Fabrication of a novel whole tissue-engineered intervertebral disc for intervertebral disc regeneration in the porcine lumbar spine

Abstract

Tissue-engineered intervertebral discs (IVDs) have been proposed as a useful therapeutic strategy for the treatment of intervertebral disc degeneration (IDD). However, most studies have focused on fabrication and assessment of tissue-engineered IVDs in small animal models and the mechanical properties of the scaffolds are far below those of native human IVDs. The aim of this study was to produce a novel tissue-engineered IVD for IDD regeneration in the porcine lumbar spine. Firstly, a novel whole tissue-engineered IVD scaffold was fabricated using chitosan hydrogel to simulate the central nucleus pulposus (NP) structure, surrounded with a poly(butylene succinate-co-terephthalate) (PBST) fiber film for inner annulus fibrosus (IAF). And, a poly(ether ether ketone) (PEEK) ring was used to stimulate the outer annulus fibrosus (OAF). Then, the scaffolds were seeded with IVD cells and the cell-scaffold hybrids were transplanted into the porcine damaged spine and harvested at 4 and 8 weeks. In vitro cell experiments showed that IVD cells distributed and grew well in the scaffolds including porous hydrogel and PBST fibers. After implantation into pigs, radiographic and MRI images indicated that the tissue-engineered IVD construct could preserve the disc height in the case of discectomy as the normal disc height and maintain a large extracellular matrix and water content in the NP. Combined with the histological and gene expression results, it was concluded that the tissue-engineered IVD had similar morphological and histological structure to the natural IVD. Moreover, after implantation for 8 weeks, the tissue-engineered IVD showed a good compressive stress and elastic moduli, approaching those of natural porcine IVD. Therefore, the prepared tissue-engineered IVD construct had similar morphological and biofunctional properties to the native tissue. Also, the tissue-engineered IVD construct with excellent biocompatibility and mechanical properties provides a promising candidate for human IDD regeneration.

Fig. 1. Schematic illustration of IVD structure composed of nucleus pulposus, inner annulus fibrosus, outer annulus fibrosus and cartilage endplates.

Fig. 2. (a) Gross morphology of the tissue-engineered IVD scaffold and SEM images showing the microstructure and morphology of (b) chitosan hydrogel, (c) PBST film and (d) PEEK ring.

Fig. 3. (a) XRD pattern, (b) FTIR spectrum and (c) XPS spectrum of PBST film, and (d) XRD pattern, (e) FTIR spectrum and (f) XPS spectrum of PEEK ring.

Fig. 4. (a) Light microscopy image of IVD cells morphology, (b) Safranin O staining, (c) collagen II immunohistochemistry staining and (d) aggrecan immunofluorescence staining of IVD cells.

Fig. 5. Live/dead staining of IVD cells seeded on (a) the PBST film and (b) chitosan hydrogel for 24 h (live cells (green), dead cells (red)); fluorescence images of cell morphologies attached (c) on the PBST film and (d) chitosan hydrogel for 3 d (cytoskeleton filaments (green), nuclei (blue)); (e) the cell survival rate and (f) proliferation of IVD cells cultured on different scaffolds.

Fig. 6. X-ray radiographic images of porcine disc after tissue-engineered IVD implantation for (a) 4 weeks and (b) 8 weeks; MRI images of porcine disc after tissue-engineered IVD implantation for (c) 4 weeks and (d) 8 weeks (hydration signal (white arrow)); bar chart showing the (e) % DHI and (f) % ST2WI of various disc samples at different time points.

Fig. 7. (a) Gross morphology of the tissue-engineered IVD retrieved from the pig after implantation for 8 weeks; (b) H&E, (c) Safranin-O, (d) collagen II immunohistochemistry and (e) aggrecan immunofluorescence staining of cross sections of the tissue-engineered IVD.

Fig. 8. RT-PCR analysis of gene expressions of (a) collagen II and (b) aggrecan of cells in tissue-engineered IVD. * indicates significant difference between the groups (p < 0.05).