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.

In vitro differentiation of airway epithelium is of interest for respiratory tissue engineering and studying airway diseases. Both applications benefit from the use of primary cells to maintain a mucociliated phenotype and thus physiological functionality. Complex differentiation procedures often lack standardization and reproducibility. To alleviate these shortfalls, we compared differentiation behavior of human nasal epithelial cells in four differentiation media. Cells were differentiated at the air-liquid interface (ALI) on collagen-coated inserts. Mucociliary differentiation status after five weeks was analyzed by electron microscopy, histology and immunohistochemistry. The amount of ciliation was estimated and growth factor concentrations were evaluated using ELISA. We found that retinoic-acid-supplemented mixture of DMEM and Airway Epithelial Cell Growth Medium gave most promising results to obtain ciliated and mucus producing nasal epithelium in vitro. We discovered the balance between retinoic acid (RA), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and fibroblast growth factor β (FGF-β) to be relevant for differentiation. We could show that low VEGF, EGF and FGF-β concentrations in medium correspond to absent ciliation in specific donors. Therefore, our results may in future facilitate donor selection and non-invasive monitoring of ALI cultures and by this contribute to improved standardization of epithelial in vitro culture.

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.

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 two-photon 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.