Drug delivery and tissue engineering division, School of Pharmacy, University of Nottingham. INTRODUCTION: Recapitulating the complex conditions that lead ...
European Cells and Materials Vol. 29. Suppl. 3, 2015 (page 63)
ISSN 1473-2262
Three dimensional printing of hydrogels, growth factors and mammalian cells to create a biomimetic environment for nervous tissue formation in vitro OA Hamid, J Yang, HM Eltaher, KM Shakesheff Drug delivery and tissue engineering division, School of Pharmacy, University of Nottingham INTRODUCTION: Recapitulating the complex conditions that lead spinal neurons formation could provide a promising approach for generating nervous tissue grafts in vitro. These conditions are mainly represented by the molecular gradient of growth factors which are responsible for neuronal differentiation during neural tube development [1]. Recent advances in three dimensional (3D) printing technology offer an interesting opportunity for the replication of the intrinsic complexity of native tissues in vitro by precise positioning of multiple cell types, hydrogels and bioactive molecules [2]. However, 3D printing of a model simulating the complex conditions that lead to spinal cord neurons development has many challenges including fabrication of a hydrogel tubular structure with clinically relevant size, the spatial delivering of bio-active molecules to generate concentration gradient and preserving cell viability during the printing process. The aim of this research is to develop a 3D hydrogel tubular model with a controlled molecular gradient using 3D printing. In addition, the impact of 3D printing process on cell viability is characterized. METHODS: An extrusion based multi-head 3D printer (Regen HU, Switzerland) was used for 3D printing a hollow tubular construct from polycaprolactone (PCL, MWT 45 kDa; SigmaAldrich, UK) and semi-cross linked alginate hydrogel (1.5%) (FMC BioPolymer, Ireland). The scaffold consisted of two concentric cylinders of PCL (12 and 8 mm in diameter) with alginate filling the annulus space. A concentration gradient of fluorescein isothiocyanate-labelled albumin (FITC-BSA), as a model, was printed in the annulus. The hydrogel annulus was printed using two printing heads, one for FITC-BSA-mixed alginate gel and the other for alginate gel. The gradient was created by gradual reduction in the number of the layers that contain FITC-BSA as a function of tube distance; consequently, the diffusion between layers will generate the gradient. The gradient was quantified as a function of tube distance by sectioning the tube into segments followed by fluorescent intensity assay. In addition, the effect of printing nozzle’s internal diameter on mammalians cell (3T3) viability was examined using live/dead assay.
RESULTS:
Fig. 1: (A) Schematic diagram of the hollow PCLalginate gel tubular construct with gradient of FITC-BSA (green colour); (B) Concentration of FITC-BSA as a function of tube distance.
Fig. 2: Pre and post printing 3T3 cell viability. DISCUSSION & CONCLUSIONS: Multi-head 3D printer provided an opportunity to fabricate a mechanically stable PCL-hydrogel tubular structure with a controlled molecular gradient. In addition, 3D printing process showed a limited negative effect on cell viability. This in vitro 3D model will help to improve the understanding of neuronal cells development for nerve regenerative applications. REFERENCES: 1Peto, H. and K. Shakesheff (2012). European Journal of neurodegenerative Diseases, 2012. 1(3): p. 385-399. 2 Zhao, Y., R. Yao, L. Ouyang, et al. (2014). Biofabrication. 6(3): p. 035001. ACKNOWLEDGEMENTS: this work was sponsored by the higher committee for education development in Iraq.
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