Control of Tissue Strain Is Essential for Enhanced Dermal Innervation in the Three-Dimensional Skin Engineering

Engineered skin models with sensory innervation are a growing and challenging field of research aimed at applications in regenerative medicine, biosensing, and drug screening. Researchers are attempting to fabricate innervated skin tissues using collagen sponges, cell culture inserts, and microfluidic devices to partially mimic the layered structure of the skin. However, innervation of the full-thickness skin model has not yet been achieved. Here, using the anchoring culture device we previously reported, which is a powerful tool to construct a full-thickness three-dimensional (3D) skin model with balanced tissue contraction forces, we drastically improved dermal layer innervation using a composite hydrogel of collagen and Matrigel (Coll:MG). To determine the preferable hydrogel matrix for neurite extension in the 3D skin construct, DRG neural spheroids were placed at the bottom of the dermal layer composed of various hydrogel scaffold, including type I collagen from different origins (dermis or tendon) and Coll:MG composite hydrogel with different compositions. We showed that the Coll:MG (2:1) composite hydrogel significantly increased vertical neurite extension in the dermal layer, concomitant with the reduced tissue shrinkage during the culture. In contrast, in the collagen-only hydrogel, neurite extension occurred mostly in the horizontal direction, and tissues sometimes detached from the anchors due to significant shrinkage, indicating that tissue shrinkage may affect the direction of neurite extension. To exemplify this idea, 3D skin constructed in the device was partially detached from the anchors to comply with the cell-induced tissue shrinkage and reduce the strain on the tissue. The data showed that the partial allowance of in-plane tissue strain remarkably increased vertical neurite extension compared to the control cultures. Collectively, our results strongly suggest that neurite extension angles can be modulated by adjusting the tissue strain during the culture. Our findings highlight the importance of controlling tissue strain for the advancement of an innervated skin model.