| Background and ObjectiveFor patients in and out in the department of orthopaedic, the prolapse of lumbar intervertebral disc is one of the most common diseases. The incidence of prolapse of lumbar intervertebral disc is high, which is a chronic recurrent disease. As the the tempo of people’s living is speeding up and aged tendency of population of our nation, the number of patients with prolapse of lumbar intervertebral disc is increasing gradually in recent years. As the high incidence and physical suffering to the patients with this chronic disease, the study about its etiology, pathogenesis, diagnosis, treatment and long-term prognosis are always hot research areas.The intervertebral disc is composed of cartilage endplate, nucleus pulposus and fibrous rings, which is pseudocartilage tissue and has poor blood supply. Furthermore, the intervertebral disc is of great importance of body’s load-bearing and shock absorbers as it’s the link of vertebral bodies and could protect our spinal cord. Among the degeneration theories of fibrous ring intervertebral disk, nucleus pulposus, cartilage endplate, centrum, ligamentum flavum and facet joint, the degeneration of nucleus pulposus cells is thought to be widely accepted as one of the main causes of the prolapse of lumbar inter vertebral disc.The nucleus pulposus is in the right middle of intervertebral disc, and was comprised of fibroblast, chondroid cells and extracellular matrix, which included of amounts of small molecule elastic glycoproteins and water. In the childhood, t he demarcation of fibrous ring intervertebral disk and nucleus pulposus was clear, however along with the growth of the age, the amount of water and elastic glue glycoprotein in nucleus pulposus will decrease and the demarcation become unclear, making the tissue white and hard. This change in the nucleus pulposus then will affect the elasticity and stress levels of intervertebral discs, resulting in increased susceptibility of lumbar disc herniation.The treatment of lumbar disc herniation is multiple, including conservative treatment, operative treatment and histological engineering et al. Among them, the conservative treatment is suitable for patients without obvious symptoms of waist, leg pain and numbness. The therapeutic options contain bed rest, physical traction, massage and nonsteroidal anti-inflammatory drugs such as celecoxib et al. However, operative treatment will be applied if the conservative treatment doesn’t work after the treatment for over three months or even a half year. The operations include the traditional way(the standard open discectomy) and the minimally invasive surgery way, which is emerging in recent years. Both surgeries could remove the degenerated nucleus pulposus tissues and relieve the related clinical symptoms. However the truth is efficiency of the minimally invasive discectomy remains controversial. At the end of last century, the emergence of tissue engineering technology brings light to resolve this clinical disease. The intervertebral disc cell transplantation therapy is a newly developed approach, which was thought to be an effective method to delay the degradation of intervertebral disc. As for the source of seed cells, the autogenous degenerative nucleus pulposus cells were believed to be the most ideal one as it’s without rejection reaction. However, the low number of nucleus pulposus cells and the invasive procedure limit the functionality available of the intervertebral disc cell transplantation therapy. Meanwhile the nucleus pulposus cells form patients with intervertebral disc degenerative disease lack cell vitality. Therefore, to find suitable seed cells become an urgent problem needed to be addressed.MethodThe cartilage endplate tissues and degenerative nucleus pulposus organization were obtained from patients who underwent discectomy and fusion for lumbar degenerative disease. These cells were cultured in the low melting point agarose, helping to select cell clones. And then the cells amplify in vitro and were collected and applied for the experiments of testing the stem cell markers using immunofluorescence and immunohistochemical assays. The third generation of CESCs(cartilage endplate stem cells) and the primary NPCs(degenerative nucleus pulposus cells) were co-cultured in transwell platea. The study was divided tito three group, including CESCs group along, NPCs group along and the co-colture group with both CESCs and NPCs. The q TR-PCR was applied to detected the expression of aggrecan, type II collagen, and Sox-9 of CESCs and NPCs after co-cultured for 0 day, 3 days, 5 days and 7 days respectively. After 7 days of co-culture, the western blotting assay was performed to determine the expression levels of the related protein. Furthermore, the flow cytometer analysis and CCK-8 assay were applied to detect the apoptosis rate and proliferation of NPCs with or without co-culture, respectively.ResultsThe selected cells in agarose in vitro exhibited positive expression of the stem cell markers(The positive rate of stem cell markers were tested by flow cytometry assay, and the results showed the percentage of CD90, CD73 and CD105 were were 98.7%, 97.5% and 98.7%). The q RT-PCR results showed higher expression levels of type II collagen, aggrecan and Sox-9 in NPCs from the co-cultured group than those in the NPCs only group from after 5 days of co-culture(P<0.05). Similarly, compare with the CESCs that without co-culture with the NPCs, the CESCs from the co-cultured for 5 days group showed statistically significant expression of type II collagen, aggrecan and Sox-9(P<0.05). After 7days of co-culture, the western-blotting assay showed the CESCs from the co-cultured with the NPCs group had a significant higher significant increase of the expression levels of proteoglycan, collagen protein Ⅱ and SOX-9. Additionally, the CESCs could increase the proliferation and decrease the apoptosis rate of the NPCs.ConclusionsDuring the co-colture of CESCs and NPCs, the existence of NPCs could assist CESCs to differentiate into NPCs-like cells; secondly, CESCs holds the potential to increase the expression of the NPCs’ s specific molecules. The interaction between the NPCs and CESCs during their co-culture demonstrates that CESCs might be a more suitable seed cell for the treatment of degenerative disc diseases’ cell transplant. |
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