3D打印羟基磷灰石/二氧化锆支架复合血管内皮细胞生长因子165藻酸钙微球缓释系统修复犬大段骨缺损

Hydroxyapatite/zirconia scaffold by three-dimensional printing compounded with vascular endothelial growth factor 165 calcium alginate microsphere slow-release system for repairing large bone defect in dogs

目的观察3D打印技术制备的羟基磷灰石/二氧化锆(HA/ZrO2)支架复合血管内皮生长因子(VEGF)165藻酸钙微球缓释系统修复比格犬股骨干骨缺损的能力。方法运用3D打印技术制备HA/ZrO2人工假体,构建复合VEGF165藻酸钙微球缓释系统共培养体系,检测藻酸钙微球包封率、载药量及缓释效果。将16只比格犬根据截取中段股骨干范围分为四组,每组4只。A组:截取犬股骨中段15 mm后,不植入生物材料,作为空白对照组;B组:截取犬股骨中段15 mm后,植入复合VEGF165藻酸钙微球的HA/ZrO2支架,作为股骨干缺损15 mm实验组;C组:同理为截取犬股骨中段25 mm实验组;D组:同理为截取犬股骨中段35 mm实验组。术后大体观察及X线影像学观察;术后12周通过Micro CT扫描、生物力学检测、墨汁染色及甲苯胺蓝染色,综合评价新型HA/ZrO2梯度生物复合材料修复骨缺损的能力。结果制备所得共培养体系中藻酸钙微球包封率为(62.4±3.6)%,载药量为(23.6±2.9)ng/mg,呈缓慢释放。术后大体观察显示四组切口均Ⅰ期愈合。术后X线影像学观察显示:A组随时间推移,形成骨不连;B组人工假体逐渐被新生骨填充,界线逐渐模糊;C组前期反应较A组缓慢,术后12周显示有连续性骨痂通过;D组新骨生成缓慢,仅在断端周围出现新生骨。术后12周新生骨量检测:B组为(238.6±19.1)mm3,C组为(223.3±13.4)mm3,D组为(110.8±6.5)mm3。三组间比较差异均有统计学意义(P<0.05),而B、C组比较差异无统计学意义(P>0.05)。术后12周极限抗压试验结果显示:B组为(49.7±2.3)MPa,C组为(49.8±2.4)MPa,D组为(46.9±3.6)MPa,三组间比较差异均无统计学意义(P>0.05)。术后12周组织切片显示:B、C组可见血管粗大,有明显分支,周围新生骨增加,期间充满血管网,各组间血管数量大小及形态区别不明显,但B组新生骨数量及血管网较多,D组也可见新生骨,其中有细小血管网,但数量较少。结论3D打印HA/ZrO2支架复合VEGF165藻酸钙微球缓释系统可以修复犬35 mm以内的大段骨缺损。

Objective To investigate the effect of hydroxyapatite/zirconia(HA/ZrO2)scaffold by three-dimensional printing compounded with vascular endothelial growth factor(VEGF)165 calcium alginate microsphere slow-release system on repairing femoral shaft defects in dogs.Methods The HA/ZrO2 artificial prosthesis was prepared by three-dimensional printing,and the co-culture system of slow-release system of composite VEGF 165 calcium alginate microspheres was constructed.Sixteen beagle dogs were divided into four groups according to the extent of femoral shaft interception,with four dogs in each group.Group A:no biomaterials were implanted into the middle femur of dogs after 15 mm of femur interception as blank control group;Group B:HA/ZrO2 scaffolds composite with VEGF165 calcium alginate microspheres were implanted into the middle femur of dogs after 15 mm of femur interception;Group C:the same method as Group B was adopted after 25 mm of femur interception;Group D:the same method as Group B was adopted after 35 mm of femur interception.General examination and X-ray imaging observation were taken after operation.The ability of new HA/ZrO2 gradient biocomposites to repair bone defects was evaluated by micro CT scanning,biomechanical testing,ink staining and toluidine blue staining 12 weeks after operation.Results The drug loading capacity of calcium alginate microspheres reached(23.6±2.9)ng/mg,and the entrapment efficiency reached(62.4±3.6)%,showing a slow rate of release.Gross examination showed surgical incision was healed in all four groups.Postoperative X-ray imaging of experimental animals showed that nonunion was formed in Group A over time;in Group B,the artificial prosthesis was gradually filled with new bone and the boundary was blurred;in Group C,the early reaction was slower than that in Group A,and the callus passed continuously 12 weeks after operation;in Group D,new bone formation was slow,only surrounding the broken end.At 12 weeks after operation,the neonatal bone mass was(238.6±19.1)mm^3 in Group B,(223.3±13.4)mm^3 in Group C,and(110.8±6.5)mm3 in Group D.There were significant differences among the three groups(P<0.05),but no significant difference was found between Group B and Group C(P>0.05).The results limit compression test at 12 weeks after operation showed no significant differences among Groups B[(49.7±2.3)MPa],C[(49.81±2.4)MPa]and D[(46.9±3.6)MPa](P>0.05).At 12 weeks after operation,the histological sections showed that the blood vessels in Groups B and C were thickened,with obvious branches,and the surrounding new bone increased.During the period,the blood vessels were filled with vascular network.There were no obvious differences in the number and shape of blood vessels between groups.However,Group B had more new bones and blood vessel networks.New bone and small vascular networks were seen in Group D.Conclusion The hydroxyapatite/zirconia scaffold by three-dimensional printing compounded with vascular endothelial growth factor 165 calcium alginate microsphere slow-release system can repair dogs'femoral bone defect within 35 mm.