McCrea, Zita (2015) Development of Jetting Techniques for the Delivery of Mesenchymal Stromal Cells for Therapeutic Applications. PhD thesis, National University of Ireland Maynooth.

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Abstract

Mesenchymal stromal cells (MSC) are an important cell source for tissue engineering and regenerative medicine (TERM) and cell therapies. Intravenous injections (I.V.) injections and scaffold implantation are currently the predominant methods of MSC administration. However, both approaches have encountered serious problems for example with I.V. injections, cells can move through the blood stream to any site in vivo provoking problems such as MSC entrapment, and failure to target the injury site. For tissue engineering (TE) applications administration of MSC to a target area include the fabrication of artificial three dimensional (3D) constructs. However, the synthesised environments of these 3D constructs have been associated with numerous limitations including, poor cellular responses, lack of cell infiltration and limited access to essential nutrients and oxygen through the scaffolds. Therefore, the overall aim of the study is to address obstacles associated with MSC administration from either I.V. injections or scaffold transplantation, by using a jetting methodology called bio-electrospraying (BES) to deliver cells directly to an injured region. A major difficulty associated with BES is reproducibility of data between different labs. This is due to the type of apparatus used as electrospray devices tend to be built “in-house”, which adversely affects reproducibility of parameters, including flowrate, potential difference (PD) and voltages. To overcome these problems we used Spraybase®, a commercially available electrospray apparatus. We aimed to establish optimised “gold standard” parameters, for a commercial electrospray instrument that would allow reproducibility between experiments/labs and/or medical environments. By modifying the shape of the ground electrode component, the commercial electrospray was transformed into a fully functional electrospinning device. 3D constructs were fabricated using our own blends of polymer mixtures involving PEO, collagen and agarose. Two distinct sets of electrospun fibres were examined in relation to surface texture, pore and fibre size. These preliminary results may benefit future applications for TE either/or by using our polymer hybrids, electrospinning apparatus and functional parameters. Finally, the BES technique was developed further to make it more clinically applicable. To achieve this, the jetting process was first explored to investigate its affects on mouse bone-derived mesenchymal stromal cells (mBMSC) at a cellular and immunological level. Known characterisations of MSC, i.e. expression of specific surface markers, suppression of T- cell activation, multilineage differentiation, and the pro-reparative properties were analysed for BES BMSC. It was determined that BES BMSC behaved similar to their non-BES counterparts in all biological aspects. The commercial electrospray was then modified to include a specially designed catheter with single needle configuration attached. This catheter fits all types of endoscopes suitable for keyhole surgeries. The only adjustment to the previous established BES parameters was to the flowrate. This was to accommodate the length fluid/cells had to travel through the catheter. We assessed if the optimized parameters were effective with the catheter by BES mBMSC onto 3D collagen-Glycosaminoglycan (CG) scaffolds, and allowing them to differentiate into chondrocytes. The results from this study indicated mBMSC remained fully functional and differentiated as normal after 21days on the scaffolds. To determine if the spray from the catheter was reaching the intended target site, differentiated chondrocytes were BES to a specific area on the scaffolds. Analysis showed chondrocyte staining only at the targeted area. These results also demonstrated huge potential for cartilage regenerative therapies. BES chondrocyte cells to an injured region using arthroscopy with this catheter can eliminate issues with immune rejection caused by scaffolds, or issues with the scaffolds themselves (mentioned above). Delivering specific number of MSC and/or other cell types directly to a specific tissue injury site, using the modified commercially available electrospray apparatus, with optimised parameters including low voltages, has revolutionised MSC delivery for therapeutic applications. This set up can potentially eliminate the complications associated with MSC entrapment, while limiting the need for artificial scaffolds in TE.

Item Type:	Thesis (PhD)
Keywords:	Jetting Techniques; Mesenchymal Stromal Cells; Therapeutic Applications;
Academic Unit:	Faculty of Science and Engineering > Biology
Item ID:	10386
Depositing User:	IR eTheses
Date Deposited:	07 Jan 2019 15:50
URI:
Use Licence:	This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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