Tissue Engineering the Inner Annulus Fibrosus: Mechanical Stimulation of a Simulated Intervertebral Disk Assembly Fabricated by a Layer-by-Layer Approach Using Silk Scaffolds and Bone Marrow Derived Mesenchymal Stem Cell Cell-Sheets

Abstract

Introduction Lower back pain is particularly predominant in 20 to 50 years olds and get progressively more serious in older people. It is linked to the degeneration of the IVD. In more severe IVD degeneration cases, spinal fusion is the standard treatment. However, short comings of this method prompt researchers to continue to look for alternative approaches using cell-tissue based constructs that can function clinically in tissue regeneration and replacement. There have been a number of reports using mechanical stimulus to try to achieve increased IVD cell proliferation, increased mRNA expression, and matrix deposition of the IVD-related proteins. It is hypothesized that stress fibres within the cells detect the mechanical stimulus and adapt accordingly. Most work involving mechanical stimulus uses hydrostatic and hydrodynamic pressure on IVD cells. However, these methods of application of mechanical stimulus do not accurately represent the physiological conditions experienced within the body. The aims of this paper are twofolds namely1 to design a axial compression bioreactor system that simulates the main physiological loading within the IVD, and2 to develop a simulated IVD-like assembly that is capable of transmitting the compressive axial load experienced in the NP into a radial compressive force to the AF using a silicone disk as the NP and BMSC cell-sheets on silk scaffolds to mimic the AF lamellae. Materials and Methods The AF of the simulated IVD-like assembly is made up of three strips of combined silk scaffold with only the center strip having two BMSC cell sheets attached to it, one on each side. The three strips of silk scaffolds would then be wrapped around the silicone NP and a suture used to stitch the loose ends together. A rehabilitative regime was chosen for the stimulation of the simulated IVD-like construct. The rehabilitative regime is based on gradually increasing the forces on the cells. The assembly was compressed at 0.25 Hz, for 15 minutes a day, for 4 weeks. The compression was increased gradually each week starting from 5% axial compression followed by 11% for the second week, 18% for the third and finally, 25% for the duration of the fourth week. Gene expression analysis, histology, and quantification of protein deposition were compared between the mechanically stimulated and a static groups of IVD-like assembly. Results After 4 weeks of dynamic culture in the bioreactor, the mRNA expression levels of Sox9, collagen type I, collagen type II, aggrecan, and decorin were upregulated when compared to the mRNA expression levels of the IVD-like assemblies in 4 weeks of static culture. However, the gene expression of biglycan did not show significant upregulation even after 4 weeks of dynamic culture. At this time point, the stimulated IVD-like assembly was sacrificed for histological analysis. Both Alcian Blue and Safranin-O staining was positive. Immunohistochemical staining analysis concluded that both collagen type I and collagen type II are present within the ECM. However, the staining for collagen type II was stronger than that of collagen type I. The results of the collagen type I and type II quantification indicate a decrease in the amount of collagen type I and an increase in the deposition of collagen type II within the ECM. After 4 weeks of dynamic culture, the decrease in collagen type I and increase in collagen type II found within the ECM was significant when compared against the 4 week static culture group. Conclusion Most studies are done using AF cells with various methods of mechanical stimulation with a wide range of magnitudes, frequencies and durations, thus it would not be feasible to compare the reported results obtained with the one we have presented. Our method of using a cylindrical silicone NP to translate an external axial force to the AF (radial compressive and circumferential tensile) to induce the BMSC cell-sheets to adopt an AF-like morphology and biochemistry is unique. However, our data is in agreement with previous reports that some form of compression or increase in pressure was able to stimulate the cells to produce more ECM to improve the regeneration of the AF. In summary, this study using BMSC cell-sheets as a cell source, fabricated into a simulated IVD-like assembly and mechanically stimulated by a bioreactor in attempt to regenerate the inner AF. We have managed to show conclusively that the cells within the BMSC cell-sheet were able to survive the rehabilitative regime in the bioreactor. Not only did the cells survive, the gene expression and protein deposition results obtained indicate that the ECM found within the simulated IVD-like assembly after dynamic culture was capable of regenerating a tissue structure similar to that found in the native inner AF. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared