Vascularized 3D-bone and cartilage tissue engineering

Introduction Modern tissue engineering (TE) focusses mainly on 3D cell culture systems. The possible size of these hydrogel constructs is limited to the diffusion of O2, cell waste and nutrients of the cell culture medium. In this project, we present a hydrogel model with embedded vascular networks, enabling the production of larger hydrogels compared to non-vascularized gels. Future approaches will focus on the design of whole organ-like-structures like bone or cartilage. Given the fact that spontaneous repair of cartilage is limited after injury compared to bone healing, cartilage tissue engineering has become more and more important in the recent years. Furthermore, we want to reduce animal experiments according to the 3R principle. Our bioreactors can be used to rebuild in vivo tissue with any kind of cells (human, mouse, rat cells of any type of tissue) to perform drug delivery tests, kinetic studies and further health research, which can´t be performed in standard 2D cell culture. Since the environment of cells in our 3D bioreactor is more related to the in vivo situation compared to standard 2D cell culture in plastic flasks, this is an alternative and improved model for advanced in vitro research.

Methods For 3D cell culture, we use the self-designed bioreactor including hydrogel and mesenchymal stem cells (MSCs). Bioreactors were 3D printed with a non-toxic and biocompatible polymer PLA (polylactic acid). 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). Self-designed silicon rings were used to seal the system and guarantee sterility. The following flow parameters were set inside the software: 15mbar, 10s unidirectional flow. MSCs were cultured at 37°C, 5 % CO2 and 95 % RH.

Results Mesenchymal stem cells were mixed successfully with scaffold material (PEG700, PEG6000 and PEG20000) 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. Cell viability showed vital cells up to 2 weeks of culture. Furthermore, a 2-cell-layer approach showed a notable cultivation of GFP positive (inner layer) and RFP positive cells (outer layer) in one hydrogel system, indicating a successful step for culturing more complex organ structures.

Discussion This new and innovative bioreactor-design and 3D culture technique provides an economical, reproducible, controlled and fast approach to produce 3D tissues with vascular structures. This research project will be the basis of further projects, achieving more complex tissue types with multiple 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 Tissue Engineering, Stem cells, Bone, Cartilage

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

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Publication History

Article published online:
05 March 2021