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