The coupled effects of microfibrillar-scaffold alignment and growth factor stimulation on MSC differentiation were investigated. Aligned microfibrillar scaffolds supported either ligamentogenesis, chondrogenesis or fibrochondrogenesis of MSCs acting when appropriately
stimulated with CTGF and/or TGFβ3. Thus, these results have broad implications for regenerating other musculoskeletal interfaces such as articular cartilage-bone, tendon-bone, and meniscus-bone. This study opens the possibility of using aligned microfibrillar scaffolds that are spatially functionalized with specific growth factors to direct MSC differentiation for engineering the bone-ligament interface. Future work will investigate the spatial incorporation of the growth factors along the length of the scaffold to compartmentalize MSC differentiation.
Electrospinning of human scale
microfibrillar scaffolds for ligament
tissue engineering
4.1 Introduction
Scaffolds produced by electrospinning of biodegradable fibres are particularly attractive for tissue engineering (TE) strategies as they provide high surface area and tailorable fibre diameter and alignment (Yang et al., 2005; Pham, Sharma and Mikos, 2006a; Li et al., 2007). However, developing porous electrospun scaffolds mimicking the size, stiffness and strength of human tissues remains a challenge, limiting their use for the TE of high load bearing tissues such as ligament and tendon. A well-documented limitation of electrospun fibre-scaffolds is limited cell infiltration to the body of the implant (Baker et al., 2008; Simonet et al., 2014). Large in-plane pores that facilitate cell migration through the body of the scaffold are reduced by the layer-by-layer deposition of densely packed fibres during electrospinning. The overall porosity of implants produced using sheets of electrospun fibres can be further reduced when they are tightly rolled (Pauly et al., 2017), stacked (multi-layered scaffolds) (Nerurkar et al., 2009; Chainani et al., 2013; Fisher et al., 2015), or braided (Rothrauff et al., 2017) to form 3D
scaffolds. Thus, cellular infiltration into these dense structures is slow, often taking between 6 and 10 weeks, with extracellular matrix (ECM) deposition restricted to the outer periphery in vitro (Baker and Mauck, 2007) and in vivo (Telemeco et al., 2005).
Strategies to enhance cell infiltration and the diffusion of nutrients and waste products include the use of bioreactors (Lowery, Datta and Rutledge, 2010) or electrospraying cells during scaffold fabrication (Stankus et al., 2006). Other strategies have aimed to enhance cell infiltration by increasing the scaffold pore size. Pore size (or interfibrillar space) is determined and constrained by the selected fibre diameter and the packing density (Eichhorn and Sampson, 2005; Simonet et al., 2014). Larger pore sizes are obtained with larger fibre diameters and low packing density (Soliman et al., 2011). Strategies include the use of micron-
size fibres in favour of nanofibers (Pham, Sharma and Mikos, 2006b), cutting holes into the implant (Sundararaghavan, Metter and Burdick, 2010; Lee et al., 2012), or introducing a sacrificial component, such as soluble fibres (Baker et al., 2008, 2012) and porogens (Kim, Chung and Park, 2008). Methods such as the use of sacrificial components or cutting holes alter the fibre morphology, lead to heterogenous fibre distributions in the scaffold, macroscopic delamination and deformation of the scaffold, while the pore size is still linked to the fibre diameter.
Realizing these limitations, others have decoupled the pore size from fibre diameter. Low-temperature electrospinning (LTE; below -30 °C) increases the interfibre distance by embedding ice particles as void spacers during fibre deposition (Simonet et al., 2007; Kim et al., 2014). The electrospinning conditions used in LTE also enhance the evaporation of the solvent (Kim et al., 2014). When interfibre fusions fail to form during electro-spinning, larger voids/pores are formed in the scaffold (Kim et al., 2014). Although this approach was found to enhance cell infiltration, cells compact LTE scaffolds to almost half its size after only 7 days in culture (Simonet et al., 2014). Others report concavities on the surface of the fibres formed due to contact with water droplets at the time of electrospinning (Kim et al., 2014). Another way to increase pore size independently of fibre diameter is the focused, low density, uncompressed nanofiber (FLUF) method. A disadvantage of FLUF is that its specific target limits the method to produce cotton ball shaped scaffolds. Other researchers have substituted the metal collector for a liquid medium to deposit electrospun fibres and then draw them to a rotating mandrel. In this way, controlled woven scaffolds composed of aligned fibres can be obtained and the thickness of these scaffolds can be controlled by varying the electrospinning time (Shang et al., 2010).
The objective of this chapter was to develop a porous microfibrillar scaffold for ligament tissue engineering with comparable size to the human anterior cruciate ligament (ACL). Poly(ɛ-
caprolactone) (PCL), a degradable polymer with FDA approval for certain biomedical and drug- delivery devices (Woodruff and Hutmacher, 2010), was used to produce electrospun scaffolds. PCL-based scaffolds have several favourable features, such as a hydrophobic surface for protein adsorption, functional groups upon simple chemical modifications, etc. During electrospinning of polymers such as PCL, there is a fraction of fibres that fuse with juxtaposed fibres (Garg and Bowlin, 2011). These welds along the length of the fibres attach fibres to one another and restrain their movement, thereby forming a membrane-like fibre-sheet. In this study, it was demonstrated that increasing the fraction of unfused fibres (i.e. less welds in juxtaposed fibres) during electrospinning reduced the flexural rigidity of the resultant
electrospun sheets, which in turn allowed the bundling of fibres into 3D scaffolds without the need for stacking or rolling. Furthermore, these unfused fibres allowed for higher interfibrillar spacing, which facilitated the rapid migration of mesenchymal stem cells (MSCs) into the body of the scaffolds. This strategy was achieved without compromising fibre diameter, alignment or mechanical integrity of the scaffolds.