Use of genipin to crosslink lyophilized matrices made from collagen nanofibrils

Collagen has been a key material in many biological studies for some time now due to the strength and stability of the material. The crosslinked collagen studied here is intended for scaffolding applications for cell growth. The fibrils, however, must first be ball-milled, dispersed, and lyophilized to create porous matrices and strengthened through crosslinking to allow for such applications. Glutaraldehyde has in the past been the most promising crosslinking agent because of the chemical´s ability to form strong and extensive crosslinks. Although a valuable crosslinker for industrial purposes, glutaraldehyde is limited in biological applications due to concerns regarding cytotoxicity. The limitations of glutaraldehyde in biotechnical applications prompted research into the development of a more natural, less toxic chemical crosslinking agent. The subject of the current research is the potential use of genipin, a natural, biodegradable molecule with low toxicity. In the present study, different concentrations of genipin were added to dispersed ball-milled bovine type I collagen and the samples were lyophilized. Collagenase resistance, scanning electron microscope (SEM) imaging and sodium dodecyl sulfate polyacrlamide gel electrophoresis (SDS-PAGE) were conducted during the experiment to study the strength of each crosslinked sample after lyophilization. Genipin crosslinking resulted in increased resistance to collagenase degradation and higher molecular size and weight while also maintaining the porosity of the material.