# Advances in Three Dimensional Bioprinting for Wound Healing: A Comprehensive Review
## 4 3D Bioprinting Techniques
### Droplet-Based Bioprinting  
Droplet-based bioprinting is mainly based on inkjet printer technology. This technology allows the accumulation of materials by passing them through a special nozzle (Figure 2D). The precise deposition of droplets flowing from the nozzle enables the high resolution of the scaffold structures with a chemical or thermal curing process. However, due to factors such as viscosity [132], surface tension, and evaporation rate in the ink structure, this printing technology possesses great difficulties. Droplet-based bioprinting technology is divided into continuous inkjet and on-demand inkjet bioprinting. Continuous inkjet bioprinting is based on the strategy of dripping from a nozzle along with the pressure caused by bioprinting. Conductive bioink tends to be in the desired position under the influence of force (Figure 2). Optional inkjet bioprinting requires thermal, piezoelectric, and electrostatic effects for pressure pulses. Droplet-based bioink technology is ideal for complex structures due to the high accuracy (100 \(\upmu\)m) of different bioinks and the ability to bioprint at a resolution of 50-500 \(\upmu\)m [157]. However, low-density bioink can be preferred for ink flow continuity and cell viability [158]. The rapid fabrication of complex structures with high accuracy is essential in many biomedical engineering applications. For this purpose, the preparation of human cell-derived artificial skin (hSKE) was introduced utilizing a unique technique based on droplet-based bioprinting technology [159]. The presented hSKE does not only imitate the dermis and epidermis of normal skin morphologically, but also mimics the keratinization process in the epidermis. Furthermore, the findings demonstrate that hSKE can enhance skin regeneration via wound grafting. Another line of research has shown the effectiveness of droplet-based bioprinting techniques for manufacturing hydrogel-fiber composites [160]. The droplet deposition of structured hydrogels onto fibers and the homogenous distribution of high cell densities are used to achieve this approach. A solution consisting of collagen-ALG-fibrin was prepared, and to achieve a cross-linking bioink, a thrombin solution was produced with a DMEM solution involving calcium chloride. In order to obtain the desired cell printing density, pelletized cells were redissolved in a cross-linking bioink. Differences between composites generated with different fibroblast densities (5 \(\times\) 10\({}^{6}\) and 30 \(\times\) 10\({}^{6}\) cells/mL) were also investigated. While 5 M cells/mL composite tissue precursors enhanced cell proliferation, 30 M cells/mL composites promoted quicker cellular migration, better fiber organization, and higher ECM synthesis. Despite the fiber-based substrate's porosity and surface roughness, the hydrogel component created an ECM-like environment that supported intimate cell-cell interactions. The needle-punched mesh served as an appropriate substrate for cells to move through the hydrogel and deposit ECM. Additionally, this method avoided filament dragging and weak adhesion problems on the initial layer of the printing surface. As a result, it is claimed that fibroblast infiltration and the creation of layered structures may imitate the skin layers of cell-hydrogel-microfiber composites. In addition to the bioink composed of synthetic materials, living cells can also be printed with this technique. For example, the encapsulation of human stem cell spheroids into ALG hydrogels was achievable using droplet-based bioprinters [161]. In this study, cryopreserved human adipose-derived mesenchymal stem cells were thawed and cultured in a flask. Later, cells were plated into hydrogels and formed into spheroids. The average droplet diameter was in the range of 330-390 \(\upmu\)m, while increasing the flow rate resulted in decreased droplet size. The encapsulated stem cell spheroids had an average diameter of 335 \(\pm\) 1 to 733 \(\pm\) 15 \(\upmu\)m, with a lower concentration of surfactant resulting in larger diameters.