【摘要】 目的:观察仿生多层纳米羟基磷灰石/壳聚糖复合支架及其结合自体骨髓后对兔腓骨缺损的修复作用。方法:实验于2005-09/2006-05在清华大学生物系生物膜与膜生物工程国家重点实验室完成。选择成年新西兰大白兔10只,按随机数字表法分为3组,阴性对照组2只,材料植入组4只,材料+骨髓植入组4只。以纳米羟基磷灰石/壳聚糖复合支架为基础材料,制备仿生多层纳米羟基磷灰石/壳聚糖复合支架。在兔腓骨造成5mm的缺损,分别旷置缝合(阴性对照组),植入仿生多层纳米羟基磷灰石/壳聚糖复合支架(材料植入组),以及植入复合自体骨髓的仿生多层纳米羟基磷灰石/壳聚糖复合支架(材料+骨髓植入组)。分别在术后8周和12周,X射线检测缺损部位的钙化情况。麻醉处死动物前10d和3d注射四环素(25mg/kg),不脱钙骨切片进行四环素荧光检测和VonKossa染色,检测实验动物骨缺损部位新生骨钙化情况。脱钙骨切片用苏木精-伊红染色,检测缺损部位的骨修复情况。结果:纳入兔10只,均进入结果分析。①术后8周时,X射线检测显示阴性对照组缺损部位无明显钙化,材料植入组和材料+骨髓植入组均有钙化。术后12周时,阴性对照组缺损部位大部分仍无明显钙化,材料植入组和材料+骨髓植入组钙化明显,材料+骨髓植入组钙化更完全。②不脱钙骨切片VonKossa染色结果表明,阴性对照组骨缺损处充满纤维组织,并有少量肌肉组织压迫侵入,无明显的钙化区。材料植入组兔的骨缺损的中心区域,出现染成黑色的钙化区,中间夹杂着中性红复染的组织细胞。材料+骨髓植入组兔的骨缺损部位的中心呈现多个“钙化岛”,成骨更加完全。③不脱钙骨切片四环素荧光检测结果表明,阴性对照组两个断端之间未见钙化荧光出现,材料植入组材料的中心有零星的小片钙化区域,材料+骨髓植入组缺损部位的中心有许多的“钙化岛”,并且这些“钙化岛”呈现出连接成片的趋势,同时伴随着更多材料的降解。④脱钙骨切片苏木精-伊红染色结果表明,材料植入组和材料+骨髓植入组缺损部位未发现成熟的纤维组织,并且观察到有大量新生血管。两个植入组均有成骨区域,材料+骨髓植入组还出现了髓样组织。结论:仿生多层纳米羟基磷灰石/壳聚糖复合支架促进骨修复的进行,自体骨髓的加入更加速修复过程。更多还原

【Abstract】 AIM: To assess the repair of bone defect with multilayer biomimetic nano-hydroxyapatite/chitosan composite scaffolds combining with or without autologous bone marrow. METHODS: The experiments were completed in State Key Lab of Biomembrane and Membrane Biotechnology, Tsinghua University from September 2005 to May 2006. Ten New Zealand white rabbits were divided into 3 groups: 2 for the negative control group, 4 for scaffold-implanted group, and 4 for autologus bone marrow seeded scaffold-implanted group. Based on the preparation of nano-hydroxyapatite/chitosan composite scaffolds, a multilayer biomimetic scaffold was fabricated. After a 5 mm bone defect was made on each rabbit fibula, the multilayer biomimetic scaffold with or without autologous bone marrow was implanted into the defect site. The rabbits without any implant in defect sites were the control. The calcification in the defect site was evaluated with X-ray respectively at weeks 8 and 12 respectively after surgery. The rabbits were injected with tetracycline (25 mg/kg) before sacrificed. Then the calcification in the defect site was analyzed with the assessment of tetracycline fluorescence and Von Kossa staining after un-decalcified sliced. The bone regeneration in the defect site was assessed after decalcified sliced and hematoxylin-eosin (HE) staining. RESULTS: Totally 10 rabbits were involved in the result analysis. ① There was no obvious calcification at the defect site of the control rabbits, while in the implanted rabbits calcification was observed at weeks 8 after surgery. There was calcification in the implanted rabbits and more in the autologus bone marrow seeded scaffold-implanted group at weeks 12 after surgery and no obvious calcification in the control group yet. ②The Von Kossa staining of un-decalcified slices showed that the bone defect sites of control rabbits were filled with fibrous tissues companied with a little muscle tissue incursion and no obvious calcified area was detected. However, in the center of bone defect of scaffold-implanted rabbits, black calcified area appeared in the middle of cells stained by neutral red. Many "calcified islands" appeared in the defect sites of the marrow seeded scaffold-implanted rabbits, which meant a higher level of bone-forming than other groups. ③The results of tetracycline fluorescence assessment showed that in the area between of the two ends no fluorescence was detected in the negative control group, while in the scaffold-implanted rabbits a few calcified areas were detected. In the defect sites of bone marrow seeded scaffold-implanted groups many "calcified islands" appeared in a trend of connecting with each other, companied with more degradation. ④The HE staining showed that in the implanted rabbits no mature fibrous tissue and many new-born blood vessels were observed. Bone regeneration was detected in the implanted rabbits and marrow-like tissue appeared in the autologus bone marrow seeded scaffold-implanted group. CONCLUSION: The multilayer biomimetic nano-hydroxyapatite/chitosan composite scaffolds facilitate the bone regeneration and the preload of autologous bone marrow enhances this process.更多还原