Effective vascularization is crucial for three-dimensional (3D) printed hydrogel-cell constructs to efficiently supply cells with oxygen and nutrients. Till date, several hydrogel blends have been developed that allow the in vitro formation of a capillary-like network within the gels but comparatively less effort has been made to improve the suitability of the materials for a 3D bioprinting process. Therefore, we hypothesize that tailored hydrogel blends of photo-crosslinkable gelatin and type I collagen exhibit favorable 3D drop-on-demand printing characteristics in terms of rheological and mechanical properties and that further capillary-like network formation can be induced by co-culturing human umbilical vein endothelial cells and human mesenchymal stem cells within the proposed blends. Gelatin was methacrylated (GelMA) at a high degree of functionalization, mixed with cells, type I collagen, and the photoinitiator Irgacure 2959 and then subsequently crosslinked with UV light. After 14 d of incubation, cells were immunofluorescently labeled (CD31) and displayed using two-photon laser scanning microscopy. Hydrogels were rheologically characterized and dispensable droplet volumes were measured using a custom built 3D drop-on-demand bioprinter. The cell viability remained high in controllable crosslinking conditions both in 2D and 3D. In general, higher UV light exposure and increased Irgacure concentration were associated with lower cell viabilities. Distinctive capillary-like structures were formed in 3D printable GelMA-collagen hydrogels. The characteristic crosslinking time for GelMA in the range of minutes was not altered when GelMA was blended with type I collagen. Moreover, the addition of collagen led to enhanced cell spreading, a shear thinning behavior of the hydrogel solution and increased the storage modulus of the crosslinked gel. We therefore conclude that GelMA-collagen hydrogels exhibit favorable biological as well as rheological properties which are suitable for the manufacturing of pre-vascularized tissue replacement by 3D bioprinting.

Three-dimensional (3D) bioprinting is a promising technology for manufacturing cell-laden tissue-engineered constructs. Larger tissue substitutes, however, require a vascularized network to ensure nutrition supply. Therefore, tailored bioinks combining 3D printability and cell-induced vascularization are needed. We hypothesize that tailored hydrogel blends made of agarose-type I collagen and agarose-fibrinogen are 3D printable and will allow the formation of capillary-like structures by human umbilical vein endothelial cells and human dermal fibroblasts. Samples were casted, incubated for 14 days, and analyzed by immunohistology and two-photon laser scanning microscopy. The 3D printability of the hydrogel blends was examined using a drop-on-demand printing system. The rheological behavior was also investigated. Substantial capillary network formation was observed in agarose-type I collagen hydrogel blends with concentrations of 0.2% or 0.5% collagen and 0.5% agarose. Furthermore, storage moduli of agarose-collagen blends were significantly increased compared to those of the corresponding single components (448 Pa for 0.5% agarose, 148 Pa for 0.5% collagen, and 1551 Pa for 0.5% agarose-0.5% collagen). Neither the addition of collagen nor fibrinogen significantly impaired the printing resolution. In conclusion, we present a tailored hydrogel blend that can be printed in 3D and in parallel exhibits cell-induced vascularization capability.

Vascularisation is essential for the development of tailored, tissue-engineered organs and tissues due to diffusion limits of nutrients and the lack of the necessary connection to the cardiovascular system. To pre-vascularize, endothelial cells and supporting cells can be embedded in the scaffold to foster an adequate nutrient and oxygen supply after transplantation. This technique is applied for tissue engineering of various tissues, but there have been few studies on the use of different cell types or cells sources. We compare the effect of supporting cells from different sources on vascularisation. Fibrin gels and agarose-collagen hydrogels were used as scaffolds. The supporting cells were primary human dermal fibroblasts (HDFs), human nasal fibroblasts (HNFs), human mesenchymal stem cells from umbilical cord's Wharton's jelly (WJ MSCs), adipose-derived MSCs (AD MSCs) and femoral bone marrow-derived MSCs (BM MSCs). The tissue constructs were incubated for 14 days and analyzed by twophoton laser scanning microscopy. Vascularisation was supported by all cell types, forming branched networks of tubular vascular structures in both hydrogels. In general, fibrin gels present a higher angiogenic promoting environment compared to agarose-collagen hydrogels and fibroblasts show a high angiogenic potential in co-culture with endothelial cells. In agarose-collagen hydrogels, vascular structures supported by AD MSCs were comparable to our HDF control in terms of volume, area and length. BM MSCs formed a homogeneous network of smaller structures in both hydrogels. This study provides data toward understanding the pre-vascularisation properties of different supporting cell types and sources for tissue engineering of different organs and tissues.

Rapid vascularization is required for the regeneration of dental pulp due to the spatially restricted tooth environment. Extracellular vesicles (EVs) released from mesenchymal stromal cells show potent proangiogenic effects. Since EVs suffer from rapid clearance and low accumulation in target tissues, an injectable delivery system capable of maintaining a therapeutic dose of EVs over a longer period would be desirable. We fabricated an EV-fibrin gel composite as an in situ forming delivery system. EVs were isolated from dental pulp stem cells (DPSCs). Their effects on cell proliferation and migration were monitored in monolayers and hydrogels. Thereafter, endothelial cells and DPSCs were co-cultured in EV-fibrin gels and angiogenesis as well as collagen deposition were analyzed by two-photon laser microscopy. Our results showed that EVs enhanced cell growth and migration in 2D and 3D cultures. EV-fibrin gels facilitated vascular-like structure formation in less than seven days by increasing the release of VEGF. The EV-fibrin gel promoted the deposition of collagen I, III, and IV, and readily induced apoptosis during the initial stage of angiogenesis. In conclusion, we confirmed that EVs from DPSCs can promote angiogenesis in an injectable hydrogel in vitro, offering a novel and minimally invasive strategy for regenerative endodontic therapy.

Stereolithography is one of the most promising technologies for the production of tailored implants. Within this study, we show the results of a new resin formulation for three-dimensional printing which is also useful for subsequent surface functionalization. The class of materials is based on monomers containing either thiol or alkene groups. By irradiation of the monomers at a wavelength of 266 nm, we demonstrated an initiator-free stereolithographic process based on thiol-ene click chemistry. Specimens made from this material have successfully been tested for biocompatibility. Using Fourier-transform infrared spectrometry and fluorescent staining, we are able to show that off-stoichiometric amounts of functional groups in the monomers allow us to produce scaffolds with functional surfaces. We established a new protocol to demonstrate the opportunity to functionalize the surface by copper-catalyzed azide-alkyne cycloaddition chemistry. Finally, we demonstrate a three-dimensional bioprinting concept for the production of potentially biocompatible polymers with thiol-functionalized surfaces usable for subsequent functionalization.

Tissue engineering is a promising approach to treat massive airway dysfunctions such as tracheomalacia or tumors. Currently, there is no adequate solution for patients requiring the resection of more than half of the length of their trachea. In this study, the best conditions for combination of three different cell types from the respiratory airway system were investigated to develop a functional ciliated and pre‐vascularized mucosal substitute in vitro. Primary human fibroblasts were combined with respiratory epithelial cells and endothelial cells. As scaffolds, fibrin gel and agarose‐type I collagen blends were used and cultured with different medium compositions to optimize both vascularization and differentiation of the respiratory epithelium. A mixture of endothelial growth medium and epithelial differentiation medium was shown to optimize both vascularization and epithelial growth and differentiation. After 28 days of co‐culture, significantly increased formation of capillary‐like structures was observed in fibrin gels with more than three times higher structure volumes compared to agarose–collagen gels. After 35 days, epithelial differentiation into a pseudostratified epithelium with typical marker expression was improved on fibrin gels. While cilia formation was shown on both scaffolds, a higher number of ciliated cells and longer cilia were observed on fibrin gels. The data elucidate the important interplay of co‐culture parameters and their impact on vascularization as well as epithelium development and provide a basis for development of functional three‐dimensional airway constructs.

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Tissue‐engineered dermo‐epidermal skin grafts could be applied for the treatment of large skin wounds or used as an in vitro wound‐healing model. However, there is currently no skin replacement model that includes both, endothelial cells to simulate vascularization, and macrophages to regulate wound healing and tissue regeneration. Here, we describe for the first time a tissue‐engineered, fully vascularized dermo‐epidermal skin graft based on a fibrin hydrogel scaffold, using exclusively human primary cells. We show that endothelial cells and human dermal fibroblasts form capillary‐like structures within the dermis whereas keratinocytes form the epithelial cell layer. Macrophages played a key role in controlling the number of epithelial cells and their morphology after skin injury induced with a CO2 laser. The activation of selected cell types was confirmed by mRNA analysis. Our data underline the important role of macrophages in vascularized skin models for application as in vitro wound healing models or for skin replacement therapy. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1340–1350, 2019.