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Biofabrication Of Cellularized And Via Av-loop Vascularized Tissue Container For The Transplantation Of Cells Producing Therapeutic Proteins
Dominik Steiner, Dr.med.1, Stefanie Heltmann-Meyer, BA1, Annika Kengelbach-Weigand, Dr.med.vet. Ph.D.1, Vanessa Trossmann2, Sophie Winkler1, Hanna Amouei3, Harald Wajant, Prof. Dr. rer. nat.4, Tobias Fey5, Peter Greil, Univ.-Prof. Dr.ing.5, Thomas Scheibel, Univ.-Prof. Dr. ing.6, Andreas Arkudas, Prof.Dr.med.1, Raymund E. Horch, Univ.-Prof.Dr.Dr.h.c. Prof.h.c.7.
1Department of Plastic and Hand Surgery and Labratory for Tissue Engineering and Regenerative Medicine, University Hospital Erlangen, FAU, Erlangen, Germany, 2Department of Biomaterials, University Bayreuth, Bayreuth, Germany, 3Department of Plastic and Hand Surgery University Hospital Erlangen, Erlangen, Germany, 4Division of Molecular Internal Medicine, Medizinische Klinik und Poliklinik II, Universitätsklinikum Würzburg, Wuerzburg, Germany, 5Department of Materials Science and Engineering, Institute of Glass and Ceramics, Friedrich-Alexander-University Erlangen-Nürnberg (FAU), Germany, Erlangen, Germany, 6Department Biomaterials, University Bayreuth, Bayreuth, Germany, 7Department of Plastic and Hand Surgery and Labratory for Tissue Engineering and Regenerative Medicine, University Hospital Erlangen, Erlangen, Germany.

PURPOSE Targeted therapies using biologicals, are of growing and overwhelming clinical importance in the treatment of cancer and autoimmune diseases. Although promising, the broad medical application is limited by several factors such as high production costs or low effective levels due to the short half-life time of the biologicals requiring frequent injections or infusions. Therefore, we want to establish a transplantable therapeutic tissue container providing a continuous secretion of biologicals by transgenic modified cells. An arteriovenous fistula (AV loop) provides oxygen and nutrition supply of the transplanted cells in the tissue container (Fig1), This study aimed at the establishment of an ideal bioink carrying the producer cells based on recombinant engineered spider silk. METHODS An engineered TNF-blocking fusion protein consisting of luciferase linked to the extracellular domain of TNFRII was used as a reporter molecule. HEK293 cells producing the reporter protein were incorporated into an hydrogel matrix formed by the recombinant engineered spider silk protein eADF4(C16). Cell adhesion and angiogenesis were further improved using a RGD-tagged eADF4(C16) silk protein. Firstly, cytocompatibility and protein diffusion studies were carried out in both spider silk bioinks [eADF4(C16) and eADF4(C16)-RGD] in vitro. Thereafter, the eADF4(C16)-RDG spider silk was implanted in vivo in the rat AV loop model. Vascularization of the spider silk matrix was induced by connecting the saphenous artery and vein with a venous interponat forming an arteriovenous fistula (AV loop). 4 weeks after implantation, the specimen were explanted and vascularization as well as biocompatibility were analyzed using histology and µCT.
RESULTS We were able to prove a continious protein secretion over a time period of 2 weeks in both spider silk based bioinks in vitro. Moreover, both spider silks demonstrated a good biocompatibility with metabolic active and vital cells in the WST-8 assay and live/dead staining, respectively. We measured a significantly higher fusion protein production of HEK293 cells incorporated in the RDG-modified spider silk. Using µCT and histology we were able to prove a stable and biocompatible matrix with incipient vascularization originating from the AV loop.
CONCLUSIONS Once determined, the optimally suitable bioink will be bioprinted with producer cells in order to generate a drug-producing container vascularized by the AV loop (Fig 2). In this regard recombinant spider silk seems to be a favourable biomaterial. This will be the first in vivo working device that aims at producing active substances in a concise chamber with arterial and venous connections. Other than systemically applied cell therapies this actively producing cell container can be safely removed at any time when no longer necessary.




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