INTRODUCTION: Type 1 diabetes is an autoimmune condition which through pathological immune system action mediates
β-cell destruction, reducing insulin production capacity1. A proposed replacement for insulin injections and pancreas transplants is the production of semi-artificial pancreas, which combines immunoisolative and oxygen permeable polymer hydrogels with mechanically resistant polymer scaffolds. Polyhydroxyalkanoates (PHAs) are becoming known in biomedical applications due to their tuneable properties, non-immunogenicity, and relative biocompatibility with multiple tissue types3. This work aims to generate a 3D printable semi-artificial scaffold for islets, using functionalized bioresorbable bacteria-derived polymers such as alginate and PHAs.
METHODS: The elastomeric medium chain length PHA (mcl-PHA), was produced via bacterial fermentation using a Pseudomonas sp. over a 24-hour period. After centrifugation and freeze drying, the cells were subjected to Soxhlet extraction to obtain pure polymer. The exact chemical structure of the polymer was confirmed using Gas Chromatography-Mass Spectrometry. A multi-material structure comprising BRIN-BD11 beta cell line-encapsulated in alginate, bioprinted into wells in a Fused Deposition Modelling (FDM) printed PHA scaffold. The biocompatibility and functionality of the prototype was measured using the insulin immunoassay, the resazurin viability assay and the LIVE/DEAD assay. VEGF containing microspheres were produced using oil-in-water emulsion method and interspersed throughout the polymer before printing. The revascularisation potential of the resulting constructs was measured using the Chorioallantoic Membrane (CAM) assay.
RESULTS: Initial findings showed that, over 7 days, the cells contained within the mcl-PHA/alginate multi-material scaffolds displayed significantly higher proliferation. compared to cells grown on alginate films, whilst displaying similar glucose-dependent insulin secretion (GSIS). Whilst survival was lower in bioprinted cells on day 1, it was not significantly different to the control by day 7. VEGF/PHA microsphere containing mcl-PHA constructs elucidated a significantly larger vascularisation response compared to the control, displaying significantly greater vascular density.DISCUSSION & CONCLUSIONS:
The cell behaviour analysis demonstrated that BRIN-BD11 cell lines can survive and produce insulin within the mcl-PHA and sodium alginate bioprinted multimaterial scaffolds in vitro, whilst the CAM assay demonstrated the ability of a functionalised construct to induce vascularisation ex ovo. Future work will focus on reducing cell death immediately post bio-printing to counteract the poor proliferative capacity of whole islets. In future, whole islets will be used both in vitro and in vivo, as well as encapsulating a variety beneficial factors within the microspheres for a multimodal benefit to any bioprinted cells.
ACKNOWLEDGEMENTS: We thank Prof. Victor Gault and Prof. Nigel Irwin from Ulster University for their advice and the gift of BRIN-BD11. We also thank the University of Sheffield and EPSRC for JH’s scholarship.
1. Atkinson et al., 2014. Type 1 diabetes. The Lancet, 383(9911), pp.69-82. 2. Nafea et al.,2011. Immunoisolating semi-permeable membranes for cell encapsulation: focus on hydrogels. Journal of controlled release, 154(2), pp.110-122. 3. Zhang et al., 2018. Polyhydroxyalkanoates (PHA) for therapeutic applications. Materials Science and Engineering: C, 86, pp.144-150.4. Nicolle e al., Shear mechanical properties of the porcine pancreas: Experiment and analytical modelling. Journal of the mechanical behaviour of biomedical materials, 26, pp.90-97.