Clinical Neural Scaffold for Olfactory Ensheathing Cells - Thesis Example

?Chapter 14 Sterilization and Surface Functionalization 14 Sterilization Effect on Fibre Morphology The relative susceptibility to the PLGA and PMMA to the chemical and irradiation steps involved in the sterilization process was studied. The sterilization of fibre mesh mounted membrane insert under three conditions was investigated to determine the effect of each mechanism individually. The effects on fibre morphology were shown in the SEM images captured after the sterilization process (Error: Reference source not found), compared with the same batch of fibre mesh prior to sterilization as a control. The UV sterilization process resulted in the melting of the polymeric fibres, deforming the nanometre features of the electrospun nanofibre morphology. It is known that UV can degrade polymer. Previous studies showed that UV wavelength of 250 nm can cleave poly (methyl methacrylate), a standard polymer to its monomer methyl methacrylate and gases (Srinivasan, Braren, & Casey, 1990). Increasing the duration of the sterilization also increased damage to the polymer structure (Shearer, Ellis, Perera, & Chaudhuri, 2005)Recently, UV has been shown to induce significant degradation of PLGC and P(LLA-CL) nanofibers (Yixiang, Yong, Liao, Chan, & Ramakrishna, 2008). The degradation by UV of electrospun nanofibers can be exploited for the fabrication of 3D nanofibrous scaffolds with micropores. For the ethanol sterilization, the global structure of the fibrous mesh shrank in size, detaching part of the fastened portion of the mesh from the membrane insert Investigation on two different scaffolds showed that ethanol sterilization also resulted in damage to the polymer (Shearer, Ellis, Perera, & Chaudhuri, 2005). Digital image analysis from SEM images, showed that average fibre diameter expanded by 21.1% (EOa= 1.08µm; EOb=0.89 µm) (Error: Reference source not found). Meanwhile, the average fibre diameter after ethylene oxide sterilization remains relatively similar to those prior to sterilization (EtOa= 0.85µm; EtOb=0.82µm). The PMMA membrane inserts were inert to all sterilization processes. Ethylene oxide sterilization proved to be superior compared to the two processes previously described for sterilizing electrospun nanofibre scaffolds. This has been the sterilization process of choice in electrospun PLGA nanofiber scaffold for inducing the continuous differentation of mesenchymal stem cells, chondrogenic and osteogThe PMMA membrane inserts were inert to all sterilization processes. enic lineages (Xin, Hussain, & Mao, 2007). Ethylene oxide sterilization had no effect on the inherent viscosity, glass transition temperature and mechanical properties of 82:18 poly(l-lactic acid)-poly(glycolic acid) used on craniofacial plates. The in vitro hydrolysis rates and mechanical strength loss were not also affected providing evidence that ethylene oxide sterilization cycle agrees well with copolymer plates and thus should not affect clinical performance (Pietrzak, 2010). 14.2 Cellular Response on Scaffold Functionalization Scaffolds to encourage cell growth and differentiation must mimic not only the biological but also the physical properties of the extracellular matrix. Functionalization of the engineered scaffolds aims to improve surface properties towards cell differentiation and proliferation. The high fidelity of the submicron scale features and dimension in the PLGA electrospun fibres mesh after coating and air-drying was verified through SEM (Error: Reference source not foundC, E, G, I). The effect of nanofibres functionalization was then studied by analysing the cellular responses on the nanofibres.After four days in culture, the responses of seeded purified OEC on the four types of functionalization were investigated using immunofluorescence microscopy. All four coatings (collagen, laminin, pLL and pDL) promoted cell attachment, when compared to the non-functionalized PLGA electrospun nanofibres mesh (Error: Reference source not foundD, F, H, J vs. Error: Reference source not foundA). All four nanotopographical functionalization maintained the native morphology of OECs as seen in the chamber slide (Error: Reference source not foundB). The fingerprint of contact guidance response was evident in the newly adopted bipolar shape by OEC in all the scaffolds, compared to the extended cell area projection in the chamber slides. However, the degree and extent of cell attachment differed significantly among the random nanofibrous mesh coated with four biomolecules, compared with those in the routine-coated chamber slides. This extent of cell attachment was highly dependent on cell density. It was observed that purified OECs cultured on the collagen and laminin-coated nanotopographic scaffold exhibited an extended bipolar projection, (shown in Error: Reference source not foundH and J), at cell density as low as 27,000 cells/cm2­. Laminin, a glycoprotein which serves as a binding agent between cell walls, has been shown to influence cell alignment of rat neural progenitor cells that were cultured on polystyrene substrates (Recknor, Sakaguchi, & Mallapragada, 2006). Differenct ECM proteins (collagen I, collagen IV, laminin, and fibronectin) influence the differentiation of human mesenchymal stem cells via a significant interplay between the ECM proteins and the elastic characteristic of the substrate used in cell culture (Rowlands, George, & Cooper-White, 2008). In contrast, pLL and pDL-coated scaffolds (Error: Reference source not foundD and F) required a higher cell density at 81,000 cells/cm2­ to have the similar cell projection profile seen in collagen and laminin. This shows that polymer coatings have to be modified to more closely mimic natural ECMs for increased cell survival. Chapter 15 Biocompatibility of OEC on Nanotopographical surface 15.1 Differential Response of Purified OEC and Fbl from WM and OB to PLGA Electrospun Fibres 158  The manipulation of the nanotopography can regulate the secretion of FN by OECs and fibroblasts. Although the contribution of FN and fibroblasts to the formation of a glial scar and the effectiveness of transplants is still under discussion (Ramer et al., 2004; Yamamoto et al., 2009), the nanotopography provides an accessible route to control this factor. Factoring differences in cell density and surface functionalization had on the morphology of same population of cells, in-depth qualitative analysis of the immunofluorescence images were performed to understand the effect of substrate nanotopography on the two main cell populations from olfactory sources: OEC and Fbl. Purified OEC and Fbl were obtained by antigenically fluorescent sorting from primary cell cultures established from two sources WM and OB that are generally used in experimental spinal cord injury repair (Error: Reference source not found). Error: Reference source not found summarizes the cellular response to be cell attachment, followed by cell elongation of each cell population from each source when confronted with submicron topography in the PLGA electrospun fibre scaffold, compared to cells seeded on flat surface in chamber slides. The coupling responses from cell density and surface functionalization were categorized in hierarchal fashion to elucidate that the alteration in cellular functions were the effect from substrate nanotopography, not due to the previously identified differences. The roughness and surface of biomaterials for biomimetic ECM have been shown to be crucial in stem cell survival and host tissue acceptance (Ravichandran, Liao, Ng, Chan, Raghunath, & Ramakrishna, 2009). The ability for OEC to attach and subsequently elongate into bipolar cells was improved when the OECs were seeded on 600-800 nm randomly non-woven fibres scaffold, at low density (81,000cells/cm2), purified OEC adhered and also adopted a much elongated shape, mimicking the bipolar morphology seen in the routine culture prior to cell sorting (unsorted) (Figure 2.5.XX). Evidence of filopodia at only two ends of OEC was verified by SEM micrographs (Error: Reference source not foundE and F). Furthermore the cells responded to the linear topography by sharply altering cellular behaviour, specifically the secretion pattern of fibronectin, as observed in WM and OB. Purified WM and OB had been shown to secrete fibronectin.(See Section 2.3). In WM, the secretion of fibronectin was continual, and colocalization was observed in both the chamber slides and scaffold (as illustrated in SEM micrograph, Error: Reference source not foundE). Meanwhile in OB, although purified OEC ceased from secreting fibronectin, the OEC continued to adhere and elongate (Error: Reference source not foundF). These results showed that substrate submicron topography had an effect on purified OEC at two levels of order: first order on the morphological response on contact guidance, and the second order on the differential fibronectin secretion pattern, which is a targeted cellular function. For purified Fbl, even though the ability to adhere was maintained, both WM and OB showed a sharp decrease in fibronectin secretion when cultured with the submicron topographical substrate. The SEM of the purified populations further illustrated an interesting phenomenon of the fibronectin secretion pattern between WM and OB. The interstitial space between the fibres seeded with purified Fbl from WM was cobbled with nets of extracellular matrix while only Fbl in OB appeared on the scaffold (Error: Reference source not found). These results showed the differential response of Fbl to the scaffold compared to OECs, where purified OECs were encouraged to adhere and elongate while purified Fbl seem to have a reduced ability to maintain its role as supportive fibroblasts. 15.2 Effect of Nanotopography on Purifed OEC from OB Feature geometry and dimensions of nanotopographic PLGA electrospun scaffold were verified by SEM (Error: Reference source not foundA and B). 200nm-400nm fibres were chosen to promote optimal contact guidance effects by reducing the features geometry using the nanocomposite electrospinning techniques and increasing the occurrence of cell elongation and alignment through the submicron features. Purified OEC from OB were seeded on the randomly oriented nanocomposite scaffold. Other than enhanced attachment and elongation, nanotopographic PLGA electrospun fibres provided further cell alignment, and regulated anisotropic growth among the purified population despite the randomness of the fibre mesh, (Error: Reference source not foundC and D). SEM image mortgage further illustrated the unidirectional growth of the purified OEC independent of the direction of the fibre with aid of filopodia (Error: Reference source not foundE and inset). It is also noticeable that cells remain proximally close at the location of seeding despite extending the culture period to seven days. These results showed that anisotropic growth, though static, was one of the second order effects of nanotopography in contact guidance. Chapter 16 Discussion and Conclusions 16.1 Suitability of PLGA Material for Clinical Use Poly (lactic-co-glycolic) acid is often known as the entrepreneurial polymeric biomaterial due to the cell phenotype and behaviour effect it shepherds selectively (Hutmacher, 2000). Its ability of controllable degradation and resorbable rates makes it a robust biomaterial for tissue engineering, especially for therapies that require no secondary surgery. The trend of the kinetic fibrous mass degradation plots are consistent with previous reports on bulk, which have elucidated the nature of PLGA biodegradation rate is dependent on the stoichiometric ratio of lactic acid and glycolic acid (Jain, 2000; Chung et al., 2006) and the molecular weight of PLGA (Park, 1994; Waeckerle-Men and Groettrup, 2005; Huang et al., 2006). In this proteolytic degradation, the mechanism of hydrolysis has been identified as random ester cleavage (in basic media) or chain-end cleavage (“unzipping”)in acidic media (Schliecker et al., 2003). The ideal PLGA for the clinical relevant spinal cord repair application is one that 1) possesses the temporal duration of approximately three weeks to allow culturing (four days), ligation (one week) and could serve as the conduit for axon regeneration thereafter1; and 2) can be processed to promote the anastomosis seen in the typical fibrin adhesive used in nerve stump ligation in the first intended spinal injury brachial plexus avulsion (Bertelli and Ghizoni, 2004); and 3) relatively inexpensive compared to the fibrin glue. The 3-week critical point of degradation PLGA 50:50 is rationalized to be sufficient under the assumption there would be higher protease activity in the cerebrospinal fluid of damaged spinal cord that undergo ischemia (Crowe et al., 1997) than a healthy one (Brylev, 2009). Such temporal degradation property is complemented with the inherent viscosity of < 1 dlg-1 and high molecular weight in PLGA 50:50 PURAC. The extended entanglement property due to the high molecular weight improves the spinnability of the polymer at a relative lower concentration, making PLGA 50:50 PURAC an attractive candidate for electrospinning to anastomose the fibrous and nerve network (Huang et al., 2003) at a relatively low cost. Therefore, PLGA 50:50 from PURAC of Holland was chosen for conducting further studies. 16.2 Nanocomposite Electrospinning (need to write discussion for Electrospinning) (below is discussion for nanocomposite electrospinning) Embedded in the PLGA fibres, the photoluminescence of various size quantum dots were evident once excited with UV light. In addition, it also enhanced the spinnability of polymer PLGA/HFIP by significantly reducing the fibre diameter. The high photoluminescence observed from the fibre is a good indication that concentration of neither PLGA nor HFIP (high volatility solvent) affects the optoelectronic property of the quantum dots, which have been previously reported have been addressed of the significant of blending polymer concentration when incorporating quantum dots (Li et al., 2007). In addition to its high signal-to-noise ratio and stability against photobleaching (Chan et al., 2002), taking into account the enhanced characteristic to the PLGA fibres, these “free-standing” nanocrystals made itself an attractive candidate as an internal monitoring device in vitro and in vivo. However such application would still need to overcome the cytotoxicity issue as recent toxicity assessments of quantum dots (Hauck et al., 2010; Fischer and Chan, 2007; Wu et al., 2010) that revealed diverse results on the response from both in vitro and in vivo system. 16.3 Development of a Clinical Relevant Neural Scaffold An effective way to reinforce the micro and nanostructure provided by the electrospun fibre mesh, while maintaining the scaffold tensile in a cell culture system is a complementary groove-ridge fit membrane insert system developed in this experiment. The ease to operate the scaffold reinforcement in membrane insert system design has allowed any form of mesh to fasten by the insert in short time, without much distortion to the inherent mechanical property of the mesh, such as mesh tensile and fibre morphology. This arrangement was important for limited cell seeding and subsequently entorhino-hippocampal slice culture. The design and structure development strategy using laser ablation in this study allows rapid production of high resolution PLGA nerve repair structure without the compromise on the scaffold intrinsic nanotopography. The laser configuration and mask-polymer arrangement employed in this strategy are compatible with the need of scaffold production and chemical and physical properties of the polymer used to fabricate the scaffold. Although the edges definition was not as clean cut, the success rate could be increased by using a more uniformed spectrum in laser radiation and by employing smaller optical demagnification to improve the projection resolution. 16.4 Biocompatibility of the To promote cell attachment and elongation, it is desirable that to maintain a scaffold with well-defined structure geometries that would provide structural cues to the elongating filopodia in the cells. Therefore this method of producing a micro scale patterned is possible with high level of ease and scalability. One obvious experimental limitation encountered was the manual delamination. A sacrificial release layer between the PLGA material and the silicon mould would have resulted in a cleaner mould release process, to prevent any compromise in the structural geometries and dimensions (Bettinger et al., 2006). Selection of mould release agent would favor properties such as waters soluble, nonreactive towards both mould substrates and dissolution under mild aqueous conditions. One example of such agent would be 90% sucrose and this solution is easily spin-coated on the microstructured silicon mould. (Lee et al., 2005; Bettinger et al., 2006) The cell adhesion on the PLGA biomaterial had elucidated the viability of this material as a cytoscaffold material for olfactory ensheathing cells and its companion fibroblasts. The topographical cues provided from the micro patterns on the mould were evidently influencing the growth pattern of the seeded cells. Although detailed difference among the various width of grooves was not able to reveal the exact dimension suited for homing OEC, it also eluded that further microscale variations may not be crucial once the minimal threshold of spatial conditions had been met. Control over the cell elongation and alignment of purified population from olfactory system have been pivotal in developing a therapeutic OEC scaffold that are both surgeon-friendly and compatible with the requirements of GMP. The observed cell response of purified OEC and Fbl to the linear substrate micro and nanotopography in this study presents OEC cytoscaffold as a clinical relevant neural scaffold with in vitro cellular tuneable and adaptive capabilities. The preservation of the nanofibrous morphology in PLGA electrospun fibre scaffold and the PMMA membrane insert using ethylene oxide sterilization present this neural scaffolding system as a suitable and attractive neural scaffold en route to clinical application. Furthermore this also potentiates the process as GMP grade procedures for other therapeutic tissue engineered scaffolding substrate. Synthetically motivating the adherence of OEC, the various biomolecules surface functionalization allow the PLGA electrospun scaffold to be biocompatible, as well characterized in other studies on OEC and other neural derivatives. Furthermore the density-dependant surface functionalization provides versatility in the much desirable control of utility of the scaffold based on the amount of purified cells successfully obtained from the primary cultures from either animals or patients and cell sorting process. In identifying the tunable cellular features, precision in nanotopography using nanocomposite electrospinning serves as a tool to modulate the effect of morphological contact guidance. The first order effect of substrate topography is morphological response to contact guidance, while the second order effect includes other cellular response, such cell elongation, directional growth and changes in cellular function (Dalby et al., 2003). Anisotropic stress generated from nanotopography is generally considered as one of the mechanism of morphological response (Bettinger et al., 2006). The manipulation of the nanotopography can be used to regulate the secretion of FN by both OECs and fibroblasts.  Although the contribution of FN and fibroblasts to the formation of a glial scar and the effectiveness of transplants is still under discussion (Ramer et al., 2004; Yamamoto et al., 2009), the nanotopography provides an accessible route to control this factor. The inclusion of cell sorting also serves to robustly select for the cell types and their optimal quantity/proportion needed for successful regeneration in repairing spinal cord injury. Although surface roughness provided by the nanocomposite non-woven electrospun fibres have encouraged favorable cell functions and unidirectional growth, static growth of purified OEC suggest that the roles of filopodia observed were not used for cell migration, as documented by other studies. Further work, such as comparison with the aligned electrospun fibres can reveal the role of filopodia in migratory mechanism adopted OEC to move along and eventually exit the scaffold, a crucial, currently still unattainable property of any neural scaffold. Read More