Non-enzymatic glycation of synthetic microtissues for three-dimensional diabetic wound healing

Abstract

BACKGROUND: Diabetes is a worldwide epidemic, and the number of those affected is only growing. Diabetes is characterized by hyperglycemia due to the body’s inability to produce or properly use insulin. Hyperglycemia contributes to diabetic complications in several ways, one of which is promoting glycation. Glycation is the non-enzymatic glucosylation of proteins, and because glycation is adventitious, the process most commonly occurs on proteins with long half-lives, such as collagen. Glycation greatly changes collagen’s mechanical and biochemical properties. Glycation leads to the production of advanced glycation end products (AGEs) that have been shown to contribute to the complications seen in diabetes in one of two ways: establishment of crosslinks between molecules in the basement membrane of the extracellular matrix, altering cellular function, or interactions between AGEs and AGE receptors on the cell surface. Diabetes greatly impairs the body’s ability to heal wounds, and it is thought that the AGEs produced by glycation greatly contribute this phenomenon. However, it is not fully understood, what direct role AGEs and glycated collagen plays in the wound healing process. Three-dimensional microtissue models have been developed for the purpose of studying wound healing, and the creation of a three-dimensional microtissue with glycated collagen allows for investigation into the specific role that glycated collagen plays on both the mechanical and biochemical properties of the wound closure and the healing process. METHODS: In order to study the effect of glycated collagen on wound healing, a protocol to make glycated collagen must first be developed. To make glycated collagen, soluble rat-tail type I collagen will be incubated with 250mM ribose at 4°C for a minimum of five days to allow the collagen to become glycated. The glycated collagen will be used to make a collagen gel, and then papain buffer will digest the gel. The extent of glycation will be determined through quantifying the digested glycated collagen gel’s autofluorescence, absorbance, and changes that can be perceived visually. Once it is confirmed that the collagen has been glycated, it will be incorporated into a microtissue model based on a previously published protocol. The microtissue will then be wounded with a micromanipulator and 16-gauge needle, and visualized via time-lapse microscopy. The rate at which the wound closes will be compared in microtissues made with glycated collagen to those made with non-glycated collagen. RESULTS: Glycation of collagen was unable to be confirmed consistently by measuring the autofluorescence of the collagen gel digests. However, the absorbance of the collagen gel digest was used to determine that the collagen was 43.16% glycated and visual changes in the collagen gels made with glycated collagen was also observed. Microtissues were able to successfully form with the glycated collagen, and were able to be used to compare wound healing in normal microtissues against those made with glycated collagen.