Optimizing glutaraldehyde crosslinking of collagen: effects of time, temperature and concentration as measured by shrinkage temperature

Abstract

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~) was determined. Temperature (20°C or 45°C), concentration (0.1% or 1.0%) or fixation time (4 or 24 h) were found to be positively correlated with the Ts of the collagen sheets. Whereas untanned collagen exhibits a T~ of around 60°C, short-term (1 or 5 min), high-temperature (50°C) fixation with a 0.1% GA solution caused the shrinkage temperature to increase to 72°C and 85.1 °C, respectively. Fixation with 0.01% GA for 5 min at 50°C appeared equally effective as 1 min with 0.1% GA (T~--70°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 Ts towards the central regions of the collagen was observed. Soaking the samples at 20°C (1 h) or at 0°C (3 h) with subsequent short-time heating to 45°C caused an almost equal rise in T, throughout the collagen samples and is therefore recommended for preparing implantable tissues.

Surface engineering of stainless steel materials by covalent collagen immobilization to improve implant biocompatibility

“…Standard chemical methods use dialdehydes, diisocyanates, azides, and carbodiimides [23]. Glutaraldehyde is the most extensively used reagent for cross-linking collagen [24], but it is known to elicit cytotoxicity in vitro [25] and in vivo [26]. The use of a water-soluble carbodiimide like EDC in conjunction with N-Hydroxysuccinimide (NHS) follows the concept of cross-linking by activation of carboxylic groups which results in the formation of amide-type cross-links under exception of any residual reactive groups [27].…”

Preparation and Properties of Gelatin Fibers Fabricated by Dry Spinning

“…After heat treatment at 100˝C for 24 h, the tensile and knot stresses of the fibers crosslinked for 3 days were enhanced from 131 and 103 MPa to 170 and 125 MPa, respectively. These results suggest that post-treatment by heat is necessary for crosslinking in this case [25,27]. respectively.…”

“…respectively. These results suggest that post-treatment by heat is necessary for crosslinking in this case [25,27]. Figure 7 shows the FT-IR spectra of the gelatin fibers.…”

Desenvolvimento de processo automático para a reticulação progressiva de matrizes de colágeno com glutaraldeído

“…Processos alternativos para minimizar os problemas da reticulação com GA tem sido introduzidos e incluem o pré-tratamento com éteres glicidílicos e hexametilenodiisocianatos [7] , glicerol [8] , azidas [9] , ou mesmo procedimentos alternativos utilizando o próprio GA [6,10] , tais como a fixação dinâmica [6] , o uso de temperaturas elevadas [11] , ou ainda, o tratamento com concentrações progressivamente crescentes de GA [12] .…”