In vivo vessel connection of pre-vascularised 3D-bioprinted gingival connective tissue substitutes

Producing oral soft tissues using tissue engineering could compensate for the disadvantages of autologous grafts (limited availability and increased patient morbidity) and currently available substitutes (shrinkage). However, there is a lack of in vitro-engineered oral tissues due to the difficulty of obtaining stable pre-vessels that connect to the host and enable graft success. The main objective was to assess the connection of pre-vascularised 3D-bioprinted gingival substitutes to the host vasculature when subcutaneously implanted in immunodeficient mice. This study produced vascularised connective tissue substitutes using extrusion-based 3D-bioprinting of primary human gingival fibroblasts (hGF) and fluorescent human endothelial cells (RFP-HUVEC) cocultures. Pre-vascularised (hGF+RFP-HUVEC -CC grids) and control (hGF only -HG grids) grids were bioprinted and pre-cultivated for 14 days to enable pre-vessels formation. In vitro vessel formation follow-up was performed. Eight-week-old female NOG mice were used for in vivo experiments. One grid per mouse was subcutaneously implanted in 20 mice (10HG/10CC). The fluorescent activity of RFP-HUVEC was monitored. Samples were retrieved at 7, 14 and 21 days. Histological, immunohistochemical, and immunofluorescent staining was performed. CC-grids formed efficient and stable pre-vessel networks within 14 days of static pre-culture. HG-grids did not contain any vessel, while CC-grids successfully connected to the host vasculature by presenting erythrocytes within the vessel lumen inside the grids starting day 7. From days 7 to 21, vessel density was stable. Human pre-vessels were present at 7 days and were progressively replaced by murine endothelial cells. This study showed that primary hGF-HUVEC co-cultures can be successfully 3D-bioprinted within biomimetic hydrogels having a close composition to the gingival connective tissue, and HUVEC organise themselves into pre-vessel networks that connect to the murine vasculature when implanted in vivo. This approach represents a promising strategy to enhance current and future oral soft tissue substitutes for prospective clinical applications.