Due to low immunogenicity and biodegradability, collagen matrices such as films, sponges and gels, are widely utilised in the medical industry for enhancing wound repair and/or drug delivery systems. Solubilised collagen, however, is limited by low mechanical strength and stability due to the degradation of natural crosslinks by chemical agents or proteolytic enzymes.
In order for the material to be applied for long term medical applications, it is subjected to crosslinking which improves the mechanical and thermal properties of the material. The material may be crosslinked using chemical agents or physical methods depending on the requirements.
The chemical method includes agents such as aldehydes, carbodiimides, diisocyanates, epoxides and acyl azides; while physical methods include UV radiation and dehydrothermal treatment. The choice of crosslinking agent is application-specific, for example whether the material would be required for short or long term use.
Potential cytotoxicity and calcification of the implant needs to be taken into account before the final decision on the choice of crosslinking agent is made. It has been found that cytotoxicity, which may be due to residual free crosslinking agents or due to enzymatic degradation products (monomers or polymers), is still not fully understood.
However, there is some reservation with respect to crosslinking collagen, especially if the matrix is to be used as a biomaterial which interacts with cells. As the material is chemically and structurally altered, adhesion and growth of cells, such as fibroblasts, on the material to aid wound healing, may be affected.
Biomaterials may be crosslinked by chemical and physical means. A few examples of those currently available for collagen are as follows:
Chemical treatment
Aldehydes: Formaldehyde and glutaraldehyde, especially the former, are slowly being phased out due to their toxicity. However, there are some industries that still utilise glutaraldehyde since it is cost-effective and the reaction rate is rapid and durable. The reaction mechanism of glutaraldehyde and collagen is complex.
The effectiveness of the crosslinking reaction is dependent on the concentration, pH and purity of the agent. A neutral pH and free unprotonated amine groups are required. Further polymerisation may occur with aldol condensation which forms covalent intermolecular bonds with crosslinking occurring in remote collagen molecules.
The aldehyde reacts with the primary amine group of lysine and hydroxylysine of the collagen molecule. Addition of lysine and heat (such as microwave treatment) enhances the crosslinking of the material. Caution needs to be applied when using this particular agent.
Calcification and cytoxicity have been known to arise in materials which use
glutaraldehyde as a stabilising agent. However, lower inflammation was found around the implant with glutaraldehyde-crosslinked biomaterials. This may be due to retarded release of the monomer or polymers from the matrix due to increased crosslinking.
Physical treatment
As chemical treatment, in some cases, causes toxicity, the use of physical methods to improve the stability of the material has been extensively researched. Treatment methods that have been used are UV and y-irradiation and dehydro-thermal crosslinking. All methods increase the stability of the material and therefore resistance to enzymatic degradation.
UV irradiation:
Crosslinking occurs using UV light at 254nm, considered to occur through free radicals and formed on the aromatic group from tyrosine and phenylalanine. As these amino acids are low in collagen, the amount of crosslinking due to this method is also limited.
Dehydrothermal crosslinking (DHT):
Optimum conditions require that the amount of water on the collagen molecule is lowered to a minimum by vacuum and heat (100°C for a few hours to a few days). It has been shown that water is responsible for the destruction of the triple helices which makes the collagen molecule more susceptible to enzymatic attack. Almost complete or complete removal of water results in the formation of amides as well as esters, linking the free amine and carboxylic groups. The search and development of alternative crosslinking agents conferring superior thermal and mechanical properties is ongoing and constantly evolving.
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