*2.2. Tissue Engineering Strategies*

#### 2.2.1. Traditional Tissue Engineering

Tissue engineering aims at producing functional and living human tissue in vitro that can be implanted to restore and to replace damaged tissues and organs [32], or can be used in vitro as living testing platforms [68]. The classical approach involves the extraction of cells from humans (primary cells or stem cells), their expansion and seeding in a biomaterial [32], followed by a dynamic culture to promote the neo tissue growth [10,38,47,49]. After cell expansion, the skin tissue engineering process involves at least three steps. (i) Fibroblasts are seeded in a biomaterial; this cellular construct acts as a dermis surrogate where the scaffold represents the temporary extracellular matrix of the fibroblast. (ii) After a variable culture period (two weeks–one month), to allow fibroblast attachment and production of endogenous extracellular matrix components, keratinocytes are seeded on the top of the engineered dermis and kept in submerged culture. (iii) After approximately two weeks, the culture conditions are switched from submerged to air liquid interface (ALI) in order to promote the stratification of the epidermis with the formation of the stratum corneum [2,69]. Such full-thickness engineered skin can be eventually implanted, but different requirements need to be satisfied [70]. The engineered skin must be safe for the patient because any cultured cell material possesses an associated risk of contamination, and viral or bacterial infection. Moreover, in the case of allogenic or xenogeneic cells and materials, there is also an associated risk of rejection. The tissue engineered skin should be effective in providing real benefit for the patient: it should attach correctly to the wound area, it should undergo normal healing limiting or discouraging the formation of a scar, and it should restore the correct pigmentation and barrier functions. Then, it should support the vascular network ingrowth [2,24] with subsequent development of other structures useful for the normal life of the patient: innervation should be promoted in order to restore the sensing properties [71] and the growth of both hair follicles and sweat glands [72]. Finally, tissue engineered skin should be cost-effective in order to achieve a concrete clinical uptake. One of the strengths of the tissue engineering approach is the possibility to have fine control over the properties of the final tissue. Indeed, by modulating the initial properties of the scaffold used to accommodate human fibroblasts and optimizing the culture conditions (culture media composition, mechanical stimulation, hydrodynamic stimulation) it is possible to obtain engineered skin with desired properties [10,38,47–49,70]. It has been demonstrated that uniaxial and biaxial stretch can induce the alignment of the de novo synthesized collagen network [73]. Furthermore, by engineering either the stiffness or the porosity of the scaffold, it is possible to control the assembly of the de novo synthesized extracellular matrix [74]. This allows, for instance, to match the final properties of the engineered skin with the properties of the patient's skin in order to lower the structural and functional differences between the restored zone and the surrounding skin [13,21,43,75–78].

#### 2.2.2. Modular Tissue Engineering: Building a Tissue from the Bottom Up

Modular tissue engineering strategy applies the concept of tissue engineering but at a sub-millimeter scale. Cells are arranged in 3D architectures in the form of micromodules or micro tissues, or building blocks having at least one dimension ranging from 50 to 200 μm. Such micrometric tissues can be either scaffold-free or scaffold-based, and they act as building blocks for the fabrication of larger structures [78]. Scaffold-free microtissues can be obtained by organizing cells in sheets [79] (with a thickness ranging from 50 to 100 μm) or spheres [80,81] (diameters up to 200 μm). Scaffold-based microtissues are obtained by entrapping cells in micrometric scaffolds, such as porous microspheres [81], non-porous microspheres [82–85], non-spherical microparticles [78] or wires of hydrogels [84]. Sheets of human fibroblast are obtained by culturing fibroblasts in flat dishes and promoting the synthesis of the extracellular matrix. When the sheets achieve confluency, they are detached from the culture dishes and, by stacking different sheets of cells, a thicker tissue can be obtained [85]. When placed in close proximity, cell–cell contacts and extracellular matrix–extracellular matrix contacts lead to the formation of continuum structures made by fibroblasts embedded in their own extracellular matrix. One of the limitations of this technique is represented by the high cell density. This induces the formation of a dermis equivalent featuring a cell: ECM ratio higher than that found in the human dermis. The presence of high traction forces exerted by fibroblasts on the immature collagen fibers induces the formation of a highly packed ECM. Moreover, the over-expressed cell density increases the metabolic request. For these reasons, the cell-sheets are often characterized by a very low thickness and by the presence of a necrotic core. The detachment of the cell sheets from the culture plate and the subsequent stacking procedures represent other issues [86–88]. The fabrication of centimeter-sized tissues can be obtained by casting spherical microtissues in molds having any shape and dimensions [83]. Fibroblasts can be entrapped in both spherical and non-spherical hydrogels under continuous conditions using microfluidic devices [89,90]. Dermal microtissues obtained using this approach have been successfully used to build up large pieces of living dermis in vitro. In this direction, fibroblasts laden hydrogel has been used as a building block to fabricate a doll-shaped dermis equivalent [83]. In this study, the possibility of building up a centimeter-sized piece of dermis was demonstrated, but the final tissue underwent sever contracture. Since fibroblasts were imbedded in collagen hydrogel, the lack of a mature cell-synthesized extracellular matrix capable of withstanding the traction force of fibroblasts caused the shrinking of the final tissue. Finally, the presence of an exogenous collagen did not guarantee the complete replication of the native extracellular microenvironment [83]. To reduce the presence of exogenous matrices and promote the synthesis and the assembly of an endogenous dermal microenvironment, human fibroblasts have been seeded in porous gelatin microspheres kept in suspension cultures [89]. Under optimized culture conditions, fibroblasts were able to produce and assemble their extracellular matrix in the inner pores of the microspheres. The microspheres were designed in order to degrade during the extracellular matrix assembly process so the final microtissue was a sort of a sub-millimeter-sized "ball of human dermis", named Dermal-μTissue (Figure 3), composed of fibroblasts and fibroblast-assembled-collagen, elastin and hyaluronic acid [83,91,92]. Dermal-μTissues, having an average diameter of about 200 μm, have been cast in centimeter-sized molds in order to promote biological sintering. The molds containing the Dermal-μTissues have been inserted in bioreactors working under engineered fluid dynamic regimes, which have been developed to improve mass transport during the assembly of the Dermal-μTissue [91,92]. In these works, shear stress and optimized fluid velocity fields were used to guide the correct assembly of the de novo synthetized ECM [92,93]. Moreover, the final dermis equivalent was completely formed by a fibroblast assembled extracellular matrix, leading to the fabrication of skin substitutes with superior functionalities compared to the engineered skin composed of exogenous ECM. For instance, when cultured in vitro in the presence of human keratinocytes and dorsal root ganglion cells, the first spontaneous formation of follicle-like structures in vitro [72] and functional innervation [35] were observed. Finally, tissue wire technology can be used to produce living fibers treated as textile and woven fibers [84]. These modular approaches lead to several advantages, such as fine control over the

final architecture, control over the final shape, and control over the spatial organization of engineered biologic structures [79–87,92,93].

**Figure 3.** The main steps for the production of a DRT composed of fibroblast-assembled/pre-vascularized human dermis substitutes, and its morphological features before and after implantation in a nude mice model. (**A**) From left to right: production of Dermal-μTissues; their molding and assembly in a maturation chamber that is kept under dynamic culture conditions; formation of a continuum of fibroblasts embedded in their own dermal extracellular matrix; epithelization and vascularization of the endogenous human dermis. (**B**) Fabrication of large pieces of endogenous human dermis (major dimension 20 cm). (**C**) Histology of the endogenous human dermis supporting the differentiation of epidermis with the formation of spontaneous rete ridge profile. (**D**) Vascularized endogenous human dermis: cell nuclei in green and capillary network in red. (**E**) Vascularized endogenous human dermis: fibroblast-assembled collagen bundles observed under label-free multiphoton microscopy in gray; capillary network in red. (**F**) Top: fibroblast-assembled hyaluronic acid in green, cell nuclei in blue; Bottom: fibroblast-assembled elastin network in yellow, cell nuclei in blue. (**G**) Implantation of a piece of the pre-vascularized endogenous human dermis. (**H**) Connection between engineered capillary network (green) and recipient capillary network (red); fibroblast-assembled collagen in gray. Figure 3B, 3D, 3E, 3G, and 3H are from reference [34] "Mazio, C. et al. Pre-vascularized dermis model for fast and functional anastomosis with host vasculature. Biomat. 192, 159–170 (2019)". Authors obtained permision from Elsevier: License Number 4681910194044.

#### Three-Dimensional Bio-Printing

Three-dimensional bio-printing [94] techniques are used to achieve correct positioning of different cell types. The aim is the fabrication of intricate biologic architectures with high spatial resolution in a standardized manner [82,95–97]. Moreover, the obtainment of a full-thickness human skin equivalent takes at least four weeks, representing an issue in the case of production of autologous skin engineering grafts that should be implanted in a shorter time frame. The positioning of different cell strata in their final configuration could reduce the time required for the development of the full-thickness graft. Other advantages of 3D printing techniques are represented by the possibility of printing a vascular network and its insertion in its final configuration in important adnexal structures such as the hair follicle precursors [97–100]. This should represent a plus in the case of deep skin damage where neither innervation nor hair follicle development during the healing process has been observed [20]. Finally, the use of 3D printing techniques aims at reducing the batch-to-batch variability by providing a standardized and controlled process. By precisely locating different matrix materials, growth factors, and different cells in a layer-by-layer assembly, functional living skin tissues would be fabricated possessing designed and personalized structures with neither size nor shape limitations, in a high throughput, and in a highly reproducible manner. In general, the equipment used to print skin tissues consists of independently controlled cell-dispensing channels. Electromechanical valves operate the dispenser, which is positioned on a three-axis robotic stage possessing high spatial resolution (below 50 μm). The dispenser can deliver pre-hydrogel solutions containing cells. Once delivered, the hydrogel solution solidifies via chemical or physical routes. According to this strategy, fibroblasts embedded in rat tail collagen have been printed layer-by-layer, forming a dermis surrogate upon which keratinocytes have been deposited in order to form the epidermal layer [101]. In this study, it was demonstrated that a 3D dermis can be formed in an automated and controlled fashion by printing nanoliter droplets of collagen containing living cells [95,99]. A printed skin equivalent can be obtained using fibrin hydrogels as bio-ink containing cells [95,100]. It was demonstrated that a dermis equivalent as large as 100 cm2 could be obtained in less than 35 minutes. After this time, a layer of keratinocytes was printed on the top of the dermis equivalent and, after confluency was obtained (24 h), the bilayer engineered skin was implanted in immunodeficient mouse. After implantation, the dermis was able to integrate with the recipient tissue and the epidermis was able to differentiate until forming the stratum corneum. Nevertheless, the dermal–epidermal interface did not present the physiological rete ridge profile. Finally, techniques for the printing of a microvasculature network have been assessed. Endothelial cells can be inserted in fibrinogen solution and printed in a gelling bath according to a prescribed 3D architecture. Together with the vascular network, a hydrogel solution containing fibroblasts can be printed by means of an independent dispenser in order to obtain a more complex dermis formed by a connective tissue equivalent containing a 3D designed microvasculature [102].
