Intervertebral Disc Tissue Engineering: An Investigation of Chitosan/PCL Scaffolds using 3D-printing and Melt-electrowriting Technology

Abstract

Intervertebral disc (IVD) degeneration is one of the major causes of lower back pain a highly common health condition that greatly effects a person’s quality of life. Due to an increasing aging population and change in lifestyles, there exists high demand for novel treatment strategies for damaged IVD. Moreover, the current treatment strategies focus on pain alleviation and restoration of mechanical properties, and do not address the underlying biological issues. Researchers have focused on IVD tissue engineering (TE), which aims to restore biological and mechanical functions by regenerating or replacing the damaged discs. To date, there is no adequate tissue-engineered IVD (TE-IVD) available, therefore a more suitable option needs to be explored. The current project aims to develop a TE-IVD utilising a naturally derived polymer, chitosan, in both the annulus fibrosus (AF) and nucleus pulposus (NP) regions of the IVD in order to encourage tissue integration and regeneration. A poly--caprolactone (PCL)/chitosan blend mimicking the AF-lamellae structure was explored using melt-electrowriting (MEW) technology, which combines the advantages of three-dimensional (3D) printing and electrospinning to fabricate micro-fibrous scaffolds with controlled architecture. PCL is commonly used for AF and displays good mechanical properties with a slow degradation rate, but has poor cellular adhesion. Chitosan was chosen to impart bioactivity to the structure. Due to chitosan’s high water absorbing capacity, a chitosan-based hydrogel was also developed to mimic the structural characteristics of the native NP. The MEW parameters were optimised to fabricate the MEW scaffolds in order to mimic the AF structure, and their physical and biological properties characterised. The biological activity of the MEW scaffolds was also investigated in vitro using human bone marrow-derived mesenchymal stem cells within a newly developed cell infiltration test. This cell infiltration test protocol will be useful for future TE research. The developed chitosan hydrogel mimicked the native NP gelatinous structure and demonstrated adequate compressive mechanical properties, degradation behaviour as well as high biocompatibility. Results demonstrated the addition of chitosan increased the cellular activity of the MEW scaffolds in a concentration dependent manner. The chitosan hydrogel fabricated was also found to be suitable for use in the NP region. The NP hydrogel and AF scaffolds showed encouraging tissue regenerative capability by addressing both mechanical and biological issues, and when combined could potentially act as a TE-IVD to replace the native IVD in its entirety. Moreover, the results obtained in this project not only demonstrated the high-potential of these developed TE-IVDs, but also provided validation of the use of the GeSiM 3.1 Bioscaffolder for the development of 3D tissue-engineered constructs.