Radial constraint of plastically compressed human dermo-epidermal skin substitutes mitigates in vitro contraction and enhances structural maturity

Abstract Dermo-epidermal skin substitutes (DESS) offer a promising approach for treating full-thickness skin defects, but prolonged in vitro culture leads to significant contraction of the engineered tissue, particularly in the presence of an epidermal layer and highly contractile donor cells. This compromises graft quality and reproducibility, posing a challenge for preclinical research. To overcome this limitation, we developed a customized anti-shrinkage device (ASD) designed to physically constrain the substitute while remaining compatible with the established fabrication process of plastically compressed DESS. Skin substitutes were cultured with and without the ASD, and their contraction behavior, morphology, and cellular organization were analyzed. Our results showed that the ASD effectively minimized tissue shrinkage (3%–8%, depending on the experimental settings), preserving morphology and reducing variability compared to non-constrained substitutes, which exhibited significant contraction (23%–36%) and irregular morphology. Fibroblasts in contraction-protected substitutes maintained an elongated, spindle-shaped morphology without pathological myofibroblast differentiation, as indicated by the absence of α - smooth muscle actin expression. Furthermore, the epidermal layer in contraction-protected substitutes exhibited improved structural organization. Overall, the ASD provides a user-friendly and effective engineering solution to mitigate contraction in bioengineered skin substitutes, enhancing their stability and reproducibility for preclinical applications. This approach may contribute to improving the reliability of advanced skin grafts for future clinical use.