# Advances in Three Dimensional Bioprinting for Wound Healing: A Comprehensive Review
## 4 3D Bioprinting Techniques
### Laser-Based Bioprinting
#### 4.1.2 Two-Photon Polymerization (TPP)  
The TPP technique is a stereolithographic method used to create nano-sized structures without going through a layer-by-layer process (Figure 2B) [101,107]. The TPP strategy is formed by infrared (IR) laser pulses of femtosecond durations. Furthermore, with the IR light threshold intensity reaching up to 200 nm, TPP enables high-resolution 3D designs compared to other 3D techniques, with its low amount of focus, which makes it favored by  
Figure 2: Schematic presentation of different types of 3D bioprinting techniques including laser-based bioprinting (**A**), two-photon polymerization (**B**), selective laser sintering (**C**), droplet-based bioprinting (**D**), extrusion-based bioprinting (**E**), fused deposition modeling (**F**), and extrusion without melting material (**G**). This figure was created using [[https://app.biorender.com/](https://app.biorender.com/)]([https://app.biorender.com/](https://app.biorender.com/)) (accessed on 1 August 2023). researchers [109; 111]. During the polymerization process, free radicals are responsible for initiating the reaction. The simultaneous absorption of two photons allows the molecule to switch to a high-energy electronic state at TPP, which causes photon voxels to increase up to 100 nm [112; 113; 114] and makes TPP a functional system for the 3D bioprinting of nano- and micro-sized materials. The speed of the TPP printing system is proven to be faster than traditional systems, with a speed of 20 mm s\({}^{-1}\)[115]. Beside all these advantages, the dependence of resolution on the laser beam diameter results in a longer bioprinting process, influencing the scaffold's size. To date, various natural and synthetic resources have been used as bioinks during the TPP 3D bioprinting of hydrogel to solve the dimensional and bioprinting time-dependent problems of the technique [109; 116]. Another disadvantage of TPP is the utilization of toxic two-photon initiators that results from organic solvent residues in the final product. A proof-of-concept study based on improvements in the bioink ingredient's biocompatibility and biodegradability was achieved using a new type of ionic carbazole water-soluble photoinitiator, 3,6-Bis[2-(1-methyl-pyridinium)vinyl]-9-methyl-carbazole diiodide (BMVMC), and cucurbit[7]urli (CB7) as the hydrogel monomer, to solve this problem [117]. A photoresist consisting of BMVMC and CB7 was prepared with a photoinitiator ratio of 0.12 wt. %. Then, an arbitrary 3D polymer structure was fabricated in the photoresist by scanning the laser beam over a predefined path over the substrate using a femtosecond laser direct writing setup. Here, the resulting material was produced with a low laser threshold at 3.7 mW and a high resolution at 180 nm. L929 mouse fibroblast cells were incubated in the [PERSON]'s modified eagle medium (DMEM) solution containing BMVMC and CB7. Additionally, another batch of L929 cells were also cultured with printed scaffolds. Moreover, the cell viability tests show that the final samples have good biocompatibility and low cytotoxicity on L929 cells, making them a potential candidate for further applications in tissue engineering. Recently, the production of photo-crosslinked silk fibroin (SF) via two-photon polymerization methods was introduced by [PERSON] et al. [118]. Here, SF was prepared by degumming the fibers, then dissolving the fibers in lithium bromide and finally diluting the SF solution to 4% \(w/v\) concentration. Later, tris(2,2\({}^{\prime}\)-bipyridyl)dichlororuthenium(II) hexahydrate (ruthenium) and sodium persulfate were added to the SF solutions. The cross-linking of the SF-based hydrogel caused an increment in Young's modulus to range between 9.6 and 47.2 kPa. Scanning electron microscope (SEM) analysis revealed that the average pore sizes were in the range of 0.25-1.13 um\({}^{2}\). In addition, normal human dermal fibroblasts (NHDF) were added to the SF solution prior to 3D printing.