多孔氧化锌/羟基磷灰石复合材料的体内生物安全性研究

In vivo biological safety study of porous zinc oxide/hydroxyapatite composite materials

目的评价多孔氧化锌(ZnO)/羟基磷灰石(hydroxyapatite,HA)复合材料的体内生物安全性。方法利用放电等离子烧结技术制备多孔ZnO/HA复合材料和多孔HA材料,首先对材料进行表征,包括扫描电镜观察材料结构、体外降解实验检测材料降解率、电感耦合等离子体发射光谱仪检测复合材料降解溶出Zn^(2+)浓度。然后制备两种材料浸提液,取15只雄性昆明种小鼠行急性全身毒性实验。小鼠随机分为A、B、C组(n=5),分别腹腔注射生理盐水或多孔HA材料、多孔ZnO/HA复合材料浸提液,记录注射前和注射后24、48、72 h小鼠体质量,72 h取肝、肾组织行HE染色,初步判断复合材料安全性。最后行动物体内植入实验,将18只雄性新西兰大白兔随机分为HA组及ZnO/HA组(n=9);建立双侧前肢桡骨1 cm长缺损模型,两组右前肢分别植入多孔HA材料和多孔Zn O/HA复合材料,左前肢不作处理作为空白对照;术后观察动物一般情况,术前1 d和术后1 d、1周、4周、8周采集兔血液进行血常规(炎症相关指标)、血生化(肝、肾功能相关指标)检测,评价材料体内生物安全性;术后4、8、12周摄X线片观察骨缺损修复情况。结果材料表征示多孔ZnO/HA复合材料具有相互连通的大、小孔结构,孔径在50~500μm之间,降解快于多孔HA材料,能持续、缓慢溶出Zn^(2+)。急性全身毒性实验示,注射后各组小鼠无异常表现,体质量均逐渐增加(P<0.05);HE染色示肝、肾组织细胞形态及结构均正常。动物体内植入实验显示,所有兔均存活至实验完成;血常规检测示各组术后均出现炎症反应,与术前1 d相比,HA组术后1 d及1、4周炎症细胞含量增加(P<0.05)、而ZnO/HA组术后1 d增加(P<0.05),两组8周时均恢复正常(P>0.05);血生化检测显示肝、肾功能指标均在正常范围;X线片示4周后Zn O/HA组骨整合优于HA组。结论多孔ZnO/HA复合材料具有良好的体内生物安全性,并初步表现良好的骨修复能力,是一种潜在的骨修复材料。

Objective To evaluate the in vivo biological safety of porous zinc oxide(ZnO)/hydroxyapatite(HA composite materials.Methods The porous ZnO/HA composite materials and porous HA materials were prepared by the spark plasma sintering technology.First,the materials were characterized,including scanning electron microscopy to observe the material structure,in vitro degradation experiments to detect the degradation rate of the materials,and inductively coupled plasma emission spectrometer to detect the concentration of Zn^(2+)dissolved out of the composite material degradation.Then the two kinds of material extracts were prepared for acute systemic toxicity test.Fifteen male Kunming mice were randomly divided into groups A,B,and C(n=5)and injected intraperitoneally with normal saline,HA extracts,and ZnO/HA extracts,respectively.The body mass of the mice was recorded before injection and at 24,48,and 72 hours after injection.The liver and kidney tissues were taken at 72 hours for HE staining to evaluate the safety of the composite material.Finally,the biological safety of the material in vivo was evaluated by implantation experiment.The eighteen male New Zealand white rabbits were randomly divided into HA group and ZnO/HA group(n=9);a bilateral radius defect model(1 cm)was established,and the right forelimbs of the two groups were implanted with porous HA materials and porous ZnO/HA composite materials,respectively;the left untreated as a blank control.The general condition of the animals were observed after operation.The rabbit blood was collected at 1 day before operation and at1 day,1 week,4 weeks,and 8 weeks after operation for routine blood test(inflammation-related indicators)and blood biochemistry(liver and kidney function-related indicators).X-ray films were taken at 4,8,and 12 weeks after operation to observe the repair of bone defects.Results Material characterization showed that porous ZnO/HA composite materials had interconnected large and small pore structures with a pore size between 50 and 500μm,which degraded faster than porous HA materials,and continuously and slowly dissolved Zn^(2+).The acute systemic toxicity test showed that the mice in each group had no abnormal performance after injection,and the body mass increased(P<0.05).HE staining showed that the cells shape and structure of liver and kidney tissue were normal.Animal implantation experiments showed that all rabbits survived until the experiment was completed;routine blood tests showed inflammation in each group(neutrophils,monocytes,and lymphocytes increased)at 1 day after operation,and all returned to normal at 8 weeks(P>0.05);compared with 1 day before operation,the content of inflammatory cells in the HA group increased at 1 day,1 week,and 4 weeks after operation(P<0.05),and the ZnO/HA group increased at 1 day after operation(P<0.05);blood biochemistry showed that the liver and kidney function indexes were in the normal range;X-ray films showed that the ZnO/HA group had better osseointegration than the HA group at 4 weeks after operation.Conclusion The porous ZnO/HA composite material has good in vivo biological safety and good bone repair ability,which is a potential bone repair material.