钛颗粒对成骨细胞增殖分化及细胞形态的影响

EFFECT OF TITANIUM PARTICLES ON PROLIFERATION, DIFFERENTIATION, AND CYTOMORPHOLOGY OF OSTEOBLASTS

目的通过观察钛颗粒对成骨细胞增殖分化和细胞形态的影响,探讨其可能的内在联系和作用机制。方法取10只新生24 h SD大鼠颅骨,采用多次酶消化法体外分离培养成骨细胞,ALP和茜素红染色进行细胞鉴定。取生长良好的第3代细胞,分别采用浓度为0.01、0.05、0.1、0.5、1 mg/mL的钛颗粒培养基(0.01、0.05、0.1、0.5、1 mg/mL组)培养,7 d后用细胞计数试剂盒8检测吸光度(A)值,比较不同浓度钛颗粒对成骨细胞增殖能力的影响,并筛选半数致死浓度;ELISA法检测细胞Ⅰ型胶原表达,比较不同浓度钛颗粒对成骨细胞分化能力的影响。另取以半数致死浓度钛颗粒培养7 d的成骨细胞(实验组)进行FITC-鬼笔环肽和碘化丙啶双重荧光染色,激光共聚焦显微镜下观察吞噬钛颗粒后细胞形态学的改变。以上观察指标均以正常第3代成骨细胞作为对照组。结果 ALP及茜素红染色鉴定提示培养的细胞为成骨细胞。培养7 d,各浓度组成骨细胞增殖、分化能力与对照组比较均有不同程度下降,差异均有统计学意义(P<0.05)。同时随浓度增加,各浓度组对成骨细胞增殖的抑制作用逐渐增强,成骨细胞Ⅰ型胶原表达逐渐降低;除0.01、0.05 mg/mL组间差异无统计学意义(P>0.05)外,其余各浓度组间比较差异均有统计学意义(P<0.05)。钛颗粒的半数致死浓度为0.5 mg/mL。激光共聚焦显微镜下示,与对照组比较,实验组吞噬了钛颗粒的成骨细胞皱缩变形,伪足收缩变短,微丝排列紊乱。结论钛颗粒可抑制成骨细胞的增殖和分化能力,这种作用可能与吞噬钛颗粒后细胞形态学的改变有关。

Objective To study the effect of titanium particles on the proliferation, differentiation, and cytomorphology of osteoblasts, and to explore the possible internal relations and mechanism. Methods Calvarial osteoblasts were separated from 10 newborn Sprague Dawley rats by repeated enzyme digestion, and were cultured in vitro. The cells were identified by alkaline phosphatase （ALP） staining and alizarin red staining. The cells at passage 3 were cultured with titanium particles culture medium at concentrations of 0.01, 0.05, 0.1, 0.5, and 1 mg/mL （0.01, 0.05, 0.1, 0.5, and 1 mg/mL groups）. The absorbance （A） values were detected by cell counting kit 8 at 7 days after cultured to compare the effect of titanium particles at different concentrations on proliferation, and median lethal concentration was screened out. The expression of collagen type I was detected by ELISA to observe the effect of titanium particles on differentiation. The osteoblasts co-cultured with titanium particles of median lethal concentration （experimental group） for 7 days, and double fluorescence staining with FITC-phalloidine and propidium iodide was performed. The cytomorphology variation of osteoblasts after swallowing titanium particles was observed under laser scanning confocal microscope. The osteoblasts at passage 3 cultured with culture medium without titanium particles served as control group. Results The cultured cells were identified as osteoblasts by ALP staining and alizarin red staining. Different concentrations of titanium particles could inhibit osteoblasts proliferation and differentiation in varying degrees, showing significant difference when compared with the control group at 7 days after culture （P 〈 0.05）. The cell proliferation and differentiation were decreased with increased titanium particles concentration; significant differences were found between the other groups （P 〈 0.05） except 0.01 and 0.05 mg/mL groups （P 〉 0.05）. The median lethal concentration of titanium particles was 0.5 mg/mL. Laser scanning confocal microscope showed cellular shrinking, microfilaments distortion, pseudopodia contraction of osteoblasts that swallowed titanium particles in the experimental group. Conclusion Titanium particles can inhibit proliferation and differentiation of osteoblasts. The effect may be related to variation of cytomorphology after swallowing titanium particles.