Simultaneous Mechanical Loading and Confocal Reflection Microscopy for Three-Dimensional Microbiomechanical Analysis of Biomaterials and Tissue Constructs

At present, mechanisms by which specific structural and mechanical properties of the three-dimensional extracellular matrix microenvironment influence cell behavior are not known. Lack of such knowledge precludes formulation of engineered scaffolds or tissue constructs that would deliver specific growth-inductive signals required for improved tissue restoration. This article describes a new mechanical loading–imaging technique that allows investigations of structural–mechanical properties of biomaterials as well as the structural–mechanical basis of cell–scaffold interactions at a microscopic level and in three dimensions. The technique is based upon the integration of a modified, miniature mechanical loading instrument with a confocal microscope. Confocal microscopy is conducted in a reflection and/or fluorescence mode for selective visualization of load-induced changes to the scaffold and any resident cells, while maintaining each specimen in a “live,” fully hydrated state. This innovative technique offers several advantages over current biomechanics methodologies, including simultaneous visualization of scaffold and/or cell microstructure in three dimensions during mechanical loading; quantification of macroscopic mechanical parameters including true stress and strain; and the ability to perform multiple analyses on the same specimen. This technique was used to determine the structural–mechanical properties of three very different biological materials: a reconstituted collagen matrix, a tissue-derived biomaterial, and a tissue construct representing cells and matrix.