Fabrication and characterization methods for investigating cell-matrix interactions in environments possessing spatial orientation heterogeneity

Fibrillar collagen is a ubiquitous structural protein that plays a significant role in determining the mechanical properties of various tissues. The constituent collagen architecture can give direct insight into the respective functional role of the tissue due to the strong structure-function relationship that is exhibited. In such tissues, matrix structure can vary across local subregions contributing to mechanical heterogeneity which can be implicated in tissue function or failure. The post-myocardial infarction scar environment is an example of note where mechanically insufficient collagen can result in impaired cardiac function and possibly tissue rupture due to post-MI cellular response and matrix interactions. In order to further develop the understanding of cell-matrix interactions within heterogeneous environments, we developed a method of heterogeneous collagen gel fabrication which produces a region of randomly oriented fibers directly adjacent to an interconnected region of anisotropic alignment. To fully capture and evaluate the degree of alignment and spatial orientation heterogeneity, several image processing and automated analysis methods were employed. Our analysis revealed the successful fabrication of an interconnected spatially heterogeneous collagen gel possessing distinct regions of random or preferential alignment. Additionally, embedded cell populations were observed to recognize and reorient with their underlying and surrounding architectures through our cell-centric analysis techniques. STATEMENT OF SIGNIFICANCE: Fibrillar collagen is a structural protein that contributes to the architecture-function relationship exhibited by various tissues where mechanically insufficient collagen architecture can lead to tissue failure. One environment where this can occur is the post-myocardial infarction scar environment where too much or too little collagen accumulation coupled with spatial fiber orientation heterogeneity can lead to environments incapable of normal mechanical functionality. While there are methodologies capable of generating aligned constructs, they do so with varying degrees of control and complexity with many producing uniform construct alignment. The presented platform is simple and produces continuous constructs possessing inherent spatial orientation heterogeneity. Coupling this with image processing and automated analysis methods enables the probing of fundamental cell-matrix interactions within heterogeneous environments.