Cell-based therapies become more and more prominent for the treatment of intervertebral disc (IVD) injuries. Different strategies are under current development and address the restoration of either annulus fibrosus (AF) or nucleus pulposus (NP). Application of such Advanced Therapy Medicinal Products (ATMPs) is strictly regulated. One requirement is to show the identity of the cells, to make sure the cells are indeed AF or NP cells and retained their IVD cell character during manufacturing process before injection to the site of injury. Therefore, we recently identified novel marker genes that discriminate AF and NP cells on tissue level. However, expression of these AF and NP tissue markers has not been investigated in cultured cells, yet. The aim of this study was to proof the tissue marker”s specificity to discriminate cultured AF and NP cells. Furthermore, we evaluated the tissue markers robustness to different cell culture conditions. AF and NP tissue was obtained from human lumbal IVD of five donors (31–45 years) with mild to moderate degenerative changes (Pfirrmann≤3). Cells were isolated by enzymatic digestion and expanded in culture medium containing 10% human serum and 1% antibiotics. To address specificity, AF and NP cells were cultured separately. To address robustness, 1) cells were cultured up to passage P2, 2) cell culture was performed using two different cell culture media and 3) cells were cryopreserved in an optional intermediate step. Gene expression analysis was performed for 11 novel AF and NP tissue marker: LDB2, ADGRL4, EMCN, ANKRD29, OLFML2A, SPTLC3, DEFB1, DSC3, FAM132B, ARAP2, CDKN2B (patent pending).Introduction
Materials & Methods
The objective of this study was to evaluate the suitability of autologous periosteal cells for spinal fusion in humans. Lumbar spondylodesis has a slow consolidation rate with a consecutive lengthy period of inability to work and the risk of non-union. This study evaluates the applicability of a cell-matrix construct for spinal fusion using clinical and radiological parameters. All experiments were approved by the university ethics committee. Lumbar spondylodesis of the segments L4/5 or L5/S1 was performed in 20 healthy patients (mean age 45 years). Indication for surgery was DDD resistant to conservative treatment. 10 weeks before fusion operation, a piece of periosteum was harvested from the proximal tibia of the patient. The material was chopped and digested. In the washed cell suspension cell number and viability were determined. The viability was greater 90% before seeding. After four passages, the cells were mixed with human fibrinogen, and soaked into polymer fleeces. Polymerization was achieved by adding thrombin. The 3D constructs were cultured for 3 weeks. The final application form were chips of 2mm thickness and 8mm diameter. Spondylodesis was performed using a ventral approach for implantation of 2 titanium cages and a dorsal approach for application of a transpedicular screw-rod system (Medtronic, Sofamor Danek). In 10 patients the chips were implanted ventrally within the cage. The other 10 patients obtained a dorsal intertransverse transplantation of the chips. Pre-operative, 3, 6, 9, and 12 months after surgery a clinical examination was performed, radiographs, and functional scores were obtained. No implant associated side effects were noted. Especially, signs of infection or allergic reaction have not been observed. The harvest sites of all patients presented symptom-free after 3 months. The rate of consolidation was 60% after 6 months, 90% after 9 months, and 100% after 12 months. No clinical or radiological signs for implant failure or malpositioning were observed. 90% of the patients were satisfied with the outcome of the surgery. Cultured autologous periosteal cells are a suitable material for anterior as well as posterior spinal fusion in humans. They may accelerate the rate of fusion and reduce the risk of non-union. Rate and velocity of osseous consolidation need to be compared to that of patients treated with iliac crest autograft. A major advantage might be the lower rate of graft site morbidity.
