**4. Improvements of stromal thickness and mechanical properties of selfassembled tissue**

The self-assembly technique has great therapeutic potential because it uses autologous cells that produce their own ECM, thus reducing allogeneic graft rejection. Although the selfassembly approach is suitable for clinical applications, the time required for tissue reconstruc‐ tion and the costs are important drawbacks hindering its wider use. Hence, many strategies to reduce tissue reconstruction time and the cost associated with cell culture were investigated. Efforts to stimulate collagen deposition and matrix reorganization are detailed in this section.

### **4.1. Mechanical stimulation**

Mechanical stimuli induce major biological modifications in the organization of the cells cytoskeleton and their ECM composition [71, 72]. For instance, the mechanical stimulation of blood flow induces the realignment of collagen fibers and strengthening of the tissue [73, 74]. At the molecular level, these changes are triggered by the activation of mechanoreceptors such as the ones containing the Arginyl-Glycyl-Aspartic acid attachment site that bind to integrins [75]. This mechanical stimulation often results in activation of extracellular signal–regulated kinase, ERK, and the c-Jun N-terminal kinase, JNK, signaling pathways that will induce cellular responses in order to adapt to new environments [75]. In some studies G-proteins seem to be also involved in the molecular signaling [76]. In response to mechanical stimuli, cells can also secrete growth factors such as transforming growth factor-beta (TGF-β) [77] that will exert paracrine or autocrine functions. Furthermore, cells can secrete and/or activate latent MMPs and other proteases [78, 79], which affect the balance between synthesis of ECM elements and their degradation. Accordingly, fibrosis was observed in mechanically overstimulated settings emphasizing that increased collagen deposition rates need to be controlled in order to remain reversible [80].

#### *4.1.1. Improved blood vessels generation*

The quality of engineered vascular tissues can be improved in a bioreactor by applying the appropriate laminar/cyclic flow. Other modification to the self-assembled blood vessel generation, such as co-seeding fibroblasts and SMCs, each at their respective half of the same sheet, before rolling around a cylindrical support [81] (**Figure 2B**) was reported. The fully autologous vascular substitutes possess high-grade mechanical strength to sustain engraftment and are readily available when needed without any immunosuppressive treatments [81–83].

#### *4.1.2. Specific culture surface can influence fiber alignment in engineered tissues*

Tissue functions can be improved using microstructured surfaces that control the interactions between cells and the ECM. With the use of a specific surface topography on an elastomeric material, it was observed that the first cell layer followed the same patterns and orientation as the material. Subsequently, this orientation influenced the second cell layer to follow a physiologically similar alignment mimicking the structure of the native tissue. Furthermore, secreted ECM followed cell orientation in every layer, resulting in very well-structured selfassembled sheets for cornea, vascular, and dermis. A micropatterned surface on which cells are seeded have the capacity to generate multiple layers, in which cells and the ECM sponta‐ neously organize in patterns consistent with the original tissue [84].
