Structural mechanism for alteration of collagen gel mechanics by glutaraldehyde crosslinking

Abstract

Soft collagenous tissues that are loaded in vivo undergo crosslinking during aging and wound healing. Bioprosthetic tissues implanted in vivo are also commonly crosslinked with glutaraldehyde (GA). While crosslinking changes the mechanical properties of the tissue, the nature of the mechanical changes and the underlying microstructural mechanism are poorly understood. In this study, a combined mechanical, biochemical and simulation approach was employed to identify the microstructural mechanism by which crosslinking alters mechanical properties. The model collagenous tissue used was an anisotropic cell-compacted collagen gel, and the model crosslinking agent was monomeric GA. The collagen gels were incrementally crosslinked by either increasing the GA concentration or increasing the crosslinking time. In biaxial loading experiments, increased crosslinking produced (1) decreased strain response to a small equibiaxial preload, with little change in response to subsequent loading and (2) decreased coupling between the fiber and cross-fiber direction. The mechanical trend was found to be better described by the lysine consumption data than by the shrinkage temperature. The biaxial loading of incrementally crosslinked collagen gels was simulated computationally with a previously published network model. Crosslinking was represented by increased fibril stiffness or by increased resistance to fibril rotation. Only the latter produced mechanical trends similar to that observed experimentally. Representing crosslinking as increased fibril stiffness did not reproduce the decreased coupling between the fiber and cross-fiber directions. The study concludes that the mechanical changes in crosslinked collagen gels are caused by the microstructural mechanism of increased resistance to fibril rotation.

Fig.1

(a) Gelation of collagen solution in mold. The mold consisted of a round upper polypropylene piece attached to a square lower base with vacuum grease. Removable needles were placed within the mold to leave holes in the gel after solidification. The mold setup was placed within a petri-dish. Strain markers were embedded in the gel before it solidified completely. (b) Anisotropic gel following two-days of cell-compaction. The upper piece of the mold was removed after the gel solidification stage in (a) to allow free media exchange. Two sets of needles on opposite sides were removed to present anisotropic constraints for cell compaction. The gel was incubated for 2 days, during which it was compacted in an anisotropic manner (c) Anisotropic crosslinked gel attached to biaxial loading device for testing. The compacted gels from (b) were exposed to the crosslinking solution and prepared for mechanical testing. Loops of silk thread were sutured along with polypropylene floats into the needle holes in the gel. The horizontal direction is the direction of predominant fiber orientation and is referred to the ‘fiber direction’. The vertical direction is referred to as the ‘cross-fiber’ direction.

Fig.2. Screening of crosslinking conditions

Shrinkage temperature vs. exposure time for collagen gels crosslinked at different glutaraldehyde concentrations. Shrinkage temperature is a measure of the extent of crosslinking.

Fig.3. Glutaraldehyde crosslinking and cross-fiber strain to preload

(a) Equi-biaxial loading to 15g for case of low-crosslinked and high-crosslinked gel, with strains referenced to unloaded state of gel. The conditions for low and high crosslinking are 1hr exposure to 0.006% glutaraldehyde, and 36 hours exposure to 0.06% glutaraldehyde, respectively. The high-crosslinked gel (black) shows decreased cross-fiber strains (E^P₂₂) to the low force preload of 0.2gm, compared to the low-crosslinked one (grey). (b,c) Trends in the cross-fiber preload strain with increased crosslinking. Crosslinking was increased by either increasing the exposure time at constant glutaraldehyde concentration of 0.06% w/v (b) or by increasing the glutaraldehyde concentration at constant exposure time of 1 hour (c). In both cases, cross-fiber strains to preload decreased progressively with increasing crosslinking. The trend suggests that crosslinking decreased the cross-fiber gel compliance at low forces.

Fig.4. Glutaraldehyde crosslinking and cross-fiber strain

(a) Equi-biaxial loading to 15g shown for low-crosslinked and high-crosslinked gels, with the strains referenced to preloaded state (E^p). The conditions for low and high crosslinking are 1hr exposure to 0.006% glutaraldehyde, and 36 hours exposure to 0.06% glutaraldehyde, respectively. The cross-fiber strain (E^P₂₂) at 15 gm is greater for the high crosslinked gel. (b,c) Trends in cross-fiber strain (E^P₂₂) with increased crosslinking. Crosslinking was increased by increasing exposure time at a constant glutaraldehyde concentration of 0.06% w/v (b), or by increasing glutaraldehyde concentration at a constant exposure time of 1 hour (c). While the trends suggest that the cross-fiber strain increases with crosslinking, they are not statistically significant (see text).

Fig.5. Glutaraldehyde crosslinking and strain-load coupling

(a,b) Biaxial strains (E^P₂₂ vs E^P₁₁) for the different loading protocols (legend) shown for the case of low crosslinked gel (a) and high crosslinked gel (b). The spread of the biaxial curves, which indicates the coupling between the biaxial strain and load anisotropy, is smaller for the high crosslink case. (c) The coupling was quantified as the slope of the biaxial strain vs. biaxial load anisotropy. (d,e) The coupling between the strain and load anisotropy decreased progressively with increased crosslinking.

Fig.6. Biochemical analysis of collagen gels crosslinked with glutaraldehyde concentrations of 0.006%, 0.06%, and 0.6%, for exposure times of 1 and 36 hours

(a) Lysine consumption, (b) Gel shrinkage temperature, (c) moles of glutaraldehyde per mole of lysine consumed, (d) % resistance to CNBr digestion, (e) % resistance to collagenase digestion. The lysine consumption, a measure of the number of crosslinked lysines, increased with glutaraldehyde concentration only. The shrinkage temperature, a measure of collagen crosslinks and glutaraldehyde polymerization, increased with both glutaraldehyde concentration and exposure time. The biochemical data in (c),(d), and (e) showed trends similar to the shrinkage temperature.

Fig.7. Relation between mechanical and biochemical trends

(a,b) Average strain-load coupling plotted against shrinkage temperature (a) and lysine consumption (b) for different crosslinking conditions. The average coupling generally decreases with both, and no localized deviations from the trend are visible due to the large standard deviations. (c,d) The average cross-fiber strain to preload (EP22) plotted against shrinkage temperature (c) and lysine consumption (d) for different crosslinking conditions. In the plot against shrinkage temperature (c), there is a significant deviation from the general trends between 56°C and 65°C. The relation with lysine consumption is more consistent (d).

Fig.8. Model predictions for effects of two microstructural mechanisms on cross-fiber strain

Increasing fibril stiffness (a) reduced cross-fiber strain at 15g relative to preload and slightly increased coupling. Increasing rotational stiffness (b) reduced cross-fiber strain at preload, slightly increased cross-fiber strain at 15g relative to preload, and decreased coupling. Experimental data (c) for 0.06% glutaraldehyde treatment for 1 hour (low crosslinking) and 36 hours (high crosslinking) showed the same trends as increasing rotational stiffness in the model but did not match model results for increased axial stiffness.