Sustained Compression On The Metabolism Of Mitochondrial And Extracellular Matrix Synthesis Of The Rabbit Nucleus Pulposus Cells

Part ⅠIsolation and culture of primary nucleus pulposus cells in intertevertebral disc(一)Isolation and culture of primary notochordal cells in intertevertebral disc Objective:To make the culture model in vitro of notochordal cells in rabbit intervertbral disc with alginate gel and observe the morphological and biological characteristics.Method:Primary notochordal cells were obtained by collagenase digestion and discontinuous density gradient centrifugation, and then embedded in1.2%low viscosity alginate gels. Cell morphology was observed under inverted phase contrast microscope, preliminary identification was analyzed by immunofluorescence staining of collagen type Ⅱ. The methods of "death and live cells" staining and cell counting kit-8(CCK-8) were used to detect the survival and proliferation of notochordal cells in alginate gels, respectively.Results:The primary notochordal cells were isolated successfully, the stable expression of collagen type Ⅱ was observed in it. And it grew well in alginate gels, but the proliferation was very slow.Conclusion:The biological characteristics of notochordal cells in vitro had a preliminary understanding, it would provide certain experimental evidence for the researches on the mechanism of disc degeneration and the seed cells for tissue engineering nucleus pulposus. (二) The isolation and culture of primary chondrocytic cells in intertevertebral disc and the establishement of co-cultureObjective:To investigate the effects of notochordal cells on the proliferation and matrix anabolism of chondrocytic cells in a co-culture system of no direct cellular interaction in vitro.Methods:The nucleus pulposus tissues were obtained from the thoracolumbar spine of 4-week-old Japanese white rabbits under the sterile conditions, and the notochordal cells and chondrocytic cells were isolated by the collagenase digestion and discontinuous density gradient centrifugation. The chondrocytic cells at second generation and the notochord cells were co-cultured in the transwell chamber device with a ratio of1:1, the chondrocytic cells cultured in monolayer as control group. After1,3,7and10days of co-culture, the morphologic feature was observed with inverted phase-contrast microscopy. The phenotype of chondrocytic cells was confirmed by immunofluorescence, CCK-8was used to determine the cell proliferation, and the expression of collagen type Ⅱ and aggrecan was detected by RT-PCR.Results:The notochord cells and chondrocytic cells were successfully isolated. The notochord cells was round or oval in shape and approximately10-15μm in diameter, which had many cytoplasmic vesicles. While the chondrocytic cells were smaller than the former, they appeared short spindle and had a diameter of4-6μm. With the along of co-culture durations, the growth of the chondrocytic cells was accelerated apparently, it was obvious for7,10days(P<0.05); and the expression of collagen type Ⅱ and aggrecan was increased gradually.Conclusion:The notochord cells could promote the proliferation of the chondrocytic cells in a co-culture system and may play an important role in preventing the degeneration of disc. Part ⅡSustained compression on the metabolism of mitochondrial and extracellular matrix synthesis of the rabbit nucleus pulposus cells with monolayer cultureObjective:The purpose of this study was to investigate whether the mitochondrial pathway is involved in compression-induced apoptosis of rabbit NP cells.Methods:The compression apparatus was used to investigate the effect of the compression in this process at one magnitude (1.0MPa) for6,12,18,24and36h. Cell viability was measured by cell counting kit-8(CCK-8). Apoptosis rate was analyzed by flow cytometry and the morphologic changes in apoptosis cells were observed by the phase-contrast microscopy. The apoptosis-related gene and protein synthesis, such as Bax, Bcl-2and Caspase-3, was analyzed by real-time polymerase chain reaction (RT-PCR) and Western-blot, respectively. The protein expression of collagen type Hand aggrecan in extracellular matrix (ECM) was detected by Western-blot in NP cells. Mitochondrial function was evaluated by analyzing the mitochondrial permeability transition pore (MPTP), as well as reactive oxygen species (ROS) and mitochondrial membrane potential (MMP).Results:The results indicated that compression at the magnitude of all time points reduced the vitality of NP cells and the protein expression of collagen type Ⅱ and aggrecan was decreased by it significantly. And it also induced apoptosis of rabbit NP cells in a time-dependent manner. Furthermore, the compression at this level profoundly suppressed the functions of the mitochondria such as the opening of MPTP, the excessive production of ROS and the decreased MMP.Conclusion:Our findings suggest that this compression not only inhibited the synthsis metabolism of ECM, but also induced the apoptosis of NP cells. Mitochondrial pathway possibly involved in the apoptosis of NP cells. And the compression-induced IVD degeneration is mediated, at least in part, via the mitochondrial apoptotic pathway in NP Part ⅢSustained compression on the metabolism of mitochondrial and extracellular matrix synthesis of the rabbit nucleus pulposus cells which cultured in alginateObjective:The purpose of this study was to investigate the effects of sustained compression on mitochondrial function and related gene expression in NP cells.Methods:The rabbit NP cells were isolated and cultured in alginate beads. The NP cells were exposed to static compression at1.0MPa for12,24,36,40and48h. The mitochondrial dehydrogenase activity of NP cells was analyzed by microplate reader. Related gene expression was detected by specific staining and real-time polymerase chain reaction (RT-PCR).Results:This compression in each time points decreased the mRNA expression of extracellular matrix such as aggrecan and induced the apoptosis of NP cells. In addition, the pressure can significantly inhibit mitochondrial function of NP cells, such as the reduced mitochondrial dehydrogenase activity.Conclusion:This static compression profoundly inhibited the extracellular matrix synthesis metabolism and mitochondria dehydrogenase activity and induced the apoptosis of NP cells. These findings suggest that the mitochondrial pathway, at least in part, is involved in this pathological progression which is initiated by compression.