Biofunctionalization and endothelialization of new cytocompatible scaffolds based on multifunctional heparin derivates for vascularized tissue constructs
Introduction: Vascular structures are important for the oxygen and nutrient supply of large tissues in vitro, since oxygen is used by surrounding cells in a distance of 200 lm. Our strategy to achieve vascularization of artificial tissues is the endothelial lining of small synthetic tubes with 2 mm inner diameter. The adhesion of the endothelial cells to the synthetic materials should be facilitated by a suitable functionalization of the tube surface using chemically modified heparin and arginine-glycine-aspartic acid (RGD) peptides. For homogenous cell seeding a protocol should be established and afterwards the endothelialized tubes should be cultured in a fluid flow bioreactor. Our long-term goal is the endothelial lining of synthetic porous polyacrylate tubes that can be perfused and allow angiogenesis to the surrounding tissue. Materials and methods: A protocol for the synthesis of thio-heparin (TH) was established in our lab. Different functionalizations (TH, RGD and TH/RGD) were evaluated on planar biocompatible polyacrylate surfaces with atomic force microscopy (AFM), contact angle measurements and X-Ray Photoelectron Spectroscopy (XPS). Endothelial cell adhesion and viability was measured by light microscopy and Alamar-Blue_. In a next step, synthetic tubes manufactured by dip-coating were functionalized with TH/RGD. Static and rotating seeding of functionalized tubes was evaluated by MTT assay. Immunohistological staining of the endothelial cell markers CD31 and von Willebrand factor (vWF) was performed. Endothelialized tubes were then cultured in a fluid flow bioreactor with a wall shear rate of 10 dyne/cm2. MTTassay and immunofluorescent stainings (Actin, CD31, vWF) were performed to evaluate cell viability and morphology. Results: The polymer was successfully functionalized with TH, RGD or TH/RGD, which was shown in changes in the roughness, contact angle and sulphur and nitrogen content compared to the control. Endothelial cells seeded on an unfunctionalized, TH- or RGD-functionalized surface adhered in a lower density compared to the control (tissue culture polystyrene, TCPS) whereas TH/RGD-functionalized polymers showed a dense cell layer. In tubular structures, rotating seeding led to a complete seeding of the tube and cells expressed typical markers. Endothelial cells cultured in a fluid flow bioreactor stayed viable and aligned along the media flow. Discussion and conclusions: These results show that polyacrylates functionalized with TH/RGD were biocompatible and able to be endothelialized completely. Endothelial cells stayed vital and aligned in a fluid flow bioreactor. In the next step, porous 3D tubes will be endothelialized and their applicability as supply system for in vitro generated 3D-tissue-constructs will be investigated. Acknowledgments: We thank the European Commission for funding the ArtiVasc 3D project under the Seventh Framework Program (grant agreement no. 263416). Disclosures: Authors have nothing to disclose.