3D打印技术制备新型HA/Zr02梯度复合材料在犬股骨干骨缺损修复中的应用

Application Of novel HA/ZrO2gradient composite materials made.by three-dimensional prin ttechnology forrepair of femoral shaft defect in dogs

目的 运用3D打印技术制备新型羟基磷灰石/二氧化锆(HA/ZrO2)梯度复合材料,分析其性能,并观察其修复比格犬股骨干骨缺损的能力.方法 随机抽取6月龄雄性比格犬1只,截去右下肢股骨中段全层15 mm制成骨缺损模型后,放入动物专用Micro CT进行容积扫描,完成数据采集、转化及后期处理,将处理后的数据导入CeraFab 7500光固化3D打印机中.根据设定参数启动打印程序,形成复合光敏树脂初胚,并对其进一步脱脂烧结,然后采用浸涂法制备HA/ZrO2梯度复合材料.运用扫描电镜、X射线衍射(XRD)和生物力学实验分析HA/ZrO2梯度复合材料的性能.制备HA/ZrO2梯度复合材料浸提液.培养小鼠成纤维细胞株L929,采用噻唑蓝(MTT)法检测HA/ZrO2梯度复合材料对体外细胞增殖和毒性的影响.将16只比格犬随机分为4组,每组4只.A组:截取犬股骨中段15 mm后,不植入任何材料;B组、C组、D组分别截去股骨中段15、25、35 mm制成骨缺损模型,然后分别植入相应规格的HA/ZrO2梯度复合材料.术后第2、4、8、12周拍摄术肢股骨正侧位X片,观察植入材料与自身骨结合情况及周围骨痂生长情况.术后12周处死实验动物,截取整段股骨,大体观察标本植入材料与周围骨生长状况;行Micro CT扫描,测定新生骨量,并对新生骨进行3D图像重建;对股骨标本进行极限抗压实验,测量极限抗压强度.结果 运用3D打印技术制备的HA/ZrO2梯度复合材料扫描电镜显示表面光滑,结构均匀,断口表面层均匀过渡结合,没有明显界限,相互之间冶金结合,结构稳定.XRD分析显示HA及ZrO2峰形明显、结晶程度较好、粉体纯度高.抗压试验显示极限抗压强度为(43.37±2.31)MPa.MTT试验显示HA/ZrO2梯度复合材料无明显细胞毒性.实验动物术后X线片显示:A组术后形成骨不连;B组有连续性骨痂通过,人工假体与宿主骨之间的界线消失;C组术后第2、4、8周连续性骨痂及新生骨长入速度较B组缓慢,但人工假体与断端间隙逐渐被新生骨填充,有连续性骨痂通过;D组新骨生成缓慢,仅在断端周围出现新生骨.术后12周取材,Micro CT 3D重建及新生骨量检测,B、C、D组单位体积新生骨量分别为(238.55±19.11)mm3/cm3、(223.31±13.41)mm3/cm3、(110.83±6.48)mm3/cm3,3组间比较差异有统计学意义(F=156.824,P<0.01),其中B、C组新生骨量明显多于D组,差异均有统计学意义(P值均<0.05).新生骨CT 3D重建显示:B组复合材料整体被新生骨包绕,无外露;C组复合材料少许外露,表面基本被新生骨填塞;D组复合材料大部外露,新生骨散在长入.B、C、D组股骨标本极限抗压强度分别为(49.72±2.33)MPa、(49.81±2.43)MPa、(46.92±3.61)MPa,3组间比较差异无统计学意义(F=1.119,P>0.05).结论 运用3D打印技术制备的纳米HA/ZrO2梯度复合材料具有可靠的生物相容性及生物力学强度,可以实现临床个体化治疗原则,能很好地修复犬股骨干35 mm内骨缺损,是一种理想的骨组织替换材料.

Objective To apply the hydroxyapatite/zirconium oxide( HA/ZrO2 ) gradient composite material by 3D printing technology, to analyze its ability to repair the defect of femoral shaft of beagle dogs. Methods Bone defect model was prepared in 6-month male beagle by truncating right leg femur after middle full-thickness 15 mm, which was put into the Micro CT scanned for volume, complete data collection, transformation and the post-processing. Then data were imported in CeraFab 7500 photocure 3D printer. Started to print program, formed a composite photosensitive resin in the early embryo, and the further defatted sintering and then used dip-coating HA/ZrO2 gradient composite materials according the parameters. The performance of the HA/ZrO2 gradient materials were taken scanning electron microscopy ( SEM) , X-ray diffraction analysis and biomechanical experiments. Prepared HA/ZrO2 gradient composite materials and cultured L929 mouse fibroblast cell lines, then MTT method was used to detect the HA/ZrO2 gradient composites cell toxicity in vitro. Sixteen dogs were divided into 4 groups, 4 dogs in each group. Group A:Dogs' 15 mm middle femur were intercepted with no biological material implanted, as the blank control group;group B, C, D were truncated middle femoral 15, 25 and 35 mm made of bone defect model, and transferred into the corresponding specifications of the HA/ZrO2 gradient composite materials. X-ray scan was taken to observe implanted biomaterials combined with own bone and surrounding callus growth after operation of 2, 4, 8, 12 weeks. Animals were executed and captured the entire length of the femur, then observed specimens implanted in biological materials and the surrounding bone growth conditions after 12 weeks. New bone mass were measured and reconstructed by Micro CT scans. The compressive experiments of femoral specimens were used to measure the ultimate compressive strength. Results The SEM showed that nano HA/ZrO2 gradient composite material made by 3D printing technology had smooth surface and even stable structure. Composite material had a metallurgical bonding and there was no obvious boundary in fracture surface. XRD analysis showed an obvious peak, better crystalline degree and better purity. Mechanical text showed that the ultimate compressive strength was ( 43. 37 ± 2. 31) MPa. MTT analysis proved that HA/ZrO2 gradient composite material had no cytotoxicity. Front and lateral X-ray examinations were taken at 2, 4, 8, 12 weeks after operation. In group A, bone nonunion was formed. In group B, continual bony callus were get through and there was no obvious boundary between artificial prosthesis and host bone. At 2, 4 and 8 weeks after operation, continuous callus and new bone growth in group C was slower than group B, but the gap between artificial prosthesis and broken ends was gradually filled with new bone, and the continual bony callus were got through at the week 12. In group D,new bone was formed in a lower speed, and appeared only around the broken ends. The specimens were taken at week 12 after operation, three-dimensional reconstruction of Micro CT showed that per unit volume new bone in group B, C, D were (238. 55 ± 19. 11) mm3/cm3, (223. 31 ± 13. 41) mm3/cm3 and (110. 83 ± 6. 48) mm3/cm3 , respectively. There were statistically significant differences among the three groups in statistics analysis (F=156. 824, P<0. 01). The new bone in group B and C were much more than group D. They were different from group D in statistics analysis (all P values<0. 05). Three-dimensional reconstruction showed that in group B, HA/ZrO2 gradient composite material was encapsulated and had no exposure; in group C, composite material had a little exposure and the surface was full of new bone; while in group D, composite material was revealable and little new bone in surface. Ultimate compressive test was showed in group B, C, D were(49. 72 ± 2. 33) MPa, (49. 81 ± 2. 43) MPa and (46. 92 ± 3. 61) MPa, respectively. There was no significant difference among group B, C and D in statistics analysis (F=1. 119, P>0. 05). Conclusions The nano HA/ZrO2 gradient composite material artificial prosthesis made by 3D printing technology has reliable biocompatibility and biomechanics that fit the individual treatment principle in clinic. As an ideal substitute for bone tissue, it could well repair femoral bone defect in 35 mm in dogs.