Synthesis and Characterization of Amine and Aldehyde-Containing Copolymers for Enzymatic Crosslinking of Gelatine

Abstract

In tissue engineering (TE), the support structure (scaffold) plays a key role necessary for cell adhesion and proliferation. The protein constituents of the extracellular matrix (ECM), such as collagen, its derivative gelatine, and elastin, are the most attractive materials as possible scaffolds. To improve the modest mechanical properties of gelatine, a strategy consists of crosslinking it, as naturally occurs for collagen, which is stiffened by the oxidative action of lysyl oxidase (LO). Here, a novel protocol to crosslink gelatine has been developed, not using the commonly employed crosslinkers, but based on the formation of imine bonds or on aldolic condensation reactions occurring between gelatine and properly synthesized copolymers containing amine residues via LO-mediated oxidation. Particularly, we first synthesized and characterized an amino butyl styrene monomer (5), its copolymers with dimethylacrylamide (DMAA), and its terpolymer with DMAA and acrylic acid (AA). Three acryloyl amidoamine monomers (11a–c) and their copolymers with DMAA were then prepared. A methacrolein (MA)/DMAA copolymer already possessing the needed aldehyde groups was finally developed to investigate the relevance of LO in the crosslinking process. Oxidation tests of amine copolymers with LO were performed to identify the best substrates to be used in experiments of gelatine reticulation. Copolymers obtained with 5, 11b, and 11c were excellent substrates for LO and were employed with MA/DMAA copolymers in gelatine crosslinking tests in different conditions. Among the amine-containing copolymers, that obtained with 5 (CP5/DMMA-43.1) afforded a material (M21) with the highest crosslinking percentage (71%). Cytotoxicity experiments carried out on two cell lines (IMR-32 and SH SY5Y) with the analogous (P5) of the synthetic constituent of M21 (CP5/DMAA) had evidenced no significant reduction in cell viability, but proliferation promotion, thus establishing the biocompatibility of M21 and the possibility to develop it as a new scaffold for TE, upon further investigations.

Synthesis and Physicochemical Characterization of Gelatine-Based Biodegradable Aerogel-like Composites as Possible Scaffolds for Regenerative Medicine

“…When implanted, the scaffold should ideally resist for the time necessary for the growth and remodulation of the new tissue and then should degrade; it should be resorbed and replaced by the proteins produced by the same cells growing on it [7]. Figure 1, from our recent paper, schematically summarizes the main steps of TE reported here [8]. Using TE, it is possible to have available effective substitutes for the liver, kidneys, and pancreas, as well as for tissues such as blood vessels, skin, cartilage, bones, ligaments, and tendons.…”

“…In this context, collagen is naturally stabilized and strengthened by crosslinking reactions through the lysyl oxidase (LO)-assisted oxidation of the amino groups of its lysine and hydroxylysine residues to aldehyde groups. Subsequent aldolic condensation reactions or imine bond formations between the oxidized residues or between the oxidized groups and amine residues, respectively (Scheme 1, in Alfei et al, 2024), lead to a mechanically improved reticulated framework [8].…”

“…In this example and based on our previous studies on LO [62], we have recently prepared three amine-containing hydrophilic copolymers, namely CP5/DMAA, CP11b/DMAA, and CP11c/DMAA (Figure 1), whose primary amine groups mimic those of the lysine residues of collagen (Scheme 1, Alfei et al, 2024), thus resulting in substrates for LO [8]. The enzymatic oxidation of their amine groups afforded the desired aldehyde functions, which promoted the reticulation of Gel B by the processes illustrated in Scheme 2 and Scheme 3 in Alfei et al, 2024 [8].…”

“…In this example and based on our previous studies on LO [62], we have recently prepared three amine-containing hydrophilic copolymers, namely CP5/DMAA, CP11b/DMAA, and CP11c/DMAA (Figure 1), whose primary amine groups mimic those of the lysine residues of collagen (Scheme 1, Alfei et al, 2024), thus resulting in substrates for LO [8]. The enzymatic oxidation of their amine groups afforded the desired aldehyde functions, which promoted the reticulation of Gel B by the processes illustrated in Scheme 2 and Scheme 3 in Alfei et al, 2024 [8]. In addition to these amine-containing copolymers, a copolymer already containing this aldehyde functionality, synthesized by commercially available methacrolein (MA) (CPMA/DMAA, Figure 1), was also used in experiments of the crosslinking of Gel B, and also in the absence and presence of LO [8].…”

“…The enzymatic oxidation of their amine groups afforded the desired aldehyde functions, which promoted the reticulation of Gel B by the processes illustrated in Scheme 2 and Scheme 3 in Alfei et al, 2024 [8]. In addition to these amine-containing copolymers, a copolymer already containing this aldehyde functionality, synthesized by commercially available methacrolein (MA) (CPMA/DMAA, Figure 1), was also used in experiments of the crosslinking of Gel B, and also in the absence and presence of LO [8]. Here, following a slightly modified protocol, we have expanded these crosslinking experiments by reticulating Gel B in further different conditions, using the copolymers shown in Figure 1.…”