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Tissue disruption and altered function of the intervertebral disc (IVD) leads to progression of degenerative disc disease. Transplantation of viable cells that can function to maintain the matrix components of the nucleus pulposus (NP) has been shown to be effective in delaying the course of degeneration. Of the various cell types that have been shown by animal models, NP cells activated by coculture with direct cell-to-cell contact with bone marrow-derived stromal cells (BMSCs) have found its way to clinical feasibility study. The major limitation of this method for application to various spinal pathologies is that cells were activated in 7-day period and performed to transplantation at that time. However, if the cells could be cryopreserved after removal, and then thawed and cultured when required by the patient's condition and extent of disc degeneration, the scope of application would be unconstrained by the short culture time and could be expanded to cover a variety of degenerative diseases that could be treated by transplantation of activated NP cells. In 2013, in vitro analysis reported by Tanaka et al showed that there were no clear differences between the noncryopreserved and cryopreserved activated NP cells in terms of cell viability, proliferation capacity, and capacity to synthesize extracellular matrix in vitro. However, it is not known whether cryopreserved cells can function effectively in in vivo. Therefore, the efficacy of activated nucleus pulposus cell transplantation after cryopreservation in canine disc degeneration model was investigated. Materials and Methods Animal experiments were performed under IRB approval. Cartilage dystrophic canine species (10-12-month-old beagles, approximately weight 10 kg, n = 6) was used. First, lateral radiographs and magnetic resonance imaging (MRI) of lumbar spine were taken, and pre-existing vertebral abnormality or disc disease was checked in all animals. Three IVDs (L2/3-L4/5) were assigned in each animal to any of the following three groups (D: degenerated control group, N: noncryopreserved activated NP cell transplanted group, and F: cryopreserved activated NP cell transplanted group). Levels were interchanged between individual animals to minimize level difference effects. L5/6 served as normal control (NC) group with no exposure. Under general anesthesia, first operation was performed through lateral approach to induce disc degeneration and to obtain NP cells from L2/3, 3/4, 4/5 discs. The mean mass of the NP aspirated from each discs was 15.9 mg. At the same time, autologous BMSCs were obtained from the iliac crest. In group F IVDs, NP cells were cryopreserved after enzymatic digestion and BMSCs were cryopreserved. Two weeks after the first operation, NP cells and BMSCs of group F were thawed, and activated by coculture system with direct cell-to-cell contact with BMSCs. In group N, NP cells were separately harvested from L1/2 disc and subsequently activated with BMSCs. In group D, no cells were transplanted after NP aspiration in the first operation. Three-weeks after the first surgery, cell transplantation was performed at the density of 1 × 106cells/100 μΛ in to group N and F discs under fluoroscopic guidance. At 4, 8, and 12 weeks after transplantation, plain radiographs and MRI were taken for evaluation of disc height and signal changes in T2-weighted image. At 12 weeks after transplantation, all beagles were euthanized and discs were harvested for histological analysis. H&E and Safranin O stained sections were studied and extent of disc degeneration was graded based on the system by Nishimura et al. All statistical evaluations among four groups were determined using the one-way ANOVA and Fisher PLSD post hoc test. Statistical significance was accepted at p < 0.05 Results On plain radiograph analysis, DHI decreased rapidly after 3 weeks after surgery in groups D (83.0 ± 3.2%), N (83.3 ± 4.6%), and F (86.8 ± 5.0%) IVDs. The average %DHI of group D was reduced to 69.4 ± 8.9% at the time of 12 weeks after surgery. In group N IVDs, the average %DHI was reduced to 84.2 ± 3.9%, whereas in group F IVDs similar effect of 82.5 ± 1.6% was confirmed. In both groups N and F IVDs, %DHI were maintained significantly compared with group D IVDs (p < 0.01). The average disc grade score using Pfirrmann classification system was 4.0 in group D IVDs, while in group N IVDs the score was 2.5 and in group F IVDs it was 2.5 Changes in MRI signal intensity using the Pfirrmann classification grading system showed that the grades of groups N and F IVDs were significantly lower than group D IVDs at 12 weeks after transplantation (p < 0.01). In histological analysis group N and F IVDs showed well preserved inner annulus structure, whereas group D IVDs showed revised contour of the inner annulus fibrosus. The average disc grade score using Nishimura and Mochida classification system was 4.1 in group D IVDs, while in group N IVDs the score was 1.5 and in group F IVDs it was 1.83 demonstrating that disc degeneration was maintained significantly in both groups N and F IVDs compared with group D IVDs (p < 0.01). Conclusion The results of the present study demonstrate that transplantation of activated NP cells inhibits progressive disc degeneration in canine disc degeneration model. The results of DHI, MRI, and histology showed that there were no significant differences in efficacy between noncryopreserved versus cryopreserved protocols in vivo. These findings suggest that by using cryopreservation, it may be possible to transplant activated NP cells upon request for patients’ needs. Disclosure of Interest None declared

Transplantation of Activated Nucleus Pulposus Cells after Cryopreservation: Efficacy Study in Canine Disc Degeneration Model