Modulation of the biophysical and biochemical properties of collagen by glycation for tissue engineering applications

Collagen type I is the most abundant extracellular matrix (ECM) protein that is responsible for conferring mechanical resilience to connective tissues [1]. To achieve this, both collagen ultrastructure and biochemistry present anatomical-specific variations. These variations ensure that collagen-rich connective tissues can accommodate the different environmental demands defined by the functional properties of the tissue as established by Wolff's Law [2]. Thus, the fine-tuning of collagen properties is critical for continued tissue homeostasis. Any significant disruption of these properties is often associated with pathological conditions such as fibrosis [3], rheumatoid arthritis [4], and cancer [5].

Tissue engineering has relied on collagen as a native structural protein to engineer scaffolds and, membranes with significant success over the last three decades [6]. The biocompatibility, accessible-chemical functionalization, and in vivo turnover of collagen are undeniable assets for collagen scaffolds and membranes to be developed for clinical applications [7, 8]. Yet, the most significant limitations of engineered collagen scaffolds are their poor mechanical property, poor structural stability, and rapid degradation. The fine-tuning of in vitro collagen scaffold properties is not yet on par with that of in vivo tissue properties. Chemical and physical crosslinking methods have been used to control the mechanical and biological stability of reconstituted collagen assemblies [9]. The most common chemical crosslinking reagents are glutaraldehyde (GTA), hexamethylene diisocyanate (HMDI), and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) [10, 11]. Photoreactive agents (e.g., riboflavin) and plant extracts (e.g., genipin) have also been used [12, 13]. These chemical exogenous collagen crosslinking methods are associated with cytotoxicity, calcification, and foreign body response, which usually overshadow their crosslinking potential [14]. The use of physical approaches such as dehydrothermal (DHT) and UV irradiation has also been evaluated to avoid the cytotoxicity associated with the chemical crosslinkers [15, 16]. However, physical crosslinking methods include heating, drying, and irradiation and cannot yield sufficient crosslinking degrees [17, 18]. Therefore, there is a need for crosslinking agents that are optimal in low toxicity, and that have the ability to confer mechanical advantages while not adversely affecting long-term tissue homeostasis. To preserve as much of the composition and structure of the ECM as possible and to mimic the collagen properties found in tissues, a possible solution would be to selectively re-engineer collagen native crosslinks in new scaffolds.

Following their synthesis in vivo, procollagen α-chains undergo a series of post-translational modifications resulting in the assembly of procollagen molecules. These include modifications of proline residues to hydroxyproline, modification of lysines to hydroxylysines, N- and O-linked glycosylation, trimerization, disulphide bonding, prolyl cis-trans isomerization and folding of the triple helix. As part of this process, the tropocollagen molecules' main stabilization is achieved by the protein disulphide isomerase (PDI) whose main function is to catalyze the formation and rearrangement of the disulphide bonds also known as intramolecular crosslinks. Following fibrillogenesis, collagen molecules are once more exposed to further stabilization within the fibril. This final step of the biosynthesis of collagen involves the formation of covalent crosslinks to stabilize the supramolecular assembly of collagen molecules into fibrils. Under physiological conditions, collagen fibrils undergo natural intermolecular crosslinking via the enzymatic lysyl oxidase (LOX), lysyl oxidase-like (LOXL), LOXL, LOXL3, LOXL4 and transglutaminase pathways, as well as nonenzymatic glycation. However, upon maturation and aging the amount of glycation-mediated crosslinks increases while the amount of enzymatic crosslinks does not decrease. This leads to an overall imbalance in crosslinking formation in favour of the glycation products as the proportion of glycation-mediated crosslinks to enzymatic-mediated crosslinks increases [19].

Glycation is the reaction of carbonyl groups of reducing sugars with free amino groups of lipids and proteins to form a Schiff base, which then undergoes a time-dependent rearrangement to form a fairly stable Amadori product [20]. These structures are still reactive and convert to stable substances called advanced glycation end-products (AGEs). AGEs formation results from long-time exposure of proteins to reducing sugars since glycation of collagen is a process without catalysis. The low turnover of collagen causes AGEs to accumulate within the collagen fibrils in our tissues and organs during normal aging or some pathological conditions such as Alzheimer's disease [21], and diabetes. In diabetic conditions, glycation is expected to proceed faster due to an increase in available free sugars that are available to react with collagen residues [22].

AGE-mediated crosslinks are known to alter the physical characteristics (elasticity, thermal denaturation, morphology) of collagen structures. A previous study showed that glucosepane (the most abundant and relevant AGE-mediated crosslink) is associated with an increased denaturation temperature, reduced density of collagen packing, and increased porosity to water molecules [23]. Gautieri et al. showed that AGEs reduce tissue viscoelasticity by severely limiting fiber–fiber and fibril–fibril sliding and brittle failure mode in tendons treated with Methylglyoxal (MGO) [24, 25]. Glycation not only results in a modification of the physical properties of the collagen but also modifies collagen interaction with key molecules like enzymes (e.g. collagenase) that lead to enzyme resistance [26].

Although the effect of glycation on collagen tissue properties has been investigated with ribose and glucose [27], [28], [29] as glycation agents, there is an evident lack of knowledge in the basic science literature explaining the biomechanical impact of AGE-mediated crosslinks on the functional and structural properties of collagen at both the nanoscale (single fibrils) and mesoscale (bundles of fibrils). Thus, in the current work, we investigate the effects of MGO-induced AGE-crosslinks on collagen structural ordering, stiffness, water sorption, and enzymatic degradability by combining multi-scale imaging and mechanical testing via Atomic Force Microscopy (AFM), Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR), and time-lapse digital imaging. This study identifies unique variations in the properties of collagen following in vitro tissue glycation by MGO and proposes this method of collagen crosslinking as a means to modulate the biophysical properties of collagen fibrils and scaffolds prior to cell seeding or clinical implantation.

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