Applications of Nanotechnology in Tissue Engineering and Human Embryonic Stem cells

Nanotechnology in tissue engineering aims to create structures at the atomic and molecular levels with a size range of 10-500 nm [Prabhakaran et al., 2012]. The natural 3D stem cell niche and ECM at the nano-scale level is very dynamic and has a complex mixture of pores, pits and a network of intricate nanofibres composed from various structural proteins including collagen fibrils which all provide fundamental cues at the cellular level that support and regulate various cell functions and activity as a consequence of topographical features [Prabhakaran et al., 2012].

Cells are highly sensitive to the local nanoscale ECM patterns and topography and can probe these features using their filopodia which can strongly encourage the retention of cell shape or induce changes resulting in subsequent differentiation via cytoskeletal arrangement modification [Howard et al., 2008; Prabhakaran et al., 2012; Stevens and George, 2005]. Typically, cells are tens of micrometers in diameter but have components such as cytoskeletal elements and transmembrane proteins that are nano-sized. Furthermore, it has been stated that stem cells have the ability to react with features as small as 5 nm and thus are highly sensitive to nanotopography [Biggs et al., 2010]. Anisotropic topography is also considered important at the nanoscale level in ECM where cells in tissues such as nerve, cardiac and tendon require to be highly organised which directs secreted ECM and tissues structure organisation from nanoscale through to macroscale levels [Lim and Mao, 2009]. With relevance to tissue engineering applications, this architecture provides an important model for the design of artificial synthetic scaffolds which can support, instruct and guide the behaviour of cells [Liao et al., 2008; Stevens and George, 2005].

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Nanotechnology enables to provide artificial templates which are able to mimic the architecture and topographical structure of the native ECM as closely and accurately as possible. This enables the expectation of a cell response and behaviour to be similar to as it would react or perform in vivo, in its natural environment. Scaffolds fabricated with a nanotexture such as nanofibres, whose topography can also be controlled are able to mimic this natural ECM architecture and provide a high surface area to volume ratio with a microporous structure [Vasita and Katti, 2006]. This has known to enhance cell adhesion and biomimetic properties, in turn attract stem cells, support stem cell activities such as proliferation, differentiation and also provide appropriate functioning of tissues (Figure 1.8) [Liao et al., 2008; Prabhakaran et al., 2012]. Various techniques are available to fabricate nanofibres including template synthesis, drawing, self-assembly, phase separation and electrospinning; the pros and cons of these methods are summarised in Table 1.10.

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Figure 1.8 Nano-scale topography and architecture influence on cell attachment abilities. Adapted from Stevens and George., 2005.

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Table 1.10 Available techniques for fabricating nanofibrous substrates [Ramakrishna et al., 2005].

Nanofibre Fabrication Technology

Description of Technique Reference

Self-Assembly Method: Autonomous organisation of individual constituents in an organised structure or pattern without human intervention. This occurs through non-covalent forces such as hydrogen bonding, electrostatic forces, or hydrophobic forces.

Advantages: Extremely fine nanofibres.

Disadvantages: Limited amphiphilic materials, random/very short nanofibres and questionable ability for large-scale production of consistent fibre dimensions.

[Liao et al., 2008; Prabhakaran et al., 2012] Phase Separation

Method: Two different phases of materials are combined and mixed together; after solidification process of this mixture the removal of one phase (solvent) leaves the remaining phase material with pores.

Advantages: Simple set-up polymer adjustable properties which allows a production of microporous substrates only.

Disadvantages: Time consuming, only applicable to a limited number of polymers that produces inconsistent fibre dimensions. Usually used for the production of microfibrous substrates.

[Ma and Zhang, 1999]

Template Synthesis

Method: Template with certain dimensions used through which a material is extruded into another non-interacting material. Advantages: Able to produce fibres with variable length and size from a variety of polymers

Disadvantages: A complicated process that has the inability to produce fibres that are continuous and feasible for small scale production

[Feng et al., 2002]

Drawing Method: Micropipette used to extrude out a fibre from a polymer droplet

Advantages: A simple set-up which allows the production of fine fibres possible

Disadvantages: Can only use viscoelastic materials, which are able to resist shear stresses applied on the material. Inconsistent production of fibres

[Nain et al., 2006]

Electrospinning Method: Use of an electrode which polarises a polymer solution drawn through a needle and deposited onto an oppositely polarised or ground electrode collector.

Advantages: Simple system, versatile, affordable and cost- effective. Long fibres can be fabricated from various materials including natural, synthetic and composite polymers from nano to micro-scale. Precise control of fibre diameter and allows large-scale production of tissue engineering scaffolds on an industrial level.

Disadvantages: fibre production rate, although considered to be faster than other production methods mentioned above. Difficult to control steadiness of the jet causing subsequent changes in fibre morphology

[Prabhakaran

et al., 2012]

[Ramakrishn a et al., 2005]

63 1.7 Electrospinning

Nanofibres have great implications for tissue engineering, in both research and industrial settings. Specifically, electrospinning has attracted great attention by the regenerative medicine field, in both academia and industry due to its unique ability to generate nanofibres from a variety of materials (including synthetic biodegradable and natural polymers). Electrospinning has the ability to form various fibrous structures that are able to provide an excellent supportive, framework for stem cell adhesion, proliferation and differentiation [Prabhakaran et al., 2012; Ramakrishna et al., 2006]. Other benefits of electrospinning include: high production rate of nanofibrous substrates, can be fabricated to fill any anatomical defect shape, a simple set-up and versatile procedure, architecture provides appropriate mechanical properties to support various cell activities, formation of highly porous mesh’, production of fibres from micro to nano-scales and their large surface area to volume ratio (100 m2/g). High surface area to volume ratio provides the ability to enhance protein adsorption activity onto the surface of these nanofibrous substrates causing subsequent enhancement in cell adhesion properties thus providing an availability of recognition sites for cells to attach, spread and expand.