3 D-bone tissue engineering with embedded vascular structures

Introduction 3 D cell culture with hydrogels have been widely used for tissue engineering. The biggest possible size of these hydrogel constructs is limited to the diffusion of nutrients, O2 and cell waste. In this project, we present a hydrogel model with embedded vascular networks, enabling the production of large hydrogels. In the near future, it will be possible to design whole organ-structures like bone or cartilage, which is one research focus of this project. Another focus of this project is the reduction of animal experiments by using these bioreactors as an alternative for standard 2 D cell culture. With these bioreactors, we want to rebuild in vivo tissues to perform drug tests and health research, which can’t be performed by standard cell culture. Since the environment of cells in our 3 D bioreactor is more related to the in vivo situation compared to standard 2 D cell culture in plastic flasks, this is an alternative and improved model for advanced in vitro research.

Methods 3 D Printing for the design of the bioreactor, we used the open source software blender or FreeCAD. For 3 D printing, we used the 3 D printer Ultimaker 3 with extra fine settings (layer height 0.06 mm, 100 % infill and 60 mm/s print speed). All bioreactors were produced with a non-toxic and biocompatible polymer polylactic acid. 3 D cell culture For 3 D cell culture, self-designed bioreactor including hydrogel and cells were used. The reactor is connected to a perfusion system on both sides under sterile conditions, and the system is filled with nutrient medium (DMEM + 10% FCS + 1% P/S). The following flow parameters were set inside the software: 15 mbar, 10 s unidirectional flow. Cells were cultured at 37 °C, 5 % CO2 and 95 % RH.

Results Mesenchymal stem cells were mixed successfully with scaffold material and inserted into a bioreactor. A pump system guaranteed medium flow and nutrient supply. Vital cells were observed by fluorescence microscopy and fabrication of vascular structures was successfully obtained by molding forms. Cross sections of hydrogels indicated a time dependent nutrient penetration of the hydrogel. Cell vitality decreased gradually with increased distance from the channel. Furthermore, a 2-cell-layer approach showed a notable cultivation of green (outer layer) and red fluorescent cells (inner layer) in one bioreactor, indicating a successful step for culturing more complex organ structures.

Discussion This new and innovative technique of 3 D tissue engineering provides a cost-effective, reproducible, controlled and fast approach to produce 3 D tissues with vascular structures inside the hydrogel. This research project will be the basis of further projects, achieving bigger tissues with more complex vessel-like structures. It will also enable research topics to labs without sophisticated equipment and no access to animal models to design and produce vascularized tissues in their own bioreactors.

Keywords 3 D Tissue Engineering, 3 D Printing, MSCs, Vascular structures, Hydrogel

Korrespondenzadresse Kai Böker, Universitätsmedizin Göttingen, Klinik für Unfallchirurgie, Orthopädie und Plastische Chirurgie, Robert-Koch-Straße 40, 37075 Göttingen, Deutschland,

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