Three-Dimensional Printing and Angiogenesis: Tailored Agarose-Type I Collagen Blends Comprise Three-Dimensional Printability and Angiogenesis Potential for Tissue-Engineered Substitutes

Abstract

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.

Combination of vascularization and cilia formation for three‐dimensional airway tissue engineering

“…Here, HDFs and HNFs were compared with respect to their vascularization potential. The pro‐angiogenic effect of HDFs was shown by our group previously (Kreimendahl et al, ), but the potential of HNFs had not been verified so far. Here, HNFs revealed no significant difference in vascularization‐supportive potential making them a promising cell source for respiratory tissue engineering as they origin from the same tissue as the ciliated cells and can support their differentiation (Kobayashi et al, ).…”

“…The vascularization potential of HNFs was tested in our standard assay in fibrin gel for capillary‐like vessel formation to prove equivalency to previously established HDFs (Kreimendahl et al, ). All parameters (structure volume, area, length, and branches) confirm comparability of nasal to dermal fibroblasts (Figure ).…”

“…Fibrin gels and agarose‐type I collagen hydrogels (FIB0.5, AGR0.5COLL0.5) were molded following a protocol described previously (Kreimendahl et al, ). In short, HUVECs and fibroblasts were trypsinized (Trypsin EDTA, Pan Biotech) and molded directly into hydrogels with final concentrations of each 3 × 10 6 cells/mL.…”

“…The samples were imaged by TPLSM using a FV1000MPE device (Olympus). Settings were set to standard parameters (Kreimendahl et al, ). Images were taken at random positions to avoid subjective choice of certain hydrogel areas.…”

“…In case vascularization was focused, it was achieved in vivo via fascial or omental flaps or heterotopic transplantation (Delaere, Vranckx, Verleden, De Leyn, & Van Raemdonck, ; Li et al, ). In terms of in vitro vascularization models, we showed successful co‐cultivation of fibroblasts and HUVECs in different hydrogel scaffolds (Kreimendahl et al, ). In our previous study, we investigated the influence of different hydrogel compositions on their mechanical stability and their effect on formation of capillary vascular systems.…”