Human skin is particularly vulnerable to external damaging influences such as irradiation, extreme temperatures, chemical trauma, and certain systemic diseases, which reduce the skin’s capacity for regeneration and restoration and can possibly lead to large-scale skin defects. To restore skin continuity in severe cases, surgical interventions such as the transplantation of autologous tissue are needed. Nevertheless, the coverage of larger skin defects caused by severe third-grade burns or extensive irradiation therapy is limited due to the depletion of uninjured autologous tissue. In such cases, many of the patient’s epidermal cells can become available using biofabricated skin grafts, thereby restoring the skin’s vital functions. Given the limited availability of autologous skin grafts for restoring integrity in large-scale defects, using bioprinted constructs as skin graft substitutes could offer an encouraging therapeutic alternative to conventional therapies for large-scale wounds, such as the transplantation of autologous tissue. Using layer-by-layer aggregation or volumetric bioprinting, inkjet bioprinting, laser-assisted bioprinting, or extrusion-based bioprinting, skin cells are deposited in a desired pattern. The resulting constructs may be used as skin graft substitutes to accelerate wound healing and reconstitute the physiological functions of the skin. In this review, we aimed to elucidate the current state of bioprinting within the context of skin tissue engineering and introduce and discuss different bioprinting techniques, possible approaches and materials, commonly used cell types, and strategies for graft vascularization for the production of bioprinted constructs for use as skin graft substitutes.

Ionizing radiation has become an integral part of modern cancer therapy regimens. Various side effects, such as radiation dermatitis, affect patients in acute and chronic forms and decrease therapy compliance significantly. In this study, primary keratinocytes were irradiated in a 2-dimensional (2D) culture as well as on a 3-dimensional (3D) collagen-elastin matrix with doses of 2 and 5 Gy. The effect of different concentrations of IGF-I, KGF, platelet lysate (PL), high and low molecular weight hyaluronic acid (H-HA, L-HA), and adipose-derived stem cell (ADSC) conditioned medium was analyzed in respect to cell viability (WST-8), wound closure (migration), and the gene expression (quantitative real-time PCR) of 2D cultures. The 3D culture was evaluated by WST-8. A mixture of H-HA and L-HA, as well as IGF-I, could significantly stimulate the keratinocyte viability and migration which were severely reduced by irradiation. The MKI67and IL6 gene expression of irradiated keratinocytes was significantly higher after H-HA/L-HA treatment. The stimulating effects of H-HA/L-HA and IGF-I were able to be confirmed in 3D culture. A positive influence on cell viability, migration, and gene expression was achieved after the treatment with H-L-HA and IGF-I. These results open the possibility of a novel therapeutic method for both the prevention and the treatment of radiation dermatitis.

The treatment of large-scale skin wounds remains a therapeutic challenge. In most cases there is not enough autologous material available for full coverage. Cultured epithelial autografts are efficient in restoring the lost epidermal cover; however, they have some disadvantages, such as difficult application and protracted cell cultivation periods. Transplanting a sprayed keratinocyte suspension in fibrin sealant as biological carrier is an option to overcome those disadvantages. Here, we studied different seeding techniques regarding their applicability and advantages on cell survival, attachment, and outgrowth in vitro and thereby improve the cell transfer to the wound bed. Human primary keratinocytes were suspended in a fibrin sealant. WST-8 assay was used to evaluate the vitality for 7 days. Furthermore, the cells were labeled with CellTracker™ CM-Di-I and stained with a life/dead staining. Cell morphology, shape, and distribution were microscopically analyzed. There was a significant increase in vitality while cultivating the cells in fibrin. Sprayed cells were considerably more homogenously distributed. Sprayed cells reached the confluent state earlier than dripped cells. There was no difference in the vitality and morphology in both groups over the observation period. These findings indicate that the sprayed keratinocytes are superior to the application of the cells as droplets. The sprayed application may offer a promising therapeutic option in the treatment of large chronic wounds.

The goal of reconstructive surgery in treating tissue defects is to achieve a stable reconstructive outcome while minimizing donor site morbidity. As a result, tissue engineering has emerged as a key focus in the pursuit of this goal. One approach is to create a tissue container that can be preconditioned and later transplanted into the defect area. The characteristics of the matrices used in the tissue container are critical to this approach’s success. Matrices generated with recombinant, functionalized spider silk (eADF4(C16)-RGD) have been reported to be biocompatible and easy to vascularize. However, the effect of exogenously added proangiogenic cells, such as endothelial cells (T17b), on the vascularization process of matrices generated with this hydrogel in vivo has not been described yet. In this study, we implanted arteriovenous (AV) loop containers filled with a spider silk hydrogel consisting of an eADF4(C16)-RGD matrix and encapsulated, differentiated endothelial T17b cells producing the reporter protein TNFR2-Fc-Flag-GpL. The histological and µCT analyses revealed spontaneous angiogenesis and fibrovascular tissue formation in the container at 2 and 4 weeks post-implantation. The reporter protein was detected after 4 weeks. No severe immune response was observed. Altogether, this study demonstrates that cell-supplemented recombinant spider silk is a highly promising hydrogel to produce matrices for tissue engineering applications.

Treating large-scale skin wounds continues to be a significant therapeutic challenge. Oftentimes, insufficient autologous material is available to achieve full coverage. Since biofabrication offers the chance of reproducible and precise large-scale production, the aim was to develop a customized 3D bioprinted skin construct containing immortalized HaCaT keratinocytes and ADSC to test the principal feasibility of this approach. Keratinocytes in different hydrogels containing varying concentrations of alginate, fibrin, collagen, gelatin, gelatin methacryloyl (GelMA) and hyaluronic acid as well as on a prefabricated collagen-elastin-matrix were cultured for a period of seven days. The metabolic activity of cultured keratinocytes was then evaluated during the co-cultivation of HaCaT and ADSC in a transwell model. The metabolic activity in all groups increased over the test period. The hydrogels containing alginate/hyaluronic acid/gelatin (Alg/HA/Gel) and GelMA demonstrated a good printability and the highest diffusion rates. There was no significant difference in pore size when comparing all hydrogels with each other. On the basis of the performed printability and diffusion assays as well as on the SEM analysis and rheological measurements Alg/HA/Gel and GelMA were chosen as hydrogels for the bioprinted 3D model. Fibrin hydrogel was integrated into the biofabricated constructs due to its excellent metabolic activity in the transwell model. As a second step, hydrogel stability, cell survival and metabolic activity in bioprinted 3D models containing keratinocytes and ADSC were evaluated over a period of 14 days. On day 14 the metabolic activity as well as the live cell count within the bioprinted constructs of the cocultured groups was significantly higher compared with day 1. The biofabricated constructs made of GelMA showed a higher cell viability compared with those made of Alg/HA/Gel. Additionally, to evaluate the cell migration out of the constructs, the metabolic activity and viability of the cells on the well bottom, was examined. After 14 days, an average of 50% of the well bottom was covered by HaCaT, which were initially printed in coculture into the constructs. These findings indicate that constructs made of GelMA containing keratinocytes and ADSC may offer a promising therapeutic option in the treatment of large chronic wounds.

The increasing demand for tissue and organ replacement has positioned biofabrication as a transformative field within regenerative medicine. Biofabrication is an interdisciplinary approach that combines cells, biomaterials, and bioactive molecules to generate biologically functioning structures and tissue models. However, the size of biofabricated constructs often remains limited due to the lack of vascular structures. Cells located in the inner regions of larger constructs suffer from a lack of oxygen and nutrients, which results in hypoxia and reduced cell viability. Furthermore, integration into the host vasculature is crucial for the long-term survival of constructs intended for in vivo implantation. Consequently, strategies to improve the vascularization of biofabricated constructs have gained significant attention. In this review, we focus on approaches for the in vitro and in vivo vascularization of biofabricated constructs. We summarize the types and sources of vascular cells used in biofabrication, as well as the signaling molecules and biomaterials that can be incorporated into biofabricated scaffolds. A special focus is placed on advanced vascularization strategies such as the in ovo chorioallantoic membrane (CAM) and in vivo arteriovenous (AV) loop models. Furthermore, we describe the role of vascularization in advanced tissue models used for disease modeling and drug screening. Although the clinical translation of biofabricated constructs is still limited by a lack of vasculature and size constraints, biofabrication offers great potential for tissue replacement, personalized medicine, and the reduction of animal experiments.