Abstract
Introduction
Back pain, a significant source of morbidity in our society, is directly linked to the pathology of the intervertebral disk (IVD). An IVD is a complex structure that is separated into the following three tissue types: the annulus fibrosus, the nucleus pulposus (NP), and the endplates. Recently, IVD engineering strategies have been increasingly focused on the regeneration or repair of the AF to increase the potential of NP engineering strategies and to mechanically assist NP replacement therapies.1,2 We have developed a novel scaffold-free three-dimensional tissue-engineered construct (TEC) derived from cultured synovial mesenchymal stem cells (MSCs) as a unique approach for cartilage repair.3,4 Previous in vitro studies reveal that growth factors and serum can modify the collagen expression and glycosaminoglycan (GAG) content of chondrogenic-differentiated MSCs.5 Therefore although there is a difference between hyaline cartilage and fibrocartilage, TEC derived from MSCs could have a potential to differentiate into the AF, a fibrocartilage in IVD. This study explores the use of TEC derived from synovial MSCs as a cell-based therapy for IVD regeneration.
Materials and Methods
Synovial MSCs were isolated enzymatically from rat synovial membranes as previously reported.3,4 For development of the TEC, cells were cultured at the density of 4.0 × 105/cm2 in HG-DMEM in the presence of 0.2 mM ascorbic acid 2-phosphate for 2 weeks. The monolayer cultured cell-matrix complex was then detached from cell-substratum interface by addition of shear stress to convert to suspension culture. The cell-matrix complex immediately starts active contraction to form the three dimensional (3D) tissue-engineered construct (TEC, Fig. 1). To explore the use of TEC in degenerative spine, rat caudal IVD were denucleated and treated with TEC (controls were denucleation only). Nine male Sprague-Dawley rats (300 to 400 g) were used, and two most cranial tail disks were denucleated in each rat, giving three disks per group per time point. At 2, 8, and 12 weeks after implantation, the animals were euthanized and disks were evaluated for disk height based on micro-CT analysis, histology including Safranin O staining, and disk grade based on a scoring system.6
Results
At the 2-week time point, there were no significant differences in disk height and disk grade between two groups (3.57 ± 0.28 vs 3.76 ± 0.46 points, p > 0.05) (462 ± 39 vs. 602 ± 49 um, p > 0.05). At the 8-week time point, the TEC-treated group showed significantly better results in disk grade (5 ± 0 vs. 3.14 ± 0.28 points, P<0.05). At the 12-week time point, TEC-treated group showed the higher disk height (234 ± 19 vs 584 ± 45 um, P<0.05) and better disk grade (5 ± 0 vs. 2.86 ± 0.46 points, p < 0.05). Untreated group experienced severe disruption of the AF and end plate, whereas such degenerative changes were alleviated with TEC implantation (Fig. 2).
Conclusion
This study shows that TEC may prevent postnucleotomy disk degeneration in vivo. Larger animals and longer time points will be necessary to further judge potential clinical impact.
Yes
None declared
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