Introduction The limitations of regenerative therapy of the tissue using stem cells are, among others, the length of time until a satisfactory amount of viable cells is obtained for the therapy and the high financial cost. The purpose of this study, was to report a low cost and very effective technique to obtain large amounts of adult mesenchymal stem cells from the degenerated intervertebral disc in a short time. Materials and Methods The methodology of the isolation in this study gave rise to an intellectual property claim deposited at INPI under number BR 10 2013 031330 0. The intervertebral disc in this study was obtained from patients with lumbar disc degenerative disease refractory to conservative treatment, who required surgery as therapy. Isolation consisted of incubating the degenerated intervertebral disc without going through enzymatic or mechanical dissociation in DMEM medium supplemented with 10% SFB and 1% P/S in a humidified oven at 37°C for 3 days. To prove that these cells were adult mesenchymal stem cells, the supernatant cells were collected with a Pasteur pipette and cultured conventionally in culture containers adding growth factors (0.02 ng/µL of EGF and FGF) for another 3 days. For this purpose, cellular adhesion tests, with fibroblastic characteristics, of the presence/absence of specific surface antigens through flow cytometry (CD 105+, CD73+, CD90+, CD45-, CD34-, CD14-, CD11b-, CD19-, HLA-DR-) and of cell-differentiation for adipogenic and osteogenic tissue were performed. Results Using this process, a confluence of ∼100% of adult mesenchymal stem cells was found in the culture containers 3 days after the patient's intervertebral disc was removed. The isolated cells presented percentages of antigens with specific surfaces in flow cytometry that were positive for mesenchymal stem cells, could differentiate into adipogenic and osteogenic tissue, and had an adhesive characteristic with fibroblastic morphology. The greater the degeneration, the smaller was the percentage of growth and isolation of cells, however its differentiation was satisfactory for adipocytes and osteocytes. Conclusion The benefits of the technique described are that it allows: (1) obtaining quickly and (2) a large amount of adult mesenchymal cells, using a methodology (3) with a low financial cost. Disclosure of Interest A. Falavigna: Conflict with CNPq, FAPERGS, AOSpine Latin America M. Peletti-Figueiró: None declared I. Aguiar: None declared M. Roesch-Ely: None declared D. Machado: None declared J. A. Henriques: None declared

Introduction Literature on histological and immunohistochemical protocols that relate degenerative discopathy and its degree of wear is scarce. The purpose of this study was to analyze the structural degeneration of the intervertebral disc of patients with different degrees of degeneration, through histopathology, immunohistochemical, and cellular proliferation and apoptosis studies. Materials and Methods The intervertebral disc was obtained from patients with lumbar disc degenerative disease refractory to conservative treatment who required surgical treatment. The material was sent for histological study with hematoxylin and eosin stain (H&E), Alcian/PAS, Masson trichrome, and safranin O/FCF, and immunohistochemistry through expression of the proteins to mark cell proliferation, Ki-67, and apoptosis, p53. MRI defined the degree of disc degeneration according to Pfirmann classification, and it was correlated with the evaluations of the different histological stains, and the positivity of the immunohistochemical markers. The present study was authorized by the ethics committee under number 0513. Results H&E was effective to evaluate fissures of the fibrous ring, necrosis, and inflammation. Alcian/PAS stain allowed a better visualization of the chondrocyte clusters and of the acid mucopolysaccharide deposits in 86.7% of the samples. Masson trichrome identified the misalignment of collagen fibers in 93.4% of the samples, with greater definition. The safranin technique was considered the most sensitive and efficient stain to study the number and diameter of chondrocyte clusters. Immunohistochemistry showed 73% of positive expression of Ki-67 and p53, which are related, respectively, to the cell proliferation followed by cellular apoptosis. Conclusion The protocol enabled a better histological and molecular characterization of the degenerative disc according to different stages of degenerative disc disease. Disclosure of Interest A. Falavigna: Conflict with CNPq, FAPERGS, and AOSpine Latin America M. Peletti-Figueiró: None declared I. S. Aguiar: None declared M. Roesch-Ely: None declared D. C. Machado: None declared J. A. P. Henriques: None declared O. Righesso: None declared