Prevascularized in vitro models for skin and tracheal tissue engineering

The field of tissue engineering offers novel and innovative treatments for tissue/organ dysfunction and disease, helping to overcome the limited supply of donor organs. The most challenging aspect is the absence of vasculature in engineered constructs, which causes cell necrosis/apoptosis and transplant rejection. Prevascularization is a promising step towards the engineering of complex and multi-layered tissue constructs supplied with sufficient oxygen and nutrients. This study reports the fabrication of in vitro skin and trachea models with regard to functional prevascularization and the optimization of co-cultivation conditions with more than two cell types involved. For the skin model, a fibrin gel scaffold was used, which was seeded with human umbilical cord vein endothelial cells (HUVECs), dermal fibroblasts (HDFs), macrophages, and dermal keratinocytes (HDKs). Skin models were cultivated for 10 days at an air-liquid interface with and without macrophages. These models were injured with a CO2 laser on day 10 and analysed 2 and 3 days after laser treatment. Vascularization was confirmed by CD31 staining followed by two-photon laser-scanning microscopy. Epidermal layer and basement membrane development were confirmed by pan-cytokeratin and collagen IV staining. A highly sophisticated skin model was created, with similarities to native skin tissue in terms of vasculature, stratified and cornified epithelium, and wound healing potential initiated by macrophages. An in vitro trachea model was also established based on 3D-printable agarose/collagen type I and agarose/fibrinogen scaffolds seeded with HUVECs and HDFs or nasal fibroblasts (HNFs). It was shown that printable scaffolds promote the formation of capillary-like vessels and support tri-cultures containing human respiratory epithelial cells (HRECs). Vascularization was analysed by CD31 staining, and HREC differentiation and pseudo-stratification was detected by pan-cytokeratin, MUC5AC and PAS staining. Scanning electron microscopy revealed cilia formation and the development of continuous HREC layers. Therefore, the first step towards a 3D-printable trachea substitute supporting angiogenesis and the pseudo-stratification of epithelium in a mechanically stable agarose/collagen type I scaffold was achieved

GelMA-collagen blends enable drop-on-demand 3D printablility and promote angiogenesis

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

New stereolithographic resin providing functional surfaces for biocompatible three-dimensional printing

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

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

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

Extracellular Vesicles-Loaded Fibrin Gel Supports Rapid Neovascularization for Dental Pulp Regeneration

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