5.1.1. Bioinks
Naturally derived biomaterials like collagen, gelatin, chitosan, etc., are generally used in TE applications since they are biocompatible/biodegradable and stimulate cell interactions. On the other hand, uncontrolled degradation, low mechanical strength and possible immunogenicity limit their broad application. To address these challenges, chemical modifications of these polymers have been performed in various cases
[55][100]. Typically, an ideal bioink should contain cell anchoring ligands and exhibit appropriate stability and elasticity for the in vitro maturation period
[45][136]. Among the abundant biomaterials, sodium alginate (SA) and gelatin (Gel) have received broad attention for the development of bioinks
[43][134].
Gelatin (Gel), a natural biopolymer produced by collagen hydrolysis, has received a lot of interest as a bioink component due to its hygroscopic nature, cytocompatibility, plentiful cell recognition sites and thermosensitivity, allowing viscosity adjustment to favor printability. On the other hand, its low printing resolution, poor mechanical properties and fast biodegradation, in the presence or not of enzymes, drastically limits its broad application
[48][51][138,141]. The methacrylation of gelatin gives an easily photocrosslinkable, non-immunogenic derivative (methacrylated gelatin, GelMA) with tunable biodegradation that has been widely used for the fabrication of constructs
[48][138].
Photocrosslinkable gelatin-methacryloyl (GelMa), produced via the reaction of gelatin with methacrylic anhydride and the introduction of substituent methacryloyl groups onto the reactive amine has also received a lot of attention for TE applications, owing to its biocompatibility and tunable mechanical properties. Lee and coworkers (2022) developed a GelMA-based bioink containing various agents for skin TE. The bioink was shown to exhibit exceptional biocompatibility and bioactivity because of increased cell adhesion and proliferation, and enhanced expression of biomarkers related to ECM remodeling (e.g., MMP9, Ki67 and decorin)
[55][100].
Alginate, an algae-extracted linear polysaccharide, is characterized by hydrophilicity, biocompatibility/biodegradability, porosity, nontoxicity and facile functionalization, as well as lack of adequate biological cues for adhesion and proliferation of cells
[48][52][56][138,143,147]. Its combination with gelatin leads to the formation of semi-interpenetrating networks mimicking ECM structure, which provide sufficient cell adhesion sites facilitating hydrogel–cell interactions
[56][147].
Silk fibroin (SF), the main protein of silk, a natural biomimetic fibrous polymer is characterized by biocompatibility (high cell proliferation and adherence, low inflammation) and biodegradability as well as increased tensile strength. It can be chemically modified by glycidyl methacrylate (Silk-GMA) and bioprinted into different structures (e.g., hydrogel, membrane, porous sponges, etc.) via photopolymerization and aqueous processing. SF bioprinting has been found to result in stable porous structures favoring cell adhesion and growth
[57][146].
Biodegradable polyurethane (PU) is a synthetic polymer with sufficient printability and elasticity, as well as adjustable mechanical properties
[51][141]. Hydrogels comprising gelatin and PU have been found to exhibit adequate printability and modifiable mechanical strength to support MSCs’ proliferation and differentiation
[51][141].
To combine manufacturability with biomimicry, Hao and coworkers (2023) developed a thermo-responsive stepwise multi-crosslinking (i.e., pre-crosslinking via the Michael addition reaction of thiol–acrylate groups before bioprinting, hydrophobic interaction during bioprinting, and “thiol-ene” click reaction of the remaining thiol–acrylate groups under UV after bioprinting) bioink comprising thiolated Pluronic F127 (PF127-SH) and methacrylated hyaluronic acid (HAMA). This strategy was revealed to significantly improve the bioink regarding rheology, printability, mechanical properties, structural integrity, cytocompatibility, etc.
[58][148].
Adipose tissue decellularized extracellular matrix (dECM), which preserves the composition of glycosaminoglycans and proteins (e.g., (micro-)fibrillar collagens, glycoproteins and proteoglycans
[59][149]) in ECM, together with a ranked nanofibrous structure
[59][149], is a thermosensitive biomaterial which could form hydrogels via self-assembly, promote accumulation, proliferation and differentiation of cells, stimulate angiogenesis and endorse skin tissue repair
[60][150].
Platelet-rich plasma (PRP) obtained via centrifugation of whole blood could, at high concentrations, speed up hemostasis through the promotion of thrombosis and coagulation at the wound site. Furthermore, increased concentrations of bioactive agents like growth factors, chemokines, miRNA and immunoglobulins, which exhibit exceptional synergistic effects, could be released by α-particles of PLTs, thus avoiding the disadvantageous application of a single growth factor. Accordingly, PRP has been used in cutaneous wound healing and regeneration/rejuvenation of skin appendages. Additionally, owing to the slow release of bioactive agents, it is anticipated to act for a long time on the wound and achieve the initiation of regenerative signals. Finally, PRP is in alignment with personalized medicine since it is a source of autologous patient-specific growth factors
[43][134].
5.1.2. Cells
Fibroblasts and keratinocytes are the frequently used cells in skin TE. Nonetheless, in cases of deep or extensive skin defects, cell extraction from patients is difficult. In this respect, stem cells (SCs) have been favored for skin tissue repair
[60][150]. SCs exhibit desirable properties (e.g., differentiation into different types of tissues and enhanced proliferation)
[44][135]. Initially derived from mesoblasts, adipose tissue-derived stem cells (ADSCs) can effortlessly be obtained in large quantities via minimally invasive methods (e.g., liposuction) in high purity. ADSCs exhibit low immunogenicity and are capable of rapid proliferation in vitro as well as multidirectional differentiation. Additionally, they are characterized by substantial self-renewalability and have been found to endorse wound healing acceleration
[60][61][150,152].
5.1.3. Bioprinting Methods
Hydrogels can be (bio)printed utilizing various 3D (bio)printing techniques such as extrusion printing, inkjet printing, laser-assisted bioprinting, etc. In extrusion printing, the most commonly used method, the bioink is deposited via the application of pneumatic or mechanical force. This technique is similar to classic hydrogel extrusion from a syringe and is thus readily applied to different hydrogel bioinks. To minimize the imposed shear force on the hydrogel bioink, as well as the required pressure, shear-thinning biomaterials can be used for hydrogel formation. Additionally, monomer solutions of low viscosity can be deposited, which could be crosslinked after printing, or alternatively, materials’ viscosity could be reduced via temperature increase
[14][15]. Inkjet printing comprises the piezoelectric or thermal deposition of small bioink droplets onto a substrate where they spontaneously crosslink and/or fuse leading to the solidification of the printed construct. Laser-assisted bioprinting uses periodic laser excitation for the heating of a donor substrate and the subsequent spatial release of defined bioink droplets adsorbed to the substrate. Digital light processing (DLP) is another method that could aid in overcoming the drawbacks of extrusion or inkjet bioprinting but has not been adequately elucidated
[57][146].
5.1.4. Encapsulation of Bioactive Agents
The encapsulation of bioactive agents and/or drug-loaded nanocarriers (NCs) in hydrogel bioinks can have additional benefits for wound healing. Various NC types have been utilized to increase drug therapeutic efficiency including polymer or lipid-based NPs, liposomes, inorganic NPs, nanohydrogels and nanofibrous structures.
Particularly, polymer-based NPs have demonstrated enhanced encapsulation efficiency, thermodynamic stability, drug protection against degradation, topical sustained release, etc. Catechol (i.e., benzene derivative exhibiting anti-inflammatory and antioxidant activity, mitigating inflammation and assisting neovascularization) bearing polymer-based NPs have received a lot of attention due to sustained drug release and reduction of side effects of antimicrobials and various anticancer drugs. In a recent study, Puertas-Bartolome and coworkers (2021) incorporated catechol-bearing polymer NPs encapsulating a model hydrophobic drug in a hydrogel comprising carboxymethyl chitosan and HA for the development of a novel bioink. The bioprinted hydrogel construct was found to exhibit shape fidelity, mechanical stability and uniform distribution of NPs, and support the proliferation of the encapsulated fibroblasts for more than 14 days
[62][154].