Department of Pharmacy (DIFAR), University of Genoa, Viale Cembrano, 4, 16148 Genoa, Italy.
Int J Mol Sci. 2024 Mar 1;25(5):2897. doi: 10.3390/ijms25052897.
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 (), its copolymers with dimethylacrylamide (DMAA), and its terpolymer with DMAA and acrylic acid (AA). Three acryloyl amidoamine monomers () 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 , , and 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 (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.
在组织工程（TE）中，支撑结构（支架）对于细胞黏附和增殖起着至关重要的作用。细胞外基质（ECM）的蛋白质成分，如胶原蛋白、其衍生物明胶和弹性蛋白，是最有吸引力的支架材料。为了提高明胶的适度机械性能，可以采用交联的策略，就像胶原蛋白那样，胶原蛋白的交联是通过赖氨酰氧化酶（LO）的氧化作用来实现的。在这里，我们开发了一种新的交联明胶的方法，不使用常用的交联剂，而是基于明胶与通过 LO 介导的氧化含有胺基残基的适当合成共聚物之间形成亚胺键或醛醇缩合反应。特别是，我们首先合成并表征了一种氨基丁基苯乙烯单体（），其与二甲基丙烯酰胺（DMAA）的共聚物，以及与 DMAA 和丙烯酸（AA）的三元共聚物。然后制备了三种丙烯酰胺基胺单体（）及其与 DMAA 的共聚物。最后开发了一种甲代烯丙基醛（MA）/DMAA 共聚物，以研究 LO 在交联过程中的相关性。用 LO 对胺共聚物进行氧化测试，以确定最适合用于明胶交联实验的基质。用 、 、和 合成的共聚物是 LO 的良好基质，并在不同条件下与 MA/DMAA 共聚物一起用于明胶交联测试。在含胺的共聚物中，用 （CP5/DMMA-43.1）合成的共聚物（M21）的交联度最高（71%）。用两种细胞系（IMR-32 和 SH SY5Y）进行的细胞毒性实验表明，与 M21 的合成成分（CP5/DMAA）类似的（P5）对细胞活力没有显著降低，但促进了细胞增殖，从而证明了 M21 的生物相容性，并且有可能进一步研究作为组织工程的新支架。
