Electrospinning Annulus Fibrosus Mimics: An In vitro Tissue Engineering Approach for Intervertebral Disc Repair

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Abstract

Treatments to alleviate chronic lower back pain, caused by annulus fibrosus (AF) tissue herniation, fail to restore intervertebral disc structure or function. Tissue engineering is a promising approach for the treatment of degenerative AF tissues. The AF lamella is oriented at 30° to adjacent layers and collagen fibres within each lamella are aligned parallel to each other. This cross-aligned fibrous structure is critical for complex mechanical behaviour. The first research objective focused on structural architecture and changes in ageing for AF tissue using porcine intervertebral discs (IVD), as these are structurally and biologically similar to human IVD. This provided a detailed understanding of the tissue's architecture and contributed to the design and development of a biomimetic scaffold. Electrospinning was selected as the optimal technique to closely mimic these highly fibrous lamellar tissues and using aligned electrospun poly(ε- caprolactone) (PCL) scaffold. However, PCL possesses low Young’s modulus, which could consequently result in functional failure of the AF. To overcome this limitation, blends of synthetic polymers – PCL and poly(L-lactic) acid (PLLA) was prepared at variable ratios PCL%:PLLA%; 80:20, 50:50, 20:80) their material and tensile properties characterised. Tensile properties of fibres angled at 0°, 30° and 60° (single layer scaffolds) and ±0°, ±30° and ±60° (bilayer scaffolds) yielded significant differences, and bilayer scaffolds considerably stronger. An overview of the data suggests PCL:PLLA 50:50 fibres were similar to human AF properties and this optimised blended scaffold blend was advanced for further study. The in vitro degradation of the optimised blend was assessed over a 6 month period with scaffolds stored in Phosphate Buffered Saline solution at 37°C. The effect of hydrolytic degradation was measured by characterising the material properties over time. The scaffolds demonstrated a slow rate of degradation and the mechanical properties generally increased over time. In addition, the biocompatibility of the optimised blended scaffold was investigated in vitro using bovine AF cells in terms of cell response. Preliminary studies on single layer showed scaffold to support cell adhesion, guide cell orientation and cellular integration. Further in vitro studies investigated the response of bovine AF cells when seeded on ±30° bilayer 50:50 scaffolds. The results demonstrated electrospun bilayer scaffold continued to guide cell orientation at criss29 cross direction, considerable collage type I production, and significant improvements in tensile properties, which were within the range for human AF lamella. However, electrospun bilayer scaffolds are essentially two-dimensional (2D) surfaces, and creating a complete 3D circular construct to replicate the AF's anatomical structure is yet to be achieved. For the first time, a custom-built Cell Sheet Rolling System (CSRS) was utilised to create a 3D circular lamellae construct that mimics the complex AF tissue and which overcomes this translational limitation. Tube-like structures (6 layers) were successfully created by rolling ±30° bilayer 50:50 scaffolds that had been seeded with bovine AF cells and cultured for three weeks to study and cell response in terms of viability, orientation, matrix deposition nano-mechanical stiffness. The resulted construct composed of six lamellae containing angle-ply aligned cells and type I collagen matrix with a mechanically stable stiffness. To the author's knowledge, this is the first work that demonstrates the application of CSRS technology to successfully fabricate a 3D tissue engineered scaffold for AF tissue. With further development and preclinical testing, this method could lead to patient-specific tissue engineered AF or IVD tissues being produced for clinical application

Keyword(s)

Annulus Fibrosus; Blend Polyester; Cell Sheet Rolling System; Electrospun Fibres

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