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目的观察伊班膦酸盐(ibandronate,Iba)与羟基磷灰石(hydroxyapatite,HA)结合后对破骨细胞骨吸收作用的影响。方法将Iba与HA复合成伊班膦酸盐-羟基磷灰石(Iba-HA)作为实验组,未结合Iba的HA作为对照组。分离、培养、纯化大鼠破骨细胞,Giemsa染色和抗酒石酸酸性磷酸酶(TRAP)染色显示破骨细胞及其细胞核的形态和数目。用两组材料与破骨细胞复合培养,透射电镜观察细胞内结构变化,SNAFL染色测定破骨细胞内氢离子浓度,骨吸收陷窝面积测定并辅以上清液内胶原C端肽片段(CTx)的浓度测定观察破骨细胞骨吸收功能。结果 Gimsa染色标本可见多核的破骨细胞、细胞核染成蓝紫色,TRAP染色标本可见TRAP酶活性的部位被染成红色,分布于破骨细胞胞浆内。透射电镜观察发现,破骨细胞与Iba-HA复合培养后,较之与HA复合培养的破骨细胞生物活性下降。SNAFL染色显示Iba-HA组破骨细胞荧光强度小于HA组,统计学有差异(P<0.05);骨吸收检测时面积显示,Iba-HA组小于HA组,统计学有差异(P<0.05),上清液CTx浓度,Iba-HA组小于HA组,统计学有差异(P<0.05)。结论与HA处于结合状态的Iba对破骨细胞的骨吸收作用仍有显著影响。
Objective To observe the effect of ibandronate (Iba) combined with hydroxyapatite (HA) on the bone resorption of osteoclasts. Methods Iba and HA were combined into ibandronate-hydroxyapatite (Iba-HA) as experimental group and non-Iba HA as control group. The osteoclasts were isolated, cultured, and purified. Giemsa stain and tartrate resistant acid phosphatase (TRAP) staining showed the morphology and number of osteoclasts and their nuclei. The changes of intracellular structure were observed by transmission electron microscopy with two sets of materials and osteoclasts. The concentration of hydrogen ion in osteoclasts and the size of bone resorption lacuna were determined by SNAFL staining and supplemented with Cx-CTX (supernatant) The concentration of osteoclasts was measured to observe the bone resorption function. Results The multinucleated osteoclasts were observed in Gimsa stained specimens. The nuclei were stained blue-violet. The TRAP-stained specimens were stained red and distributed in the cytoplasm of osteoclasts. Transmission electron microscopy showed that the osteoblasts cultured with Iba-HA had a lower biological activity than the osteoclasts cultured with HA. SNAFL staining showed that the fluorescence intensity of osteoclasts in Iba-HA group was lower than that in HA group (P <0.05). The area of bone resorption assay in Iba-HA group was smaller than that in HA group (P <0.05) , The concentration of CTx in supernatant was lower in Iba-HA group than in HA group (P <0.05). Conclusion Iba, which binds to HA, still has a significant effect on the bone resorption of osteoclasts.