Stiffness distribution patterns in inner and outer tissue of the skin reversed during mouse growths

A research group led by Graduate Student Itsuki Shioka, Graduate School of Frontier Science Initiative and Associate Professor Satoru Okuda of the Nano Life Science Institute at Kanazawa University has announced that they successfully developed a novel atomic force microscopy (AFM) technique to measure the spatial distribution of stiffness within living tissues and revealed dynamic changes in the stiffness distribution within the skin during growth. Collagen is a major component of stiffness. Measurements of the stiffness distribution within the mouse skin showed that the inner tissue was softer than the outer tissue before or immediately after birth, whereas this relationship was reversed in adult skin. The findings are expected to contribute to the fields of regenerative medicine and tissue engineering. The results were published in the international journal Acta Biomaterialia on October 7.

Tissue stiffness is determined by cell behaviors, including type and density of cells and the molecules produced by them. Conversely, changes in stiffness have been reported to play an important role in controlling cell behaviors. For example, tissue stiffness changes noticeably during development and cancer progression processes. Meanwhile, while outlines of stiffness differences and changes in different tissues have been clarified, it remained impossible to relate stiffness dynamics to cell behaviors due to the lack of a technique to measure the detailed stiffness distributions within living tissues.

In this study, the group developed "ex vivo structured illumination microscopy (SIM)-AFM," a measurement technique to relate stiffness distributions within living tissues to molecular distributions. The ex vivo SIM-AFM technique combines an ex vivo culture technique, which allows for the maintenance of living tissue slices, an upright SIM, which can acquire molecular distributions at high resolution, and AFM, which can acquire detailed stiffness distributions by scanning the sample surface with a probe. By virtue of the combined features, the ex vivo SIM-AFM technique can provide stiffness distributions aligned with clear molecular distributions in living tissue slices under culture conditions. This measurement technique was then applied to mouse skin tissue.

The results showed that the mouse skin was composed of multiple tissue structures, including the outer epidermis, inner dermis, and hair follicles, and had a highly heterogeneous stiffness distribution corresponding to these structures. The dermis was softer than the epidermis in mice before and immediately after birth, whereas the relationship was reversed in adult mice. They further investigated what molecules were involved in the stiffness.

In the experiment, an enzyme that degrades collagen molecules, which are considered important for skin elasticity, was added to the mouse skin tissue to observe changes in stiffness distributions. One hour after the enzyme was added, the skin tissue was softer than before the addition. This result indicates that collagen is the main component responsible for skin stiffness. Collagen contents showed a correlation with stiffness.

As collagen increases rapidly in the dermis in mice after birth, the increase in skin stiffness before and after birth was considered to be attributable to the amount of collagen molecules. The stiffness distributions of the molecular origin may be a cause of differences in skin texture. The developed measurement technique can be applied not only to skin but also to various organs, embryonic tissues, and tissue slices for histopathology.

Okuda said, "It was not easy to obtain the stiffness values inside the living tissue, and we needed to exercise our ingenuity to maintain the tissue condition during the measurement. Moving forward, I would like to apply the technique to the spatiotemporal dynamics of stiffness in various organs and clarify its role and function."

Journal Information
Publication: Acta Biomaterialia
Title: Ex vivo SIM-AFM measurements reveal the spatial correlation of stiffness and molecular distributions in 3D living tissue
DOI: 10.1016/j.actbio.2024.09.023