A system approach for identifying connective tissue degeneration in diabetic analogues (SAPIENT)

Collagen is the most abundant protein in the human body and its correct interaction with sugars is vital to human health. The understanding of collagen-sugar interactions is severely limited by the lack of adequate tools to comprehend it in a 3D environment and particularly in vivo. Glycation is the non-enzymatic reaction between glucose and proteins, lipids or nucleic acids. It is associated with ageing and diabetes mellitus (DM). In DM, the glycation is expected to influence the collagen orientation and is likely to disrupt the normal cell interactions. Different alterations of the collagen structure are expected to occur in the different tissues, at the different length scales (atomic and nanoscale). In particular, in bone tissue a non-enzymatic glycation of collagen determines an alteration of bone remodeling by inhibiting osteoblast differentiation. Indeed, the accumulation of Advanced Glycation End-products (AGEs) in bone decreases toughness and increases stiffness, therefore contributing to skeletal fragility. It has also been demonstrated that neuropathies are one of the most common complications of DM, with a prevalence of approximately 60%. Neuropathies basically involve progressive degeneration of the nerve fibers in a dying back pattern, and seem to be related to deleterious effects of glycation both on nerve cells and on the extracellular matrix structure.
To evaluate the effect of glycation on bone/nerves tissues, here we will reproduce the same conditions of the disease in bone and nerves engineered 3D tissue analogues in order to study and compare the etiology of Diabetes Mellitus at the tissue level, both on natural (murine model) and on engineered tissues. In detail, we will use db/db obese insulin resistant type 2 Diabetes mellitus (DM) mice, to investigate first ex vivo the different structure of collagen in healthy and type 2 DM mice, across bone and nerves tissues. Indeed, the animals will be sacrificed to extract the tissues of interest which will be studied first ex vivo by different characterization techniques such as scanning small, wide angle X-ray scattering (SAXS/WAXS) microscopy, mechanical analysis (static/dynamic conditions), Atomic force microscopy (AFM), Nuclear Magnetic Resonance (NMR), etc. and the results compared with the in vitro findings from tissue analogues (Fig.1). Modelling of ex vivo and in vitro data collected on type 2 DM tissues will be realized through a synergic combination of structural and morphological information at the atomic, nanoscale and microscale. The main objective of the project is the development of human engineered tissues to study the in vitro effects of Diabetes Mellitus. In vitro analogues could represent a valid alternative way to animal models. They allow to investigate the effects of glycation on tissues and to test anti-diabetes drugs in a substantially more ethical, economic and safe way. Indeed, the use of animal models in biomedical research presents several issues, in particular ethical and economic concerns.
From an ethical point of view, the use of animals for in vivo testing is limited because, according to “3Rs” guiding principles for a more ethical research, it is recognized the importance of animal welfare, and alleviation of stress and pain to the animals. Furthermore, even though several human diseases are currently induced on animal models, the driving mechanisms in the development and progression of pathologies could be different with respect to humans and the genetic variability (interspecies variations) negatively affects the reproducibility of data.

SAPIENT will aim to elucidate the mechanical, structural, chemical and biological implications of collagen-glucose interactions over the full range of (patho-)physiological glucose concentrations. To this end, we have assembled leading groups in the areas of materials science, polymer, medicine together with excellent tissue engineering facilities to generate relevant murine and human tissue analogues, and mouse models. This partnership will deliver enhanced tools for investigating collagen-sugar interactions across molecular, fibril, engineered tissue matrix to ex vivo and in vitro models.