Control of crosslinking for tailoring collagen-based scaffolds stability and mechanics.
Control of crosslinking for tailoring collagen-based scaffolds stability and mechanics.
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DOI：
10.1016/j.actbio.2015.07.034
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发表时间：
2015-10
影响因子：
9.7
通讯作者：
Cameron RE
中科院分区：
文献类型：
--
作者：
Davidenko N;Schuster CF;Bax DV;Raynal N;Farndale RW;Best SM;Cameron RE
我们提供的证据表明，在组织工程中用于交联胶原基支架的标准反应物浓度比使用中的机械完整性和在水性环境中抗降解的稳定性所需的浓度高100倍。我们通过比较由（a）来自两个不同供应商的胶原蛋白，（B）明胶（部分变性胶原蛋白）和（c）50%胶原蛋白-50%明胶混合物制成的支架进行了详细和系统的研究来证明这一点。使用冻干法对材料进行处理，以产生具有各向同性结构和孔径范围为130至260 μm的均匀、高度多孔的支架。使用碳二亚胺处理交联支架，以确定交联条件的变化（低至非常低的浓度）对支架的形态、溶胀、降解和机械性能的影响。将11.5 mg/ml的碳二亚胺浓度定义为标准品（100%），并逐渐稀释至0.1%。发现碳二亚胺含量降低10倍导致游离胺基（主要在胶原蛋白赖氨酸残基上）的量显著增加（几乎4倍），而不损害所有所得支架的力学和水中稳定性。这一发现的重要性在于，通过减少交联，对整合素介导的结合至关重要的相应细胞反应性羧酸根阴离子（胶原谷氨酸或天冬氨酸残基）保持完整。事实上，碳二亚胺交联的10倍减少导致接近天然样细胞附着于胶原支架。我们已经证明，控制交联程度，从而保留天然支架化学，提供了一个重要的一步，在胶原蛋白和明胶为基础的组织工程支架的生物性能。本工作开发了具有结构、材料和生物学特性的胶原和明胶基支架，适用于心肌组织再生。本研究的新奇和意义在于阐明了胶原的组成、来源和交联浓度对支架物理和细胞结合特性的影响。我们证明，用于交联胶原支架的标准碳二亚胺浓度比使用中机械完整性和溶解稳定性所需的浓度高出100倍。这一发现的重要性在于，通过减少交联，相应的细胞反应性羧酸根阴离子（对于整合素介导的结合至关重要）保持完整，并且保留了天然支架化学。这为组织工程支架的生物学性能提供了重要的一步。
We provide evidence to show that the standard reactant concentrations used in tissue engineering to cross-link collagen-based scaffolds are up to 100 times higher than required for mechanical integrity in service, and stability against degradation in an aqueous environment. We demonstrate this with a detailed and systematic study by comparing scaffolds made from (a) collagen from two different suppliers, (b) gelatin (a partially denatured collagen) and (c) 50% collagen–50% gelatin mixtures. The materials were processed, using lyophilisation, to produce homogeneous, highly porous scaffolds with isotropic architectures and pore diameters ranging from 130 to 260 μm. Scaffolds were cross-linked using a carbodiimide treatment, to establish the effect of the variations in crosslinking conditions (down to very low concentrations) on the morphology, swelling, degradation and mechanical properties of the scaffolds. Carbodiimide concentration of 11.5 mg/ml was defined as the standard (100%) and was progressively diluted down to 0.1%. It was found that 10-fold reduction in the carbodiimide content led to the significant increase (almost 4-fold) in the amount of free amine groups (primarily on collagen lysine residues) without compromising mechanics and stability in water of all resultant scaffolds. The importance of this finding is that, by reducing cross-linking, the corresponding cell-reactive carboxylate anions (collagen glutamate or aspartate residues) that are essential for integrin-mediated binding remain intact. Indeed, a 10-fold reduction in carbodiimide crosslinking resulted in near native-like cell attachment to collagen scaffolds. We have demonstrated that controlling the degree of cross-linking, and hence retaining native scaffold chemistry, offers a major step forward in the biological performance of collagen- and gelatin-based tissue engineering scaffolds. This work developed collagen and gelatine-based scaffolds with structural, material and biological properties suitable for use in myocardial tissue regeneration. The novelty and significance of this research consist in elucidating the effect of the composition, origin of collagen and crosslinking concentration on the scaffold physical and cell-binding characteristics. We demonstrate that the standard carbodiimide concentrations used to crosslink collagenous scaffolds are up to 100 times higher than required for mechanical integrity in service, and stability against dissolution. The importance of this finding is that, by reducing crosslinking, the corresponding cell-reactive carboxylate anions (essential for integrin-mediated binding) remain intact and the native scaffold chemistry is retained. This offers a major step forward in the biological performance of tissue engineered scaffolds.