Regeneration of the Intervertebral Disc With Nucleus Pulposus Cell-Seeded Collagen II/Hyaluronan/Chondroitin-6-Sulfate Tri-Copolymer Constructs in a Rabbit Disc Degeneration Model

Abstract

Advancement in tissue engineering provides a promising approach to recover the functionality of the degenerated intervertebral disc. In our study, a nucleus pulposus (NP) cell-seeded collagen II/hyaluronan/chondroitin-6-sulfate (CII/HyA/CS) tri-copolymer construct was implanted into the disc space directly after nucleotomy in a rabbit model. The aim of this study was to investigate whether the NP cell-seeded CII/HyA/CS tri-copolymer constructs could regenerate the degenerated disc in vivo after implantation into the rabbit nucleotomy model. Nucleotomy is one of the most prevalent surgical modalities to treat degenerative disc disease, which could achieve good short-term effects of pain relieve, whereas removal of the entire or partial NP changes the biomechanical characteristics of the remaining disc and the adjacent vertebral segments and a series of long-term complications such as accelerated annulus and the facet joints degeneration may ensue. Therefore, it is necessary to think about possible procedures immediately after the primary nucleotomy surgery to avoid these complications. NP cells isolated from thoracic and lumbar spines of New Zealand White rabbits of approximately 3 weeks of age and 1 kg in weight were labeled with a 5- (and-6) -carboxyflurescein diacetate succinimidyl ester (CFDA-SE) fluorescent dye and seeded within the CII/HyA/CS scaffold by a centrifugation method. After in vitro culture for 1 week, NP cell-seeded CII/HyA/CS tri-copolymer constructs were allografted into the disc defects of recipient rabbit immediately after nucleotomy of the lumbar spine. The Bradner Disc Index and the T2-weighted signal intensity index were determined using lateral plane radiographs and magnetic resonance imaging at 4, 12, and 24 weeks after the operation. Finally, the operated discs were explanted for gross morphological observation, histological evaluation, and cell viability assessment. Animals with only nucleotomy and cell-free CII/HyA/CS scaffold implantation served as controls. In our study, we could demonstrate that the T2-weighted signal intensity index of the operated discs decreased in all three groups 1 month after surgery and the index of the cell-containing scaffold insertion group was significantly higher than that of the other two groups. After 24 weeks, the index of the cell-containing scaffold insertion group increased significantly. However, further decline was observed in both the noninsertion group and the scaffold insertion group. In radiographic analysis, the narrowing of the intervertebral disc space was significantly retarded by the cell-scaffold hybrids implantation up to 24 postoperative weeks. Furthermore, the gross morphology and histological evaluation indicated that the allografted NP cells were viable and showed extracellular matrix production. In our study, we had constructed rabbit NP cell-seeded CII/HyA/CS tri-copolymer implants in vitro. Immediately after nucleotomy of the recipient rabbit, we allografted the precultured cell-scaffold hybrids into the lacuna of the disc. Results documented survival of the allografted NP cells and extracellular matrix deposition, which finally resulted in maintenance of disc height and restoration of T2-weighted signal intensity on magnetic resonance imaging.