraldehyde-treated materials. Despite the differences in crosslinking mechanism, EDC and glutaraldehyde-treated materials are very similar. Subtle but intriguing differences in biochemical structure remain to be investigated.

Biaxial mechanical/structural effects of equibiaxial strain during crosslinking of bovine pericardial xenograft materials.

We have investigated the effect of biaxial constraint during glutaraldehyde crosslinking on the equibiaxial mechanical properties of bovine pericardium. Crosslinking of cruciate samples was carried out with: (i) no applied load, (ii) an initial 25 g ( approximately 30 kPa) equibiaxial load, or (iii) an initial 200 g (approximately 250 kPa) equibiaxial load. All loading during crosslinking was done under a defined initial equibiaxial load and subsequently fixed biaxial strain. Load changes during crosslinking were monitored. Mechanical testing and constraint during crosslinking were carried out in a custom-built biaxial servo-hydraulic testing system incorporating four actuators with phase-controlled waveform synthesis, high frame-rate video dimension analysis, and computer-interfaced data acquisition. The paired biaxial stress strain responses under equibiaxial loading at 1 Hz (before and after treatment) were evaluated for changes in anisotropic extensibility by calculation of an anisotropy index. Scanning electron microscopy (SEM) was performed on freeze-fractured samples to relate collagen crimp morphology to constraint during crosslinking. Fresh tissue was markedly anisotropic with the base-to-apex direction of the pericardium being less extensible and stiffer than the circumferential direction. After unconstrained crosslinking, the extensibility in the circumferential direction, the stiffness in the base-to-apex direction, and the tissues anisotropy were all reduced. Anisotropy was preserved in the tissue treated with an applied 25 g load; however, tissue treated with an applied 200 g load became extremely stiff and nearly isotropic. SEM micrographs correlated well with observed extensibility in that the collagen fibre morphology changed from very crimped (unconstrained crosslinking), to straight (200 g applied load). Biaxial stress-fixation may allow engineering of bioprosthetic materials for specific medical applications.

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.

HMDC crosslinking of bovine pericardial tissue: a potential role of the solvent environment in the design of bioprosthetic materials

The need for alternative crosslinking techniques in the processing of bioprosthetic materials is widely recognized. While glutaraldehyde remains the most commonly used crosslinking agent in biomaterial applications there is increasing concern as to its biocompatibility-principally due to its association with enhanced calcification, cytotoxicity, and undesirable changes in the mechanical properties of bioprosthetic materials. Hexamethylene diisocyanate (HMDC), like glutaraldehyde, is a bifunctional molecule which covalently bonds with amino groups of lysine residues to form covalent crosslinks. Evidence within the literature indicates HMDC-treated materials are less cytotoxic than glutaraldehyde-treated materials; however, there is limited characterization of the material properties of HMDC-treated tissue. This study uses a multi-disciplined approach to characterize the mechanical, thermal, and biochemical properties of HMDC-treated bovine pericardial tissue. Further, to facilitate stabilization of the HMDC reagent, non-aqueous solvent environments were investigated. HMDC treatment produced changes in mechanical properties, denaturation temperature, and enzymatic resistance consistent with crosslinking similar to that seen in glutaraldehyde treated tissue. The significantly lower extensibility and stiffness observed under low stresses may be attributed to the effect of the 2-propanol solvent environment during crosslinking. While the overall acceptability of HMDC as a crosslinking agent for biomaterial applications remains unclear, it appears to be an interesting alternative to glutaraldehyde with many similar features.