# Bioengineered Skin Substitutes: Advances and Future Trends
## 3 Cell-Based Skin Substitutes
### Bioengineered Dermo-Epidermal Skin Substitutes (Under Development)
#### 3.2.2 3D Bioprinting of Cell-Laden Hydrogels for Wound Dressings
Moreover, histological characterization demonstrated the formation of dermal and epidermal skin layers comparable to the native skin, which is accompanied by the presence of new microvessels in the mouse tissue. The authors concluded that the neoangiogenesis was triggered mainly by the presence of endothelial cells that were seeded in the skin graft. In addition, human keratinocytes which are known to secrete various angiogenic growth factors stimulated vascularization [107]. Further, [PERSON] et al. [108] developed a handheld skin printer that allowed in situ formation of skin tissue sheets of different homogeneous and architected compositions (Figure 5). They demonstrated that this system is compatible with dermal and epidermal cells incorporated with ionic crosslinkable alginate, enzymatically crosslinkable proteins (e.g., fibrin), and their mixtures with collagen type I and hyaluronic acid. Additionally, in a study by [PERSON] et al. [109], a cell-laden alginate/gelatin temperature-dependent hydrogel was used as a bioink for an extrusion-based 3D bioprinting using amniotic epithelial cells and Wharton's-jelly-derived mesenchymal stem cells. In this study, the hydrogel bioink was engineered by changing the alginate/gelatin concentration ratio to achieve optimum rheological properties for 3D printing. There are other recent research examples on developing cell-laden hydrogel bioinks to print skin layers or substitutes, focusing on natural hydrogels [110; 111; 112; 113; 114; 115; 116]. However, it is vitally important to design specific physical, mechanical, and biological properties of hydrogel bioinks by modification of the gel composition, concentration, and ratios to provide promising biomaterials for future skin tissue engineering applications. Generally, an appropriate hydrogel bioink should be cell compatible and able to incorporate/encapsulate cells before and after crosslinking. Moreover, hydrogels should have an optimum viscosity and shear-thinning behavior to maintain the steady state of the gel and protect cells during printing, especially for extrusion-based 3D printers. Moreover, the bioink hydrogel should mimic the physical and mechanical features of native skin after printing to produce appropriate cues for cells to differentiate and proliferate. Figure 5: Handheld skin printer. (**a**) Schematic diagram demonstrating the working principle of handheld bioprinter. One or several bioink solutions (green color), containing premixed biomaterials and cells are arranged. The handheld bioprinter transforms bioinks into homogenous or architected biomaterial sheets or tissues directly within a culture dish or a wound site. (**b**) Rendered image of handheld bioprinter. A handle (1) enables positioning above the target surface or wound. A stepper motor, pulley, and drive mechanism (2) define the deposition speed, V. Two on-board syringe pump modules (3) control the dispensing flow rates for bioink (4) and crosslinker solution (5). 3D printed microfluidic cartridge (6) for the spatial organization of solutions and sheet formation. (**c**) Photograph of 3D printed microfluidic cartridge. Scale bar 10 mm. (**d**) Schematic side-view image demonstrating sheet formation between the moving microfluidic cartridge and the deposition surface or wound. (**e**) Representative photograph showing in situ deposition of \(\delta=250\) μm thick fibrin-hyaluronic acid/collagen sheet on top of a full-thickness excisional porcine wound using a handheld skin printer. (top) Close-up view of sheet formation within wound bed with a 2 cm microfluidic cartridge (bottom) (**f**) (Control, not printed) on day 0 and printed 5 min after in situ formation of biomaterial sheet [108].