| Background：Osteoarthritis, which occurs mainly in knee that under heavy weight-bearing, is acommon chronic disease of joints. It was caused by multiple mechanical and biologicalfactors and the specific etiology and pathogenesis of osteoarthritis are still unclear. Theknee joint is the most complex and largest joint of the body, which plays a very importantrole in lower limbs movement and load supporting. Compared with other cells of humanbody, articular cartilage has a unique structure and mechanical property. As an importantpart of the joints, the metabolism and function of chondrocytes are regulated by thepressure environment of intra-articular. Many animal models of osteoarthritis haveconfirmed the progression of osteoarthritic disease was closely related to pressure,especially the impact of hydrostatic pressure on metabolism of chondrocytes. Cytokinesand proteins that are highly relevant to the occurrence and progression of osteoarthritissuch as type Ⅱ collagen, IL-1β, TNF-α and MMP-13would have great impact on thestudy of the correlation between pressure and pathogenesis of osteoarthritis, thus we can demonstrate the link between pressure and osteoarthritis.Objective：This study aims to establish an effective human chondrocytes pressure model tosimulate the pressure environment inside human weight-bearing joints so as to lay aexperimental basis for the research of pressure and chondrocytes furtherly next steps.We intend to investigate the relationship between high-intensitive pressure anddegeneration of articular cartilage and osteoarthritis through the high-intensitivehydrostatic pressure experiments.Methods：1. The normal and osteoarthritic articular tissue of human knee joints obtained fromclinical operations, and get primary normal and osteoarthritic by two steps enzymaticdigestion. Chondrocytes were subcultured and observed; Toluidine blue staining andimmunohistochemical staining of type II collagen were employed to identify thechondrocytes; Human chondrocytes pressure experimental model was establish based onthe multifunctional thermostatic high-intensitive hydrostatic pressure loading device.2. The chondrocytes of experimental groups were applied0,3.0,7.0, and10.0MPahydrostatic pressure for2h per day with the multifunctional thermostatic high-intensitivehydrostatic pressure loading device,.5days later, CCK-8, MTT assay and flow cytometrywere employed to detect apoptosis and proliferation of chondrocytes. The humanchondrocytes pressuremodel was assessed base on the experimental data.3.The normal and osteoarthritic chondrocytes were respectively applied10.0MPahydrostatic pressure for2h per day with the multifunctional thermostatic high-intensitivehydrostatic pressure loading device. After5days treatment, the expression of Type Ⅱcollagen, IL-1β, TNF-α and MMP-13in chondrocytes of each groups were detected byqRT-PCR and Western-Blot.Results：1. Energetic normal and morbid osteoarthritic chondrocytes were obtained from primary culture, which is more in line with the biological characteristics of the human.The multifunctional thermostatic high-intensitive hydrostatic pressure loading device caneffectively simulate the hydrostatic pressure loads in physiological conditions withinhuman knee joints.2. The results of CCK-8, MTT assay and flow cytometry show that: compared with thecontrol group,3.0MPa pressure promoted proliferation of chondrocytes and inhibitedapoptosis; The proliferation activity of7.0MPa group chondrocytes was decreased, andthe apoptosis was increased; The10.0MPa group showed significant trend in inhibition ofproliferation and promotion of cell apoptosis. The pressure experimental model is stable,reliable and repeatable and it can effectively simulate the pressure environments withinhuman joints through experimental evaluation.3. qRT-PCR and Western-Blot analysis showed that: after the treatment ofhigh-intensitive hydrostatic pressure, the normal and osteoarthritic chondrocytes bothshowed the inhibition of expression of Type Ⅱ collagen, and the promotion ofexpression of IL-1β, TNF-α and MMP-13. In addition, compare with normal chondrocytesafter the treatment, osteoarthritic chondrocytes showed that the expression of Type Ⅱcollagen was reduced, the expression of MMP-13and IL-1β were increased, and theexpression of TNF-α has no significant differences.Conclusion：1. In this study, we obtain cartilage tissue of human knee joints from clinicaloperations. Through the primary culture, we got normal energetic chondrocytes andmorbid osteoarthritic chondrocytes, which is more in line with the biologicalcharacteristics, and the experimental results are reliable. The pressure model of humanchondrocytes that we established can simulate the pressure environment more effectivelywithin human load-bearing joints and has more advantages than animal models.2. We preliminarily determined the pressure gradients in pressure experiments withhuman chondrocytes and confirmed the different intensities of hydrostatic pressure mayhave different effects on proliferation and apoptosis of chondrocytes in human knee joints with this pressure model. This pressure model can safely and stably apply hydrostaticpressure to culture cells and also useful for relevant experiments with various hydrostaticpressure.3. High-intensitive hydrostatic pressure over physiological levels not only directlyaffects metabolism, induce apoptosis and injury of chondrocytes, but also indirectly leadsto acceleration of articular cartilages degeneration, which could transform normalchondrocytes to osteoarthritic chondrocytes, or aggravate the osteoarthritic pathologyprocess by inhibition of Type Ⅱ collagen synthesis and promotion of expression ofIL-1β, TNF-α and MMP-13. |
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