3D打印羟基磷灰石-明胶支架联合BMSCs和HUVECs修复兔颅骨缺损的组织学评价

Histological evaluation of 3D printed HAP-GEL scaffold combined with BMSCs and HUVECs in repairing skull defects in rabbits

目的通过组织病理学方法评价3D打印技术制备的羟基磷灰石-明胶(HAP-GEL)支架联合骨髓间充质干细胞(BMSCs)和脐静脉内皮细胞(HUVECs)修复兔颅骨缺损的效果。方法将3D打印HAP-GEL支架与BMSCs和HUVECs细胞联合培养,构建组织工程骨。24只新西兰大白兔随机分为空白对照组、阳性对照组、HAP-GEL材料组及BMSCs+HUVECs+HAP-GEL支架共培养组,每组6只,于缺损区随机植入3D打印HAP-GEL支架、HAP-GEL支架+BMSCs和HUVECs细胞,自体颅骨设为阳性对照组,空白缺损设为空白对照组。分别于模型复制后4周和12周通过组织学观察骨愈合情况。结果镜下各组均未见急慢性炎症细胞浸润。术后4周,HE染色显示空白对照组仅有少量纤维结缔组织生成;HAP-GEL材料组有少量新骨生成;BMSCs+HUVECs+HAP-GEL支架共培养组可见部分新骨开始彼此融合,新生骨量与阳性对照组相似;术后12周,HE染色显示空白对照组新生骨量少;HAP-GEL材料组新生骨呈较大面积的板层状;BMSCs+HUVECs+HAP-GEL支架共培养组缺损区几乎全部被新生骨充填,新生骨量较前两组明显增多,且与阳性对照组相似。术后4周,Masson染色显示空白对照组仅有少量纤维组织;HAP-GEL材料组内有蓝染新骨、少量红染成熟骨形成,但着色均较浅;BMSCs+HUVECs+HAP-GEL支架共培养组红染成熟骨面积较HAP-GEL材料组大,修复效果与阳性对照组相似。术后12周,Masson染色显示空白对照组出现蓝染新骨;HAP-GEL材料组成熟骨面积进一步扩大,支架被分割包裹、吸收;BMSCs+HUVECs+HAP-GEL支架共培养组可见大片红染成熟骨生成,夹杂少量蓝染新生骨,支架残存少量,成骨效果与阳性对照组相似,优于HAP-GEL材料组与空白对照组。结论3D打印HAP-GEL支架联合BMSCs和HUVECs对兔颅骨缺损有较好的修复效果。

Objective To evaluate the effect of hydroxyapatite-gelatin(HAP-GEL)scaffold prepared by 3D printing technique combined with bone marrow mesenchymal stem cells(BMSCs)and human umbilical vein endothelial cells(HUVECs)in repairing rabbit skull defect with histopathological method.Methods The 3D printed HAP-GEL scaffolds were co-cultured with BMSCs and HUVECs to construct tissue-engineered bone.Twenty-four New Zealand white rabbits were randomly divided into HAP-GEL scaffolds group,HAP-GEL scaffolds+BMSCs and HUVECs group,blank control group and positive control group with 6 rabbits in each group.The 3D printed HAP-GEL scaffolds or HAP-GEL scaffolds combined with BMSCs and HUVECs were randomly implanted in the defect area.The autogenous skull was set as positive control,and the blank defect was set as negative control.Bone healing was analyzed by histopathological observation at 4 and 12 weeks after modeling.Results Microscopically,no acute or chronic inflammatory cell infiltration was found in all groups.HE staining showed that 4 weeks after operation,only a small amount of fibrous connective tissue was formed in the blank control group,while a small amount of new bone was formed in the HAP-GEL scaffolds group.Besides,part of the new bone began to fuse in the HAP-GEL scaffolds+BMSCs and HUVECs group,and the new bone mass was similar to that in the positive control group.At 12 weeks after operation,there was little new bone mass in the blank control group,and the new bone in the HAP-GEL scaffolds group was lamellar-shaped.In addition,the defect area in the HAP-GEL scaffolds+BMSCs and HUVECs group was almost filled with new bone,the mass of which was significantly more than that in the former two groups and similar to that in the positive control group.Masson’s trichrome staining showed that 4 weeks after operation,only a small amount of fibrous tissue was found in the blank control group.Blue-stained new bone and a small amount of red-stained mature bone were formed in the HAP-GEL scaffolds group,but the staining was faintly colored.In the HAP-GEL scaffolds+BMSCs and HUVECs group,the area of red-stained mature bone was larger than that in the HAP-GEL scaffolds group,and the repairing effect was similar to that in the positive control group.At 12 weeks after operation,blue-stained new bone appeared in the blank control group as shown in Masson’s trichrome staining.The mature bone area in the HAP-GEL scaffolds group was further enlarged,and the scaffolds were segmented,wrapped and absorbed.In the HAP-GEL scaffolds+BMSCs and HUVECs group,a large area of red-stained mature bone was formed,mixed with a small amount of blue-stained new bone,with a small amount of scaffold remained,and the osteogenic effect was similar to that of the positive control group but better than that of the HAP-GEL scaffolds group and the blank control group.Conclusions The 3D printed HAP-GEL scaffold combined with BMSCs and HUVECs exhibited great efficacy in repairing skull defects in rabbits.