聚碳酸亚丙酯/壳聚糖纳米纤维复合三维多孔支架修复骨缺损的实验研究
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摘要
目的
分离培养新西兰大白兔骨髓间充质干细胞(bone mesenchymal stem cells, BMSCs),制作聚碳酸亚丙酯／壳聚糖纳米纤维(propylene carbonate/chitosan nanofibers, PPC/CSNF)复合三维多孔支架并评价支架与兔BMSCs的体外相容性、体内组织相容性。体外制作细胞-支架复合物并植入兔股骨髁缺损处,以评价其修复局部骨缺损的效果,初步探讨治疗骨缺损的组织工程学新方法。
方法
1、运用全骨髓贴壁法分离、培养兔BMSCs。
2、通过形态学和流式细胞等方法法观察BMSCs的表面抗原、细胞周期和细胞活力等。
3、用成骨诱导液将BMSCs向成骨细胞定向诱导分化,通过形态学观察、碱性磷酸酶(alkaline phosphatase, ALP)染色、矿化结节染色、Ⅰ型胶原染色等进行分析鉴定,了解所分离培养的BMSCs骨向分化能力。
4、用溶液浇铸／粒子沥滤法制备PPC支架,再用相分离法原位复合三维壳聚糖纳米纤维制备PPC/CSNF支架,测定支架孔隙率,并进行扫描电镜观察。
5、BMSCs分别与两种支架材料联合培养,比较两种支架的细胞相容性,并进行扫描电镜观察。
6、PPC/CSNF多孔支架植入兔皮下,于术后第1,2,4周取出材料,大体观察和苏木精-伊红(hematoxylin-eosin, HE)染色观察材料的组织相容性。
7、制作兔股骨髁部骨缺损模型,分四组实验,实验组缺损处植入PPC/CSNF/BMSCs复合物、标准组移植自体骨、对照组植入单纯PPC/CSNF.空白组不做处理,术后第4、8、12周取材,通过大体观察、放射学检查和组织学检查等方法评价其修复缺损情况。
结果
1、全骨髓贴壁法能获得兔BMSCs,可在体外分离扩增。
2、经流式细胞仪分析,BMSCs细胞表面抗原CD29表达为89.23%,CD44表达为90.57%,CD34表达为0.53%,CD45表达为0.61%；G0-G1细胞占92.32%。
3经诱导后BMSCs的细胞形态发生变化,由梭形逐渐转化为三角形或多角形等, ALP染色和Ⅰ型胶原染色阳性,钙化结节染色可见红色致密结节。
4、PPC支架孔径为300-500μm且孔连通性好；PPC/CSNF支架中的壳聚糖纳米纤维分布均匀,其直径为50-500nm；孔隙率为92%。
5、扫描电镜观察BMSCs可在支架材料上较好的粘附生长。
6、将材料植入皮下后无明显组织排斥反应,HE染色未见明显炎症和排斥。7、术后观察,实验组修复缺损处有骨组织形成,支架降解,修复情况接近标准组；对照组少量骨组织形成,材料部分降解；空白组缺损主要由纤维结缔组织填充。
结论
1、全骨髓贴壁法可获得兔BMSCs,可在体外长期、稳定培养,可向成骨细胞诱导分化,是理想的组织工程种子细胞。
2、PPC/CSNF多孔支架具有较好的细胞和组织相容性,在体内可逐渐降解。
3、PPC/CSNF多孔支架复合兔BMSCs可加速新骨形成,达到修复兔股骨髁部骨缺损的目的。
Objective
To isolate and cultivate BMSCs from New Zealand rabbits, fabricate three dimensional PPC/CSNF composite porous scaffolds, and then evaluate the compatibility of the PPC/CSNF composite porous scaffolds and the BMSCs of rabbits in vitro and in vivo. To plant cell-scaffold compound fabricated in vivo to repair bone defect of rabbits, evaluate the effect and conduct a preliminary study on this new tissue engineering method.
Methods
1. BMSCs were isolated from New Zealand rabbits by the whole bone marrow adherence method and cultured.
2. The surface antigen, cell cycle and viability of BMSCs were observed with morphology and flow cytometry methods.
3. BMSCs were induced directionally and differentiated into osteoblast by using the induction culture medium. Their capability of osteogenic differentiation is worked out through morphologic observation, ALP staining, mineralized nodule staining, type I collagen staining, etc.
4. PPC porous scaffold was firstly fabricated by combining a solution-casting and a porogen-leaching technique, followed by in situ phase separation of chitosan solution within the PPC scaffold to form PPC/CSNF composite porous scaffold. The scaffolds were observed by a scaning electron microscope (SEM). And the porosity of the scaffolds were characterized.
5. The two tpyes of scaffolds were co-cultured with BMSCs. They were observed by scanning electron microscope.
6. PPC/CSNF scaffolds were planted into the subcutaneous tissue of rabbits, and were taken after 1,2 and 4 weeks later separately. General observation and HE staining were conducted to analyze the histocompatibility of the materials.
7. The rabbits'femoral condyle defects were modeled and experimented in four groups. PPC/CSNF/BMSCs compound was planted into the defected position in the experimental group; autogenous bone in the criterion group; PPC/CSNF scaffolds in matched group; and the control group was left without any treatment.4,8 and 12 weeks after the operations, general observation, radiology examination and HE staining were made to evaluate the status of defect repairing.
Result
1. BMSCs of rabbit can be obtained through whole bone marrow adherence method and can be cultured in vitro.
2. BMSCs expressed CD29 and CD44 at 89.23% and 90.57% respectively while expressed CD34 and CD45 at 0.53% and 0.61% respectively. The cell cells of G0-G1 phase were 92.32%.
3. The cells morphology changed from spindle to ellipsoid and triangle after been induced. Positive reactions were showed in ALP and collagen typeⅠstaining. Red dense nodules were seen in the mineralized nodules staining.
4. The pores of PPC scaffolds feature diameters varying from 200 to 500μm and have good connectivity. The diameter of chitosan nanofibers in the PPC/CSNF scaffold varied from 50nm to 500nm. All the scaffolds showed a high porosity of 92%.
5. Observed with the scanning electron microscope, the BMSCs had good adhesion with the scaffold.
6. No tissue rejection was seen after the planting of the materials, and no distinct inflammation and rejection was found in the HE staining.
7. The following results were observed after the operation: in experimental group, bone tissue was formed in the defected position, the scaffolds degraded and the effect of repairing was similar to that of the criterion group; in matched group, some bone tissue was formed and the scaffold degraded partly; in control group, the defect was mainly filled with fibrillar connective tissue
Conclusion
1. The BMSCs of rabbit can be obtained through the whole bone marrow adherence method.. They can be cultured stably for long term and be induced into osteoblast. They are the ideal choice for tissue engineering.
2. PPC/CSNF porous scaffold is characterized with good adhesion and tissue compatibility and can degrade gradually in vivo.
3. PPC/CSNF porous scaffold composited with BMSCs of rabbit have a good abliity to accelerate the formation of new bone, and repair the bone defect finally。
分离培养新西兰大白兔骨髓间充质干细胞(bone mesenchymal stem cells, BMSCs),制作聚碳酸亚丙酯／壳聚糖纳米纤维(propylene carbonate/chitosan nanofibers, PPC/CSNF)复合三维多孔支架并评价支架与兔BMSCs的体外相容性、体内组织相容性。体外制作细胞-支架复合物并植入兔股骨髁缺损处,以评价其修复局部骨缺损的效果,初步探讨治疗骨缺损的组织工程学新方法。
方法
1、运用全骨髓贴壁法分离、培养兔BMSCs。
2、通过形态学和流式细胞等方法法观察BMSCs的表面抗原、细胞周期和细胞活力等。
3、用成骨诱导液将BMSCs向成骨细胞定向诱导分化,通过形态学观察、碱性磷酸酶(alkaline phosphatase, ALP)染色、矿化结节染色、Ⅰ型胶原染色等进行分析鉴定,了解所分离培养的BMSCs骨向分化能力。
4、用溶液浇铸／粒子沥滤法制备PPC支架,再用相分离法原位复合三维壳聚糖纳米纤维制备PPC/CSNF支架,测定支架孔隙率,并进行扫描电镜观察。
5、BMSCs分别与两种支架材料联合培养,比较两种支架的细胞相容性,并进行扫描电镜观察。
6、PPC/CSNF多孔支架植入兔皮下,于术后第1,2,4周取出材料,大体观察和苏木精-伊红(hematoxylin-eosin, HE)染色观察材料的组织相容性。
7、制作兔股骨髁部骨缺损模型,分四组实验,实验组缺损处植入PPC/CSNF/BMSCs复合物、标准组移植自体骨、对照组植入单纯PPC/CSNF.空白组不做处理,术后第4、8、12周取材,通过大体观察、放射学检查和组织学检查等方法评价其修复缺损情况。
结果
1、全骨髓贴壁法能获得兔BMSCs,可在体外分离扩增。
2、经流式细胞仪分析,BMSCs细胞表面抗原CD29表达为89.23%,CD44表达为90.57%,CD34表达为0.53%,CD45表达为0.61%；G0-G1细胞占92.32%。
3经诱导后BMSCs的细胞形态发生变化,由梭形逐渐转化为三角形或多角形等, ALP染色和Ⅰ型胶原染色阳性,钙化结节染色可见红色致密结节。
4、PPC支架孔径为300-500μm且孔连通性好；PPC/CSNF支架中的壳聚糖纳米纤维分布均匀,其直径为50-500nm；孔隙率为92%。
5、扫描电镜观察BMSCs可在支架材料上较好的粘附生长。
6、将材料植入皮下后无明显组织排斥反应,HE染色未见明显炎症和排斥。7、术后观察,实验组修复缺损处有骨组织形成,支架降解,修复情况接近标准组；对照组少量骨组织形成,材料部分降解；空白组缺损主要由纤维结缔组织填充。
结论
1、全骨髓贴壁法可获得兔BMSCs,可在体外长期、稳定培养,可向成骨细胞诱导分化,是理想的组织工程种子细胞。
2、PPC/CSNF多孔支架具有较好的细胞和组织相容性,在体内可逐渐降解。
3、PPC/CSNF多孔支架复合兔BMSCs可加速新骨形成,达到修复兔股骨髁部骨缺损的目的。
Objective
To isolate and cultivate BMSCs from New Zealand rabbits, fabricate three dimensional PPC/CSNF composite porous scaffolds, and then evaluate the compatibility of the PPC/CSNF composite porous scaffolds and the BMSCs of rabbits in vitro and in vivo. To plant cell-scaffold compound fabricated in vivo to repair bone defect of rabbits, evaluate the effect and conduct a preliminary study on this new tissue engineering method.
Methods
1. BMSCs were isolated from New Zealand rabbits by the whole bone marrow adherence method and cultured.
2. The surface antigen, cell cycle and viability of BMSCs were observed with morphology and flow cytometry methods.
3. BMSCs were induced directionally and differentiated into osteoblast by using the induction culture medium. Their capability of osteogenic differentiation is worked out through morphologic observation, ALP staining, mineralized nodule staining, type I collagen staining, etc.
4. PPC porous scaffold was firstly fabricated by combining a solution-casting and a porogen-leaching technique, followed by in situ phase separation of chitosan solution within the PPC scaffold to form PPC/CSNF composite porous scaffold. The scaffolds were observed by a scaning electron microscope (SEM). And the porosity of the scaffolds were characterized.
5. The two tpyes of scaffolds were co-cultured with BMSCs. They were observed by scanning electron microscope.
6. PPC/CSNF scaffolds were planted into the subcutaneous tissue of rabbits, and were taken after 1,2 and 4 weeks later separately. General observation and HE staining were conducted to analyze the histocompatibility of the materials.
7. The rabbits'femoral condyle defects were modeled and experimented in four groups. PPC/CSNF/BMSCs compound was planted into the defected position in the experimental group; autogenous bone in the criterion group; PPC/CSNF scaffolds in matched group; and the control group was left without any treatment.4,8 and 12 weeks after the operations, general observation, radiology examination and HE staining were made to evaluate the status of defect repairing.
Result
1. BMSCs of rabbit can be obtained through whole bone marrow adherence method and can be cultured in vitro.
2. BMSCs expressed CD29 and CD44 at 89.23% and 90.57% respectively while expressed CD34 and CD45 at 0.53% and 0.61% respectively. The cell cells of G0-G1 phase were 92.32%.
3. The cells morphology changed from spindle to ellipsoid and triangle after been induced. Positive reactions were showed in ALP and collagen typeⅠstaining. Red dense nodules were seen in the mineralized nodules staining.
4. The pores of PPC scaffolds feature diameters varying from 200 to 500μm and have good connectivity. The diameter of chitosan nanofibers in the PPC/CSNF scaffold varied from 50nm to 500nm. All the scaffolds showed a high porosity of 92%.
5. Observed with the scanning electron microscope, the BMSCs had good adhesion with the scaffold.
6. No tissue rejection was seen after the planting of the materials, and no distinct inflammation and rejection was found in the HE staining.
7. The following results were observed after the operation: in experimental group, bone tissue was formed in the defected position, the scaffolds degraded and the effect of repairing was similar to that of the criterion group; in matched group, some bone tissue was formed and the scaffold degraded partly; in control group, the defect was mainly filled with fibrillar connective tissue
Conclusion
1. The BMSCs of rabbit can be obtained through the whole bone marrow adherence method.. They can be cultured stably for long term and be induced into osteoblast. They are the ideal choice for tissue engineering.
2. PPC/CSNF porous scaffold is characterized with good adhesion and tissue compatibility and can degrade gradually in vivo.
3. PPC/CSNF porous scaffold composited with BMSCs of rabbit have a good abliity to accelerate the formation of new bone, and repair the bone defect finally。
引文
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