In articular cartilage tissue, the ECM is composed of a collagen network, essentially nano-scale collagen II fibres organized in different directions, which acts as a natural scaffold to provide mechanical and structural support as well as promote cell attachment and proliferation (Camarero- Espinosa et al., 2016). Nano-fabricated techniques make it possible to create nanofibers which closely mimic the nanofibrous collagen matrices that are found in articular cartilage ECM (Smith and Ma, 2004).
In the field of tissue engineering, the term “nanofiber” is usually used to describe fibers whose diameters are between 1 and 1000 nanometres (Kumbar et al., 2008). These fabricated nanofibers possess the structural and mechanical properties of ECM, which promote the formation of 3D tissue structures (Jayakumar and Nair, 2012). Typically, nanofibres have large surface area per unit volume (Kumbar et al., 2008), which supports cell adhesion and proliferation (Dalby et al., 2002; Glass‐Brudzinski et al., 2008). Nanofibers have been observed to have higher rates of protein absorption than macro-scale surfaces, which are a key mediator in cell attachment to a biomaterial surface (Baharvand, 2014). Furthermore, the nanofibrous constructs have been found to selectively enhance the absorption of specific proteins such as fibronectin and vitronectin, (Woo et al., 2007) which is significant as fibronectin is a protein known to enhance cell adhesion and bind many growth factors.
1.5.1 Production methods
There are different methods to fabricate polymeric nanofibers, fore example; electrospinning, phase separation, drawing, and template synthesis (Barnes et al., 2007). Electrospinning is a highly efficient method of producing nanofiber, and as such, it is used in this project (Dahlin et al., 2006).
31 Phase Separation
Phase separation is a technique that has long been used to create porous polymer membranes and scaffolds (Van de Witte, 1996, Mikos, 2000). To produce a porous nanofiber structure, a polymer is dissolved in a proper solvent and rapidly cooled to induce phase separation. Then, the solvent is later exchanged with water, and the construct is freeze-dried (Van de Witte, 1996). Nanofibers can be obtained by selecting the appropriate gelling temperature. Higher gelling temperatures have been shown to produce microfiber formation while lower gelling temperatures reduce the diameter to nanofiber dimensions (Zhao et al., 2011).
The advantages of this method include the fact that it does not require specialized equipment. In addition, constructs can be produced in a mould to achieve a specific geometry. However, this process can only be carried out with a limited number of polymers and would be difficult to scale- up to a commercial setting (Barnes et al., 2007).
Drawing
In this technique, the fibres are obtained when the polymer droplet on the flat surface comes in contact with a micropipette. The pipette is withdrawn from the surface of the droplet, and a fine fibre is pulled from the bulk (Ondarcuhu and Joachim, 1998). Unfortunately, the fibre formation appears inconsistent because the surface tension at the bulk material surface during drawing increases due to the evaporation of the solvent over time. However, this method is considered to be a time consuming and discontinuous technique though it is simple and requires minimum equipment (Ramakrishna et al., 2005).
Template Synthesis
This technique is generally considered to be simple as it basically involves forcing the polymer solution through the specified dimensions and shape pores, and as a result, fibres having dimensions of the pores of the template are generated. The major drawback of this method is that it is limited to only a few number polymers .i.e conductive polymers like poly (p-phenylene
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vinylene), polyphenylenes and poly (acetylene), and as such, it fabricates on a small scale (Ramakrishna et al., 2005).
Electrospinning
Electrospinning is a time and cost-efficient technique for producing polymer fibres and is the most commonly used method for producing fibre meshes in tissue engineering. It has the capacity of producing long, continuous fibres ranging from 3 nm to 10 μm in diameter (Pham et al., 2006). Moreover, a 3D architecture for cell culture and tissue construction is provided by nanofibrous scaffolds which in turn promote 3D tissue formation (Barnes et al., 2007).
1.5.2 Principles of electrospinning
There are basically three essential components involved in the electrospinning technique; a syringe pump, a high voltage generator and a collector. As shown in Figure 1.7, an electrical field has been generated between the collector and needle on the syringe pump due to the potential difference between them. There are two basic forces that affect the solution drop in the fabrication of electrospun fibres, and these are the surface tension force and the applied electric field. The strength of the electric field causes the solution to drop from the needle in a conical shape manner known as Taylor Cone. If the surface tension of the polymer solution is overcome by the electrical force, the charged droplet forms a jet that arises from the tip of the Taylor Cone. As the jet extends, it is drawn into a thin fibre which undergoes a whipping motion as it travels towards the collector. The jet splits into smaller fibers due to the instability and repulsive forces created within it. During this process, the solvent gradually evaporates into the traveling space between the needle and the collector, which eventually leads to the formation of continuous and thin fibres on the collector (Teo and Ramakrishna, 2006).
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Figure 1.7: The principles of electrospinning (Taken from Zhu et al., 2013).
1.5.3 Electrospinning nanofibers for cartilage tissue engineering
In the field of tissue engineering, electrospinning is considered as a favourable technique for fabricating 3D scaffolds. Electrospun scaffolds are very effective in facilitating cartilage repair in articular cartilage tissue engineering simply because electrospun fibres are very similar in size to collagen fibres in native articular cartilage tissue (Braghirolli et al., 2014). As a result, the influence of nanofibers in cartilage tissue engineering has been investigated by many researchers (Yang et al., 2011).
It has been observed that electrospun scaffolds fabricated from PCL have the ability to proliferate and preserve the phenotypic characteristics of chondrocytes. Moreover, combining of nanofiber scaffolds with growth factors, human mesenchymal stem cells could be effectively differentiated into chondrocyte phenotype (Li et al., 2003).
Sonomoto et al., (2016) demonstrated that PLGA electrospun scaffold induce MSCs derived from healthy donors and patients with OA to differentiate into chondrocytes with chondrogenic markers
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(production of proteoglycan and collagen II). Other study reported that PCL /gelatin scaffolds fabricated using electrospinning processes were biocompatible with articular cartilage. In addition, the scaffold enhanced the chondrogenesis of MSCs and showed evidence of rabbit articular cartilage defect repair, resulting in an enhanced gross appearance cartilage-specific gene expression, suggesting a possible application in the treatment of articular cartilage defects (Liu et al., 2014). The behaviour of chondrocytes was investigated by Wimpenny et al., (2012) with the use of nanofiber composites (poly (L, Dlactide) (PLDLA) nanofibre coatings on PLDLA film). Electrospun nanofibers were found to enhance chondrocyte attachment as well as maintain the rounded phenotypic nature of chondrocytes.
Steele et al., (2014) created a multi-zone cartilage construct by using electrospun polycaprolactone nanofibers. Analysis of the multi-zone scaffolds demonstrated region-specific variations in chondrocyte number, ECM composition, and chondrogenic gene expression.
Mirzaei et al., (2017) have been provided a nanofibrous glucosamine - poly(L-lactide) acid / polyethylene glycol scaffolds which enhanced the biological properties such as cell adhesion, proliferation and protein absorption rate, and induction of chondrogenesis (collagen II and prtoglycan production).