Study On Cyclic Tensile Stress Regulation Osteogenic Differentiation Of Human Intervertebral Disc Cartilage Endplate-derived Stem Cells

Background:Degenerative disc disease(IDD) is the prerequisite and basis for a series of pathological spinal disorders occurred in the major clinical manifestations of neck pain, spinal stenosis, disc herniation, vertebral instability and other illnesses. Studies have shown that disc degeneration caused by neck pain at all accounted for the first two patients to the hospital, second only to respiratory infections infectious diseases. In the process of disc degeneration, intervertebral disc tissue components, disc tissue structure and function have changed, including the gradual loss of the nucleus pulposus proteoglycan composition and moisture disorder structural arrangement of the annulus and endplate cartilage calcification and vascularization, adjacent vertebral osteophyte formation.The intervertebral disc is spinal motion segment in the key structure, and early degenerative changes in the organization. Hypoxia, abnormal force and acidic environment are closely related to degeneration of the intervertebral disc, which stress plays a very important role. A large number of studies show that the continuing role of abnormal mechanical factors is one of the main causes of disc degeneration. However, hidden in the process of specific pathophysiological mechanism is unclear.In the previous study, we found similar between human degenerative mesenchymal stem cell-like cells present in cartilage endplate and successfully separated, named human degenerative cartilage endplate stem cells(cartilage endplate- drived stem cells, CESCs). In recent years, stress and related mechanisms in the process of disc degeneration are widespread attention and study. But the effect of mechanical stress on CESCs differentiation is unclear. This study aims to on the basis of previous studies, based on the use of periodic stress means to CESCs for the study, preliminary study stress on the CESCs osteogenic differentiation.Objectives:Get degenerative disc endplate clinical specimens obtained cartilage endplate digested primary adherent cell culture, amplification, using our previous method, application agarose suspension from the first generation of cell culture systems screened endplate cartilage stem cells. Through the observation of stress on cartilage endplate stem cells differentiation, and to investigate its significance in the process of intervertebral disc degeneration, in order to provide new ideas and theoretical basis for the prevention and treatment of degenerative disc disease.Methods:Get degenerative disc endplate clinical specimens(separation in lumbar fusion surgery), into a clean bench within two hours after the separation of the part of tissue for immunohistochemical staining to detect endplate tissue BMP-2 protein expression in the presence or absence. To obtain the endplate cartilage tissue under a dissecting microscope, using sterile ophthalmic instrument package of cartilage endplate were again separated to remove pollution of the surrounding ligaments organizations, after rinsing phosphate buffer. Wash the tissue is cut into a volume of about 1mm × 1mm × 1mm tissue block size, the minced tissue blocks were transferred into 25cm2 culture flask, approximately 5 times the volume containing 0.15% Ⅱ collagenase serum-free DMEM / F12 medium, allowed to stand in 37 ℃, 5% CO2 incubator for 12 hours. Digestion is completed, with a pore size of endplate tissue cell strainer 70μm filter after digestion, and the filtrate centrifuged at 1000 r / min for 5 minutes. Supernatant, containing 10% fetal bovine serum(FBS), 1% of penicillin and streptomycin in DMEM / F12 culture re suspended cells, is placed at 37 DEG C, 5% CO2 culture box in culture, when the primary culture of cells up to 90% confluence after agarose suspension culture system for further screening CESCs culture. Then filter out cell line induced to differentiate the three-line identification and phenotypic analysis by flow cytometry.Select growing well CESCs, will CESCs with 2 × 105 / mL were seeded on collagen Ⅰ coated 6-well plates BioFlex flexible silicone membrane, placed in 37 ℃, 5% CO2 incubator cultured. After adherent cells were grown to 80-90% confluence, and replaced with 1% FBS volume of DMEM / F12 medium cultured for 12 hours, to make each group were synchronized. DMEM / F12 medium and then replaced with containing 10% FBS, according to pre-set program in the Flexcell-4000 TM system stress load is applied separately 1h, 6h, 12 h, 24 h elongation of 10%, a frequency of 1Hz pull stretch stimulation, while doing static control experiments After the drawing is complete, the cells were collected, applied Western blot to detect the expression of BMP-2, was measured using q PCR part osteogenic gene expression changes in cartilage. The data using SPSS13.0 software to do statistical analysis, P < 0.05 said there were statistically significant differences.Results:1. Endplate cartilage cells obtained were screened by flow cytometry and induce differentiation and identification of the three-line prompt screening out cells with stem cell properties.2. The screened CESCs periodic should force loading, discovery and into the expression of osteogenesis related genes(BMP-2, ALP, Runx2) compared with the control group increased significantly, and the difference was statistically significant. With chondrogenic related gene(SOX9) expression with stretching time, expression is gradually reduced.Conclusions:1. By flow cytometry and three-line induced differentiation experiments confirmed, we screened the resulting cell with stem cell properties, This result is consistent with our previous2. Cyclic tensile stress can promote cartilage endplate Stem cells expression of BMP-2, while raising a bone-related gene expression, prompting the osteogenic differentiation of CESCs.