钛颗粒负荷对切应力作用下骨髓间质干细胞F-actin表达和DNA含量的影响

假体磨损碎屑颗粒是引起假体一骨界面无菌性炎症和骨溶解而致全关节置换术失败的主要原因之一。磨屑颗粒所诱发的骨溶解须有周围骨组织中成骨细胞分泌足够的骨基质以弥补丢失的骨量，而成骨细胞正常的数量和质量有赖于其来源骨髓祖细胞—骨髓问质干细胞的正常增殖分化能力的维持。为了探讨磨屑钛颗粒对大鼠骨髓间质干细胞(Rat MSCs，rMSCs)产生细胞毒性的可能细胞分子机制，选用健康3月龄SD雄性大鼠，采用Percoll等密度梯度离心法分离获取rMSCs，经体外传代纯化培养后，与不同直径、不同负荷浓度、不同负荷作用时间的钛颗粒悬液共孵育，再采用精准的流室系统对钛颗粒负荷的rMSCs施加一定的流体剪切应力(Fluid shear stress，FSS)后立刻固定细胞，经免疫荧光抗体染色，结合激光共聚焦显微镜技术和图像分析软件定性定量分析rMSCs F—actin表达和DNA含量的变化情况。同时设置相应的未经钛颗粒孵育的rMSCs细胞为对照组细胞。结果显示，切应力作用可上调rMSCs胞内F—actin的表达。亚微(Submieron)直径(0．9μm)的钛颗粒负荷对rMSCs F—actin表达和DNA含量的抑制作用最为显著，并伴有凋亡小体出现；直径为2．7μm的钛颗粒负荷产生的抑制作用略为减弱，而较大直径(6．9μm)的抑制效应最弱。相同条件下，钛颗粒负荷对F—actin的抑制效应有一定的时间和浓度依赖性：以0．1wt％浓度对F—actin表达和DNA含量的抑制效应最为明显，亦有凋亡小体的出现；随着浓度的降低，抑制作用亦减弱，以0．01wt％浓度最弱；随着作用时间的延长，F—actin表达和DNA含量逐渐降低，至实验中的32h达到最低值。提示：磨屑颗粒对rMSCs活力的抑制作用是假体无菌性松动的可能分子机制，对其具体细胞分子机制进行深入研究，必将有助于有效防治假体松动药物的研发应用以及人工关节材料的优化设计，从而为全关节置换术患者真正带来福音。

Alterations in Expression of F-actin and DNA of Fluid Shear Stress Treated-mesenchymal Stem Cells Affected by Titaninum Particles Loading

Particulate wear debris within the bone-prosthesis microenvironment generated by normal wear and corrosion of orthopaedic implants is considered to be one of the main factors responsible for chronic aseptic inflammation and development of osteolysis in the long-term instability and failure of total joint arthroplasty. While the decrease in bone volume caused by wear debris-induced osteolysis could have been compensated by enough new bone matrix secreted by osteoblasts. Actually, the normal osteoblastic population depend on the regular differentiation and proliferation of their progenitor cells--bone marrow mesenchymal stem cells (MSCs). This study aims to investigate the potential mechanism for the rat MSCs cytotoxicity upon exposure to Titanium (Ti) particles. Rat mesenchymal stem cells (rMSCs) isolated from 3-month-old male Sprague-Dawley rats by Percoll intensity gradient method were cultured in DMEM medium (low glucose) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 micrograms/ml streptomycin in a humidified incubator with 5% CO2 at 37 degrees C. In order to gain the homogenous cell population, rMSCs were passaged to 3-4th subpassage which were used in all the experiment groups. Then rMSCs were seeded in the 6 well culture plates and exposed to three different circle diameters (mean size, TD1: 0.9 micron, TD2: 2.7 microns, TD3: 6.9 microns) with three different concentrations (0.1 wt%, 0.05 wt%, 0.01 wt%, W/V) at different durations (8 h, 16 h, 24 h,), respectively. Unexposed rMSCs were used as control. In the given periods of Ti loading, fluid shear stress (FSS) was applied to each group cells. The expression of F-actin and DNA of the rMSCs at the indicated time were determined with laser confocal scanning microscopy and image analysis software. The results showed that there was up-regulation expression of F-actin in the rMSCs without Ti particles loading but in the presence of FSS. Ti particles loading can suppress the expression of F-action and DNA of rMSCs, but this down-regulation response varied with the three circle diameter, concentrations and durations of Ti particles. Among three kinds of diametrically different Ti particles, submicron Ti particles (0.9 micron) had the greatest suppressive response on rMSCs, together with some apoptosis bodies. Under the same diameter condition, the inhibition induced by Ti particles loading was in a manner dependent on the particles concentration and exposure duration. The reductive effects produced from 0.1 wt% Ti was the greatest and earliest among the responses from Ti particles at three different concentrations; and the lower the concentration, the weaker the repressive influence. Furthermore, with the elongation of exposure to Ti particles, the expression of F-actin and DNA decreased gradually, the lowest level was at 32 h. These findings demonstrated that Ti particles loading can attenuate rMSCs' viability in a manner dependent on the circle diameter, particles concentration, treatment period, suggesting that a reduction in the number of viable MSCs together with a compromise of the their differentiation into functional osteoblast may exacerbate aseptic loosening of total joint implant. Further investigation into particles-mediated suppression of MCSs viability may reveal novel mechanism of implant loosening and aid in development and application of osteolytic drug therapy and the optimization of design and selection of future orthopaedic biomaterials, thereby improving long-term compatibility and stability for arthroplasty patients.