Neurite extension in 3 dimensional space engineering platform to study neurite guidance

EURITE EXTE SIO I 3-DIME SIO AL SPACE: E GI EERI G PLATFORM TO STUDY EURITE GUIDA CE CHE WE HUI (B. Eng. Hons.) US A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY US Graduate School for Integrative Sciences and Engineering ATIO AL U IVERSITY OF SI GAPORE i Acknowledgements I would like to thank my supervisor, Associate Professor Tong Yen Wah, for his guidance, support, and confidence in me. I would also like to thank my TAC members, Assistant Professor Evelyn Yim and Associate Professor Li Jun, for their guidance and help in monitoring my progress. This work was funded by the National University of Singapore (NUS) under the grant numbers R279-000-330-592 and R279-000-328-112. I received guidance from Associate Professor Tang Bor Luen (National University of Singapore/Biochemistry), and logistical support from his lab. I was taught by Ms Yanan Chen for the cell cultures and immunofluorescence staining parts. My scholarship is funded by NUS Graduate School for Integrative Sciences and Engineering. I would also like to thank Dr Shirlaine Koh, Dr Xie Wenyuan, Ms Youyun Liang, Ms Wang Honglei, Ms Niranjani Sankarakumar, Mr Yiren Chen, Mr Anjaneyulu Kodali, and Mr Luo Jingnan for their valuable inputs on various parts of this work. ii Table of contents Title page .i Acknowledgements ii Table of contents .iii Summary vii List of Tables .ix List of Figures .ix List of symbols, definitions and terms specific to this study, and abbreviations .xii Chapter 1: Hypotheses, objectives and motivations .1 Chapter 2: Literature review on neural tissue, neural tissue engineering, and the rationale for studying PHBV microspheres .5 2.1 Neural tissue biology 2.2 The need for intervention after neural tissue damage 2.3 Limitations of current therapies in matching the complexity of the neural tissue environment 2.4 Tissue engineering scaffolds as a platform to combine therapies and provide a more comprehensive recovery environment 10 2.5 Delivery of soluble cues by scaffold 11 2.6 Presentation of bound cues by scaffold surfaces 12 2.7 Other cues provided by scaffolds .14 2.8 Forms of scaffold, with focus on microspheres 14 2.9 Biomaterials for fabricating scaffolds, with focus on PHBV .18 iii 2.10 Overall benefits of PHBV microspheres for neural tissue engineering 19 2.11 Neuronal models for assessing PHBV microspheres' suitability for neural tissue engineering 20 2.12 Lack of neurite bridges as a critical problem for microspheres .21 2.13 Neurite extension mechanism and its dependence on substrate adhesion …………………………………………………………… .…22 2.14 Neurite suspension and the possibility of connection between separated microspheres .22 2.15 Hydrogel's use for studying neurite bridging between microspheres .23 2.16 Guiding neurites to bridge microspheres 24 2.17 Analysis methods for bridges .26 2.18 Bottom-up tissue assembly as an added advantage of using microsphere supported by hydrogel for neural tissue engineering .27 Chapter 3: Materials and methods 28 3.1 Materials .29 3.2 Microsphere fabrication .29 3.3 Scanning electron microscope imaging of microspheres .30 3.4 PC12 cell culture 31 3.5 Mouse fetal cortical neuron harvesting and culture .31 3.6 Neurosphere culture and differentiation .32 3.7 Cell seeding 32 3.8 Neuron-microsphere-hydrogel (NMH) assembly 33 3.9 MTT assay 33 iv 3.10 Total DNA quantification (TDQ) .34 3.11 Immunoflourescent Staining 34 3.12 Axon-dendrite segregation analysis .36 3.13 Neurite counting 36 3.14 Statistical analysis 38 Chapter 4: PHBV microspheres as neural tissue engineering scaffold support neuronal cell growth and axon-dendrite polarization .39 4.1 Fabrication of PHBV microspheres .40 4.2 PHBV microspheres support PC12 growth and proliferation 41 4.3 Laminin-coated PHBV microspheres supported high degree neuronal maturation 44 4.4 PHBV microspheres promoted greater axon and dendrite segregation 48 4.5 PHBV microspheres supported differentiation of NPC into neurons .50 4.6. Conclusion on the potential of PHBV microspheres as neural tissue engineering scaffolds .52 Chapter 5: Study of neurite bridging between PHBV microspheres 54 5.1 Various neuronal cell types formed bridges between PHBV microspheres .55 5.2 Optimization of NGF concentration .55 5.3 PC12 on PHBV microspheres in Ln-collagen hydrogel 58 5.4 Demonstration of the lack of bridges between microspheres, and proposal of bridging mechnisms 62 Chapter 6: Characterization of suspended bridging .64 v 6.1 Seeding cells into the hydrogel space did not affect suspended bridging but increased overall bridging 65 6.2 Neurite extension from microsphere surface into gel-space observed for different neuronal types .67 6.3 Increasing amounts of gel-space neurites increased suspended bridging 69 6.4 Decreasing permissiveness of microsphere surface further increased suspended bridging .72 6.5 Permissiveness difference between the microsphere surface and gel-space controls suspended bridging 75 6.6 Conclusion on suspended bridging mechanism .78 Chapter 7: Surface bridging and its relationship with suspended bridging 79 7.1 Increased amounts of continuous surfaces increased surface bridging 80 7.2 Suspended and surface bridges can be promoted simultaneously 83 7.3 Conclusion on the relationship between surface and suspended bridging .87 Chapter 8: Conclusion and future prospects .88 8.1 Meeting objectives and with deeper understanding of PHBV microspheres as neural tissue engineering scaffold .89 8.2 Development of better scaffolds .90 8.3 New perspectives 90 8.4 Future development 92 References 94 Publications 113 vi Summary Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) microspheres, with properties such as injectibility and more efficient drug delivery properties, have important benefits for neural tissue engineering but have not been well studied. Thus, PHBV microspheres’ suitability as neural tissue engineering scaffolds was investigated using PC12 cells, cortical neurons (CN), and neural progenitor cells (NPC) to cover a variety of neuronal types for different applications. Microspheres were fabricated using an emulsion-solvent-evaporation technique. DNA quantification, cell viability assays, and immunofluorescent staining were carried out. PC12 cultures on PHBV microspheres showed growth trends comparable to 2D controls. This was further verified by cell spread staining. Also, CN expressed components of the signalling pathway on PHBV microspheres, and had greater axon-dendrite segregation (4.1 times for axon stains and 2.3 times for dendrite stains) than on coverslips. NPC were also found to differentiate into neurons on the microspheres. These results indicate that PHBV microspheres are suitable scaffolds for neural tissue engineering. However, neurons on microspheres (neuron-microspheres) were isolated compared to well-integrated neural tissue. It was found that neurites bridged the microspheres but these connections were fragile. Thus, neuron-microspheres were encapsulated in a laminin-collagen hydrogel to promote and protect the bridging formations. Bridges were found across the continuous surface between microspheres in contact (surface bridges), and in the gel space between microspheres (suspended bridges). This neuron-microsphere-hydrogel construct increased the proportion of bridge-forming neurites by 31% as compared to neuron-microspheres alone. Furthermore, the neuron- vii microsphere-hydrogel was found to increase the proportion of suspended bridges by 3.5 times. The surface bridges were subsequently verified to form from neurites extending across continuous surfaces, and thus packing microspheres closer to generate more continuous surfaces increased surface bridging by 70%. However, seeding cells into the gel space did not increase the proportion of suspended bridges but this still increased the overall proportion of bridges by 21%. Images of neurites in the gel-space suggested these suspended bridges could have formed instead from neurites extending out of microsphere surfaces, into the gel-space, then onto other microspheres. Neurites in the gel-space resembled a key step here. Varying gel-space permissiveness increased such gel-space neurites by 36% which then increased bridging by 54%, verifying the hypothesis. Reducing microsphere permissiveness further increased bridging by 30%. Permissiveness-difference between the gel-space and microsphere was found to be driving bridging. There were also times more nonbridging gel-space neurites when cells were seeded into the gel-space instead, demonstrating how permissiveness-difference guided neurites away from microspheres. Surface bridging involves neurites on microsphere surfaces while suspended bridging involves neurites in gel-space. This seemingly competing relationship was investigated and it was found that they could be enhanced simultaneously, although the latter can be further increased (46%) by reducing the former. This study showed PHBV microspheres' suitability for neural tissue engineering, improved bridging between microspheres, elucidated bridging mechanisms, further justified bridging study, introduced new perspectives such the need to minimise scaffold permissiveness instead, and uncovered injectible guidance cues in the form of continuous surfaces and permissiveness difference that are different from common preformed ones. viii List of Tables 3.1. List of major settings and hardware specifications used to obtain immunofluorescent images 35 4.1. Summary of the antibodies chosen to stain for each major functional structure of the signalling pathway 44 List of Figures 2.1. Environment in the neural tissue .7 2.2. Summary of biomolecule delivery formulations for nanosphere/ microsphere scaffolds formed from emulsions by solvent evaporation 18 2.3. Mechanism of suspended neurite bridging characterized by Goldner et al 23 3.1. Overview of methods used in this work 28 3.2. Water-in-oil-in-water double emulsion solvent evaporation technique for fabricating PHBV microspheres 30 3.3. Illustration of how an image was processed to get the colocalization percentages 37 4.1. Fabrication and characterization of PHBV microspheres .40 4.2. Comparable growth of neuronal cells on PHBV microsphere compared to coverslips 43 4.3. PHBV microspheres support high degree neuronal maturation 46 4.4. Differentiation and axon-dendrite polarity establishment of primary cortical neurons cultivated on PHBV microspheres 47 4.5. PHBV microspheres increased axon-dendrite segregation .49 4.6. PHBV microspheres supported growth and differentiation of NPC .51 ix 4.7. Summary of the advantages of PHBV microspheres and their capacity to support neuronal cells 53 5.1. Formation of neurite bridges by different neuronal types .56 5.2. Optimization of culture condition for neurite extension .57 5.3. Verification of neurite bridges in the microsphere-hydrogel constructs .59 5.4. Comparison of the proportion of neurite bridges between neuronmicrosphere (NM) and neuron-microsphere-hydrogel (NMH) samples .61 5.5. The proposed mechanisms for neurite bridging in neuron-microsphere-hydrogel .63 6.1. Comparison of the amount of surface and suspended bridges between neuron-microsphere-hydrogel samples with different cell seeding densities .66 6.2. Proposed mechanism for suspended bridging in neuron-microsphere-hydrogel .68 6.3. Increasing laminin concentration increased hydrogel permissiveness and the amount of gel-space neurites 70 6.4. Increasing the amount of gel-space neurites increased suspended bridging .71 6.5. 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PHBV microspheres as neural tissue engineering scaffold support neuronal cell growth and axon-dendrite polarization. Acta Biomater 2011; DOI 10.1016/j.actbio.2011.09.026 Chen W, Tong YW. Mechanisms and promotion of 3D neurite bridging between PHBV microspheres in a microsphere--hydrogel hybrid scaffold. Soft Matter 2011; 7(24): 11372 – 11379. Chen W, Tong YW. Engineering of connected neurons through bridging by neurites in a microsphere-hydrogel scaffold system. (Submitted) 113 [...]... receptors are NCAM and α4β1-integrin that bind to external substrates such as laminin and fibronectin The cytoskeleton, that is also coupled to the receptors, then uses the strong linkage between the receptors and adhesion molecules to pull the growth cone forward Finally, the cytoskeleton behind the growth cones reorganizes into a stable shaft, thus extending the neurite Adhesion of the neurite to substrate... neural tissue engineering, and the lack of studies on PHBV microspheres as will be described in chapter 2.10 Chapter 4 will be dedicated to objective 1 3 Objective 2: To investigate neurite bridging, a critical limitation, of PHBV microspheres • PHBV microspheres with neurons on them will be encapsulated in laminin-collagen to support and study neurite bridging The rationale for choosing laminin-collagen... various shapes Furthermore, guiding neurites to form signalling networks is crucial to generating functional neural tissue, but there is a lack of neurite bridging between microspheres that has not been well investigated This work first verified the suitability of PHBV microspheres as neural tissue engineering scaffolds, then studied 3- dimensional neurite bridging in an engineered neuron-microsphere-hydrogel... unlikely that present therapies focusing on one or some of these factors would be effective Tissue engineering scaffolds could be a key to solving this 2.4 Tissue engineering scaffolds as a platform to combine therapies and provide a more comprehensive recovery environment Tissue engineering aims to restore, maintain or improve tissue function, as well as tackle current challenges such as donor tissue shortage,... for encapsulating biomolecules These techniques include layer-by-layer assembly, enzyme-induced crosslinking, thermo-induced crosslinking, electrospinning, emulsion formation coupled with solvent evaporation, and emulsion formation coupled with crosslinking [28] Besides providing solutions to the challenges, it was found that biomolecules can be incorporated into the scaffolds before, during, or after... solution [ 43] , and dichloromethane from PLGA and PHBV solutions [ 73, 74] 15 Biomolecules, such as insulin [ 43] , basic fibroblast growth factor (bFGF) [49], prostaglandin E2 (PGE2) [ 73] , bone morphogenetic proteins BMP-2 and BMP-7 [74], bovine serum albumin (BSA) and hepatocyte growth factor (HGF) [40], as well as recombinant human bone morphogenetic protein-2 (rhBMP2) [35 ] have been incorporated by adding... factor limiting their use in neural tissue engineering Possible means of improving bridging include stabilising currently reported suspended bridges, and using a hydrogel to provide mechanical support Hypothesis 3: Improved bridging between PHBV microspheres would generate constructs that are more useful for neural tissue engineering • Explanation for this, with literature support, will be presented in. .. from emulsions where the emulsion droplets were formed into microspheres by crosslinking of molecule chains in the droplets, or by removing the solvent to let the droplets solidify Examples of the first method include crosslinking gelatin using glutaraldehyde (GTA) [38 ,49], and dextran using glycidyl methacrylate (GMA) [35 ] Solvent removal methods include removal of chloroform from PHBV and PLGA solutions... the effects of neurite extension cues, and some could in fact switch the effects of guidance cues [16] For neuronal cell types, various biomolecules in the substrate and in soluble forms have been found to affect neurite extension, such as laminin and collagen in the ECM, and soluble factors such as nerve growth factor (NGF) and retinoic acid [9,16,21] These biomolecules are grouped into several families... and restoring functional neural networks, and their being a tissue engineering challenge due to the complex environment required were discussed in chapter 2 .3 [11- 13] PHBV microspheres with neurons on them (neuron-microspheres) are essentially isolated constructs, in contrast to the in vivo neurophysiology of well connected and functioning neural tissue [1,4] described in chapter 2.1 Neurite extension . vi 6.1 Seeding cells into the hydrogel space did not affect suspended bridging but increased overall bridging 65 6.2 Neurite extension from microsphere surface into gel -space observed. non- bridging gel -space neurites when cells were seeded into the gel -space instead, demonstrating how permissiveness-difference guided neurites away from microspheres. Surface bridging involves neurites. bridging in neuron-microsphere-hydrogel 68 6.3. Increasing laminin concentration increased hydrogel permissiveness and the amount of gel -space neurites 70 6.4. Increasing the amount of gel-space