Characterization of murine excisional wounds based on atomic force microscopy indentation

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Murine excisional skin wounds represent a widely applied model to investigate factors influencing the healing process. Among those, mechanical factors are receiving increasing attention, for instance concerning the role of fibroblasts' activation in contracting the wound and forming a fibrotic scar. Atomic Force Microscopy (AFM) represents a useful tool for the mechanical characterization of biological tissues at the micrometer length scale. We recently used AFM indentation to characterize healthy murine dermis for animals of different ages. In this study, we performed AFM-based indentation on different regions of a wound and the adjacent unwounded skin at two time points of the healing process, i.e. during new tissue formation (day 7 after wounding) and early remodeling (day 14). Data analysis of the earlier time point indicates that the hyperproliferative epithelium is much stiffer than the underlying regions of the granulation tissue and the latter are softer than adjacent skin. These differences are reduced at the later time point. Different stiffness measures are extracted from the data and compared in their capability of discriminating between tissue regions. A finite element analysis of the indentation experiments implementing a biphasic constitutive model was performed to investigate the influence of constitutive model parameters and surface roughness. Compared to the conventional readout of AFM measurements, which assumes that tissues behave as incompressible linear elastic materials, the shear stiffness can be up to 50 % higher. Simulation of the local topography, quantified using AFM contact mode imaging, showed that local stiffness may be underestimated by up to 50 % due to surface roughness. The present data and protocol can be used in future studies for a quantitative investigation of mechanobiological factors influencing physiology and pathology of wound healing. Statement of significance The healing of tissue injuries, in particular skin wounds, places a substantial burden on the global health care system. Although it is widely accepted that the local mechanical environment of the extracellular matrix represents an important aspect of the tissue repair process, its quantitative characterization during the course of healing is largely incomplete. In this study, we use AFM-based indentation to map the mechanical properties of structural compartments in a healing skin wound at two timepoints. Our results pinpoint key differences between relevant compartments, resolving previously reported contradictions on the deformability of wounds, and providing important insights for mechanobiological studies. Furthermore, we rationalize the influence of different parameters, including the surface topography, using a bi-phasic computational model. The results have general implications for the interpretation of force-indentation data widely used to characterize biological materials.

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