Bioprinting as a Novel Tool for Osteochondral Tissue Engineering ... assessed both pre- and post-printing, by trypan blue exclusion and calcein AM/ethidium ...
Bioprinting as a Novel Tool for Osteochondral Tissue Engineering 1,2
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Michael J. Sawkins , Bryan N. Brown , Lawrence J. Bonassar , Felicity R.A.J. Rose , Kevin M. Shakesheff 1
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University of Nottingham, Nottingham, UK, Cornell University, Ithaca, NY, USA
Introduction The field of tissue engineering has typically focused on controlling the temporal release of growth factors and other cell signalling molecules, with little consideration of the spatial presentation pattern. The creation of more complex signalling environments would allow the development of more sophisticated and more effective tissue engineering therapies. Additionally, both normal and abnormal developmental and repair processes could be modelled and 1 studied in vitro with a high degree of precision. Here, the Fab@Home solid freeform fabrication platform is evaluated for its ability to print scaffolds containing zonal distributions of poly(lactic-co-glycolic acid)-based (PLGA-based) microspheres, which can be used for controlled release of growth factors. Structurally, the scaffolds are composed of a second type of PLGA-based microparticle, which is capable of sintering to form porous constructs which are 2 mechanically competent for bone repair . Consideration is also given to the inclusion of an alginate hydrogel phase 3 for cell delivery and patterning . The ultimate aim is to show that bioprinting can deliver ‘all-in-one’ scaffolds for osteochondral repair, which provide appropriate mechanical support, offer spatially and temporally controlled release of multiple proteins and support proliferation, differentiation and extracellular matrix production of multiple cell types. Materials & Methods Thermoresponsive microparticles were produced via melt blending of PLGA with poly(ethylene glycol) (PEG), followed by cryomilling and fractionation by sieving. Microspheres were produced via a W/O/W double emulsion technique from a mixture of PLGA and a PLGA-PEG-PLGA triblock copolymer (TB). The Fab@Home system was used to print scaffolds composed of PLGA-PEG microparticles (with or without PLGA-TB microspheres), calcium sulphate crosslinked alginate hydrogel, or combinations of the two. In some cases, live cells (MC3T3-E1 murine preosteoblasts or bovine articular chondrocytes) were suspended in the alginate prior to crosslinking and printing, and their viabilities assessed both pre- and post-printing, by trypan blue exclusion and calcein AM/ethidium homodimer-1 live/dead staining respectively. PLGA-PEG containing scaffolds were sintered for 24 hours after printing to produce solid scaffolds. Results
Figure 1 – Pre-/post-sinter (left/right) images of 15 x 15 mm scaffolds composed of – (a) PLGA-PEG microparticles and (b) microparticles alongside crosslinked alginate. Post-print viabilities for MC3T3-E1 preosteoblasts and primary articular chondrocytes were found to be 78 ± 9% and 80 ± 2% respectively, both as proportions of pre-print viability.
Discussion & Conclusions Results so far demonstrate that PLGA-PEG microparticles can be printed using the Fab@Home platform and successfully sintered after printing, and also that PLGA-TB microspheres can be incorporated into these scaffolds without disrupting the process. Finally, these microparticulate materials can be printed alongside crosslinked alginate in dual material scaffolds, and the scaffolds sintered with full retention of the alginate phase. Criteria for judging success here include retention of scaffold size and shape, and ability of sintered scaffolds to withstand manual handling and manipulation. Early results have also shown the ability, in principle, to pattern multiple instances of both materials simultaneously. Further work will focus on further mechanical characterisation of printed scaffolds, and on examining the response of a number of (potentially) osteogenic cell types to single and dual material scaffolds which provide zonal growth factor delivery
Acknowledgements Thanks to Omar Qutachi, Helen Cox, Carol Bayles and Jeff Lipton for training and advice. This work was funded by the EPSRC, EMDA, BBSRC, ERC and Cornell University.
References 1. Malone, E. & Lipson, H. Rapid Prototyping Journal 13, 245-255, (2007) 2. Hamilton, L. et al. Journal of Pharmacy & Pharmacology 62, 1498-1499, (2010) 3. Cohen, D. L. et al Tissue Engineering 12, 1325-1335, (2006)
