OPTIMIZING GLUTARALDEHYDE CROSS-LINKING OF COLLAGEN - EFFECTS OF TIME, TEMPERATURE AND CONCENTRATION AS MEASURED BY SHRINKAGE TEMPERATURE

Glutaraldehyde (GA) crosslinking (fixation) of collageneous tissues is a widely used method for the preparation of implantible tissues to be used as biomaterials. In an attempt to optimize the fixation process, experiments were carried out with two types of collagen (native collagen membrane and synthetic collagen sheet) to study the effect on crosslinking of temperature, GA concentration and fixation time. Secondly, stimulation of GA diffusion was studied and finally, a procedure of low T-presoaking followed by brief exposure to high temperatures was investigated. As a measure of the degree of crosslinking the shrinkage temperature (T(s)) was determined. Temperature (20-degrees-C or 45-degrees-C), concentration (0.1% or 1.0%) or fixation time (4 or 24 h) were found to be positively correlated with the T(s) of the collagen sheets. Whereas untanned collagen exhibits a T(s), of around 60-degrees-C, short-term (1 or 5 min), high-temperature (50-degrees-C) fixation with a 0.1% GA solution caused the shrinkage temperature to increase to 72-degrees-C and 85.1-degrees-C, respectively. Fixation with 0.01% GA for 5 min at 50-degrees-C appeared equally effective as 1 min with 0.1% GA (T(s) = 70-degrees-C). Microwave irradiation showed to be slightly more effective in enhancing the crosslinking process compared with conventional heating. Surprisingly, at any combination of temperature, concentration and fixation periods of 4 h or 24 h, an increased T(s) towards the central regions of the collagen was observed. Soaking the samples at 20-degrees-C (1 h) or at 0-degrees-C (3 h) with subsequent short-time heating to 45-degrees-C caused an almost equal rise in T(s) throughout the collagen samples and is therefore recommended for preparing implantable tissues.