Purpose

To develop a novel model composed solely of Col I and Col III with the lower and upper limits set to include the ratios of Col I and Col III at 3:1 and 9:1 in which the structural and mechanical behavior of the resident CM can be studied. Further, the progression of fibrosis due to change in ratios of Col I:Col III was tested.

Methods

Collagen gels with varying Col I:Col III ratios to represent a healthy (3:1) and diseased myocardial tissue were prepared by manually casting them in wells. Absorbance assay was performed to confirm the gelation of the gels. Rheometric analysis was performed on each of the collagen gels prepared to determine the varying stiffnesses and rheological parameters of the gels made with varying ratios of Col I:Col III. Second Harmonic Generation (SHG) was performed to observe the 3D characterization of the collagen samples. Scanning Electron microscopy was used for acquiring cross sectional images of the lyophilized collagen gels. AC16 CM (human) cell lines were cultured in the prepared gels to study cell morphology and behavior as a result of the varying collagen ratios. Cellular proliferation was studied by performing a Cell Trace Violet Assay and the applied force on each cell was measured by means of Finite Element Analysis (FEA) on CM from each sample.

Results

Second harmonic generation microscopy used to image Col I, displayed a decrease in acquired image intensity with an increase in the non-second harmonic Col III in 3:1 gels. SEM showed a fiber-rich structure in the 3:1 gels with well-distributed pores unlike the 9:1 gels or the 1:0 controls. Rheological analysis showed a decrease in substrate stiffness with an increase of Col III, in comparison with other cases. CM cultured within 3:1 gels exhibited an elongated rod-like morphology with an average end-to-end length of 86 ± 28.8 µm characteristic of healthy CM, accompanied by higher cell growth in comparison with other cases. Finite element analysis used to estimate the forces exerted on CM cultured in the 3:1 gels, showed that the forces were well dispersed, and not concentrated within the center of cells, in comparison with other cases.

Conclusion

This study model can be adopted to simulate various biomechanical environments in which cells crosstalk with the Collagen-matrix in diseased pathologies to generate insights on strategies for prevention of fibrosis.

中文翻译：

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研究 I 型和 III 型胶原蛋白不同比例对心肌细胞的生物力学影响的模型

目的

开发一个仅由 Col I 和 Col III 组成的新模型，其下限和上限设置为包括 Col I 和 Col III 的比例为 3:1 和 9:1，其中常驻 CM 的结构和机械行为可以被研究。此外，测试了由于 Col I:Col III 比率变化引起的纤维化进展。

方法

具有不同 Col I:Col III 比例的胶原凝胶代表健康 (3:1) 和患病的心肌组织，通过手动将它们浇铸在孔中来制备。进行吸光度测定以确认凝胶的凝胶化。对制备的每种胶原蛋白凝胶进行流变分析，以确定用不同比例的 Col I:Col III 制成的凝胶的不同刚度和流变参数。进行二次谐波生成 (SHG) 以观察胶原蛋白样品的 3D 表征。扫描电子显微镜用于获取冻干胶原凝胶的横截面图像。在制备的凝胶中培养 AC16 CM（人）细胞系，以研究不同胶原蛋白比例导致的细胞形态和行为。

结果

用于成像 Col I 的二次谐波生成显微镜显示，随着 3:1 凝胶中非二次谐波 Col III 的增加，获得的图像强度降低。与 9:1 凝胶或 1:0 对照不同，SEM 显示 3:1 凝胶中富含纤维的结构具有分布均匀的孔隙。流变学分析表明，与其他情况相比，基板刚度随着 Col III 的增加而降低。在 3:1 凝胶中培养的 CM 表现出细长的棒状形态，平均端到端长度为 86 ± 28.8 µm，这是健康 CM 的特征，与其他情况相比，伴随着更高的细胞生长。用于估计在 3:1 凝胶中培养的 CM 上施加的力的有限元分析表明，与其他情况相比，力分散良好，没有集中在细胞中心。

结论

该研究模型可用于模拟各种生物力学环境，在这些环境中，细胞与患病病理中的胶原蛋白基质串扰，从而产生对预防纤维化策略的见解。