纳米羟基磷灰石/壳聚糖复合人工骨的成骨及再血管化

Osteogenesis and revascularization of nano-hydroxyapatite/chitosan artificial bone

目的:通过纳米羟基磷灰石/壳聚糖(N-HA/CS)复合骨形态发生蛋白(BMP)制备N-HA/CS-BMP复合人工骨,初步了解其植入后成骨与血管长入之间的关系,以及复合人工骨的孔径对再血管化的影响。方法:①材料的制备:采用共沉淀法、粒子沥滤法制备N-HA/CS多孔复合材料,孔隙率为85%,孔径为100￣500μm;通过N-HA/CS与氧化锌粉末按质量比为8∶1的方式混合制备成致密复合材料;然后分别复合BMP制备N-HA/CS-BMP复合人工骨。②实验过程:20只新西兰兔,在兔双后肢胫骨近段内侧用直径为3.5mm手摇钻头钻2个孔制备骨缺损模型。随机取15只兔,右侧植入2块多孔N-HA/CS-BMP复合人工骨为多孔N-HA/CS+BMP组,左侧植入2块致密N-HA/CS-BMP复合人工骨为致密N-HA/CS+BMP组;另5只兔右侧植入2块多孔N-HA/CS为多孔N-HA/CS组,左侧植入2块致密N-HA/CS为致密N-HA/CS组。③观察指标:术后4,6,8周麻醉后墨汁灌注处死动物取出标本,行大体观察、X射线检查、组织学观察、Ⅰ型胶原免疫组化染色、显微计算机图像采集分析,了解各组成骨能力、血管化程度、复合人工骨的成骨与血管化之间的相互关系。结果:①一般情况:术后死亡2只动物,2只一侧肢体发生骨折,伤口均于2周左右愈合,未发生感染。②X射线检查:术后4,6周显示植入孔明显,材料与骨之间有密度减低的透光环,8周材料与骨结合紧密,透光环消失。③组织学观察:术后4,6周的材料内的炎性反应较重,主要是白细胞和巨噬细胞,8周的材料内炎性反应减轻;8周壳聚糖大部分降解,从而显现出羟基磷灰石的多孔结构。④显微计算机图像分析:多孔N-HA/CS+BMP组血管密度和新生骨小梁面积大于其他3组(P<0.05),多孔N-HA/CS+BMP组血管密度与骨小梁面积呈直线正相关关系(r=0.483,P=0.003)。⑤Ⅰ型胶原免疫组织化学染色显示结果支持显微计算机图像分析结果。结论:复合人工骨的成骨与血管化呈直线正相关关系,复合人工骨的成骨与血管化在早期是随着材料降解而完成的,多孔结构在晚期对血管化和成骨有利。

AIM： To prepare compound artificial bone through compounding nano-hydroxyapatite/chitosan （N-HNCS） with bone morphogenetic protein （BMP）. It is expected to primarily discover the relationship between osteogenesis and revascularization of this compound artificial bone, and investigate the influence of big aperture of N-HNCS-BMP on revasculadzation. METHODS： ①Preparation of compound materials： The co-precipitation method and the particle leaching method were employed to prepare N-HA-CS porous compound materials, with a porosity of 85% and an aperture diameter of 100- 500 μm; the compound materials and zinc oxide powder were mixed at a weight ratio of 8：1 to produce dense materials, which was then applied to compound with BMP. ②Experimental methods： Twenty New Zealand white rabbits were adopted, and an incision was cut on each rabbit medialis proximal tibia. Then tibias were broached with a bit of 3.5 mm in diameter, with 2 bores each side. All the rabbits were divided into four groups： Group A： Fifteen rabbits were implanted porous N-HNCS-BMP into the right side tibia; Group B： Those 15 rabbits were implanted dense N-HNCS-BMP into the left side tibia; Group C： Five rabbits were implanted porous N-HNCS into the right side tibia; Group D： Those 5 rabbits were implanted dense N-HNCS into the left side tibia. ③ Detecting index： After being anaesthetized in the 4^th, 6% 8^th weeks post-operation, all the animals were executed by Chinese ink perfusion, and the specimens were taken out for the general observation, radiography, histological observation, Type I collagen immunohistochemical staining, computerized image capturing and analysis, and statistic processing was carried out to understand the relationship between osteogenesis and vascularization of compound artificial bone in each group. RESULTS： ①General observation： Two rabbits died post-operation, and fracture of one-side tibia occurred in 2 rabbits. Wound of all the rabbits healed about 2 weeks later, and no infection appeared.②X-ray examination： Implanting bores were obvious in the 4^th and 6^th weeks post-operation, and there was a low-density ring between materials and bones. In the 8^th week, materials and bones were combined tightly and the low-density ring disappeared.③Histological observation： There were many leucocytes and macrophages in materials in the 4^th and 6^th weeks, but they decreased in the 8^th week, indicating the inflammatory reaction was relieved. As chitosan degraded, the porous structure of hydroxyapatite appeared in the 8^th week.④Computerized image analysis： There was a linear positive correlation between the vascular density and the area of bone trabecula in Group A （r =0.483, P =0.003）, and the osteogenesis and vascularization in Group A excelled that of Group B, C and D （P 〈 0.05）.⑤Results of Type I collagen immunohistochemical staining supported the computerized image analysis result. CONCLUSION： There is a linear positive correlation between osteogenesis and vascularization of compound artificial bone, and the two indexes may complete along with chitosan degradation in the early stage. The porous structure is helpful for vascularization and osteogenesis in the advanced stage.