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Design and modification of implants to harness endogenous neural precursor cells for brain repair
thesisposted on 16.02.2017, 03:57 by Fon, Yee Kee Deniece
This thesis describes the design and modification of existing biomaterials to assist neural regeneration in the damaged adult brain. A novel aspect of this work is the targeting of implants towards the subventricular zone (SVZ), a stem cell niche within the adult mammalian brain, enabling the use of endogenous neural precursor cells (NPCs) as a source of cell replacement. Implant performance was assessed in terms of the inflammatory response elicited upon implantation, and the ability to re-direct the migration and support the survival of NPCs in and around the implant. The utility of an injectable form of a gelatin-based hydrogel (Gtn-HPA) for implantation into the brain was assessed. An investigation of the in vivo biocompatibility of this material within the caudate putamen of adult rats over 60 days revealed an astrocyte response that was typical of a stab wound injury incurred during implantation. Reactive astrogliosis in the surrounding tissue reached a maximum after 7 days, which coincided with the formation of a glial scar surrounding the implant. Thereafter, reactive astrogliosis became localized at the tissue-implant interface with the consolidation of the glial scar. The gelatin hydrogel degraded relatively slowly, with some material remaining at 60 days. Although Gtn-HPA hydrogels did not exacerbate the astrogliosis typically associated with surgical trauma, the presence of a glial scar can act as a barrier to tissue-implant integration. In order to modulate the astrocyte response, porosity was introduced into the Gtn-HPA hydrogels (pGtn) by the addition of a second polymer component. These hydrogels were implanted towards the SVZ as a means of attracting endogenous neuroblasts towards the implant. In order to promote the survival of neuroblasts that have been re-directed to the implant, glial cell line-derived neurotrophic factor (GDNF) was incorporated into the hydrogel. The presence of porosity and/or GDNF within the hydrogel effectively attenuated reactive astrogliosis, and prevented glial scar formation at both 7 and 21 days post implantation (dpi). Some neuroblasts were diverted away from their natural migratory pathway and were present at the boundary of all Gtn-based implants at 7 dpi, but not in lesion only animals. However, no neuroblasts were present at the implant boundary at 21 dpi, indicating that additional signaling is required to maintain neuroblasts at the implant site. Electrospun PCL scaffolds have already been shown to be biocompatible in the brain and can support infiltration of parenchymal neurites and exogenous neural progenitors. Therefore, the ability of PCL scaffolds with partially aligned nanofibers to support endogenous neuroblasts was investigated. A synthetic, small molecule non-peptide ligand has been reported to mimic the trophic effects of brain-derived neurotrophic factor (BDNF). This BDNF-mimetic was incorporated into electrospun PCL scaffolds (PCL+mB) as a means of enhancing neuroblast migration towards the implant and potentially improving neuroblast survival. Neuroblasts were able to infiltrate both PCL and PCL+mB implants at 8 dpi, but were only present in PCL+mB scaffolds at 21 dpi. In vitro release studies showed that only negligible amounts of the BDNF-mimetic were released from the scaffolds after 14 days, which would indicate that BDNF-mimetic was not directly responsible for the persistence of neuroblasts at 21 dpi. There was a significant reduction in the amount of astrocyte infiltration into PCL+mB scaffolds, suggesting that the maintenance of neuroblasts up to 21 dpi may be mediated by a modulated glial response. The ability of implanted PCL+mB scaffolds to support robust endogenous neuroblast infiltration up to 21 dpi is highly significant for neural regeneration. However, the ability to control the behavior of infiltrated cells will require the presentation of biological cues in a more sophisticated manner, for example, in the form of an immobilized gradient to guide neurite outgrowth and/or cell migration via chemotaxis. The final study was an examination of the ability to co-opt existing inkjet printing technology to deposit biomolecular gradients onto solid-state porous substrates such as electrospun PCL scaffolds. Although commercially available desktop inkjet printers can be modified for printing biomolecules, it suffered from poor process reliability and printed gradients were often highly pixelated. Therefore, a laboratory-scale, piezoelectric-driven, fluid deposition system (Dimatix Materials Printer, DMP) was employed to deposit biomolecular gradients onto electrospun scaffolds. The DMP offered user control over jetting parameters, which allowed bio-inks to be more reliably printed. This system also enabled user-control over the positioning of individual droplets. The generation of a continuous biomolecular gradient also depended on adequate ink-substrate interactions. This interaction was optimized by subjecting scaffolds to air plasma treatment, to increase surface hydrophilicity and improved the ability of printed bio-ink to penetrate and spread into the scaffold, creating more continuous features. The use of increasing number of overprints was used as to further increase feature continuity through increased bio-ink surface coverage, as well as reliably control the amount of biomolecules deposited. The application of these strategies enabled the successful deposition of a continuous gradient of poly-L-lysine onto electrospun PCL scaffolds.