# 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  
There are various approaches to produce cell-laden hydrogels for skin engineering [99]. Recently, 3D printing of cell-laden hydrogels emerged as a novel fabrication technique. This method involves the printing of hydrogel with cells in a layer-by-layer manner to fabricate a complex bioscaffold [100]. The main advantage of this method in skin engineering is the ability to develop clinically relevant skin constructs that closely mimic the native skin architecture and heterogeneity. However, the success of bioprinting for skin regeneration is strictly dependent on the engineering of appropriate printable bioinks to support the function of cells and stimulate the fabrication of new ECM after printing. Hydrogel-based materials are one of the most promising bioinks for skin regeneration applications, as they have some unique properties such as their tunable rheological behavior, which is vitally important for delivering cells by printing [89; 98]. Several review papers have extensively discussed the main aspects of 3D bioprinting and its different techniques [101; 102; 103]. Here, we mention some recent studies regarding cell-laden hydrogel-based skin substitutes developed via 3D bioprinting. Although there are various hydrogels used for bioprinting, they are mainly restricted to natural polymers such as alginate, collagen, gelatin, fibrin, and hyaluronic acid [104; 105]. In a study by [PERSON] et al. [106], the authors used a 3D bioprinter to produce a human-plasma-derived bilayered skin for the treatment of burn injuries and traumatic and surgical wounds. The authors used primary human fibroblasts and keratinocytes that were obtained from skin biopsies [106]. The dermal part was formed by printing human fibroblasts embedded within a plasma-derived fibrin hydrogel. Their results showed the production of skin equivalents with structural resemblance to the human skin, confirmed by the presence of fibroblasts spread within the dermal compartment and the terminal differentiation of keratinocytes. In another study, [PERSON] et al. [107] employed the 3D bioprinting technology to integrate capillary-like endothelial networks into a dermo-epidermal skin graft including neonatal human epidermal keratinocytes (NHEKs) and neonatal human dermal fibroblasts (NHDFs), both embedded in a fibrin-collagen hydrogel matrix. In this work, human dermal microvascular endothelial cells) were mixed with thrombin and printed on top of a manually plated layer of collagen-NHDF cells containing fibrinogen. After synthesis of the fibrin hydrogel, a layer of collagen-containing NHEK cells was pipetted on top of it to create a bilayered network. In order to take into account in vivo considerations, printed structures were implanted into skin full-thickness wounds on the back of athymic nude mice to examine the healing process. Wound treating behavior was compared with control (no treatment) and Apligraf (discussed previously) groups. Wounds healed with printed substitutes needed 14-16 days to heal, contrasting with 21 days in the control group and  
Figure 4: (**a**) Schematic demonstrating metacylated gelatin (GelMA) acid methexylated hyaluronic acid (HAMA) chain integration to prepare polymer solution. Furthermore, the addition of a photoinitiator to prepare a UV-crosslinkable hydrogel containing adipose-derived stem cells (ADSCs) to produce a cell-laden hydrogel wound dressing. Gel solution before (left picture) and after (right picture) 50 s of UV exposure. And (**b**) Angiogenic characteristics of the bicomponent network (BCN) hydrogel in the chick chorioallantoic membrane (CAM) test. Micrographs (upper pictures) and semi-automatic treated images (lower pictures) of the hydrogel and hydrogel-loaded ADSCs, taken on day 14 of embryonic development [98]. 28 days in the group implanted with Apligraf [107].