包裹BMSCs壳聚糖水凝胶复合CPC骨组织工程支架的构建及治疗骨缺损的实验研究
发布时间：2018-09-01 09:12
【摘要】：目的研究温敏型壳聚糖／β-甘油磷酸钠(chitosan／β-glycerol phosphate C/GP)水凝胶的制作以及作为细胞载体对其中的兔骨髓间充质干细胞（bone marrow mesenchymestem cells, BMSCs）粘附、生长、和增殖的影响。 方法将壳聚糖(chitosan, CS)与β-甘油磷酸钠(β-glycerol phosphate，GP)按照3:1的比例混合后放置于37℃温度条件下可形成固态水凝胶。将体外培养、扩增的兔BMSCs与C/GP溶液混合、固化形成水凝胶后制作冰冻切片行苏木素-伊红(HE)染色观察其粘附和生长情况；通过CCK8法和死/活细胞荧光染色法检测BMSCs在C/GP水凝胶中的生长、增殖情况。 结果BMSCs／C／Gp混合溶液置于37℃培养箱内l0mins可固化形成水凝胶。BMSCs与C/GP溶液混合培养14天后，细胞增值数量多，其形态为长梭形，伸展良好。冰冻切片HE染色显示：C/GP水凝胶呈现均质红染，有孔隙样结构；BMSCs分布较均匀，形态大部分为圆形，胞核蓝染，呈圆形或椭圆形。CCK8法测定实验组光吸收值与对照组比较，除第2天有显著性差异（P 0.05）外，第4、6、8天比较均无显著性差异（P0.05）。死/活细胞荧光染色显示：培养14天后，实验组中BMSCs绝大多数被染成绿色，极少量死细胞被染成红色。细胞形态为多边形，伸展良好。活细胞比率和活细胞密度与对照组比较均无显著性差异（P0.05）。 结论C／Gp温敏型水凝胶在接近于人体内体温的条件下可长期保持水凝胶状态，C／Gp温敏型水凝胶对BMSCs无细胞毒性且适合细胞粘附和生长，为进一步试验奠定了基础。 目的探讨新型大孔隙磷酸钙骨水泥（calcium phosphate cement，CPC）材料支架的细胞毒性和对细胞粘附、生长和增殖的影响。 方法新型CPC在固相的混合过程中，首先将细化后的磷酸四钙（tetracalciumphosphate, TTCP）和磷酸氢钙(dicalcium phosphate anhydrous, DCPA)粉末按1:1（摩尔比）的比例配制成CPC固体粉末；再将质量比为50%的水溶性甘露醇晶体加入到CPC固体粉末中用来制造大孔隙。应用磷酸盐缓冲液为固化液。将CPC固体粉末与固化液按照2g:1ml的比例在研钵中混合均匀，得到糊状混合物，即CPC面团。通过CCK8法检测细胞在新型CPC材料浸提液中的生长增殖情况；通过电子扫描电镜测试材料孔径；应用力学三点弯曲实验测试新型CPC的生物力学性能。 结果通过扫描电镜观察和测量，新型CPC材料的孔径值达到267.43±118.01μm，而传统CPC材料的孔径值只有6.66±2.58μm，两者比较具有显著性差异(p0.05)；新型CPC材料孔隙率为66.15±6.91%，传统CPC材料孔隙率为35.02±4.71%，两者比较具有显著性差异(p0.05)。新型CPC材料的最大负荷、抗弯强度和坚韧度较传统CPC均增加了约1倍(p0.05)，明显提高了其负荷承载能力。CCK8法检测出新型CPC材料浸提液与细胞共培养不同时间后其光密度值（optical density value, OD）值与阴性对照组均无显著性差异(p0.05)。新型CPC材料细胞毒性评级为１级，即该材料对BMSCs无明显毒性。 结论新型CPC材料具有强大的生物力学性能、大孔隙、高孔隙率和良好的生物相容性，有望成为理想的骨组织工程支架。 目的研究温敏型C/GP水凝胶对包裹其中的BMSCs在CPC自凝固化过程中的保护作用以及BMSCs在新型CPC混合材料（包含包裹骨髓间充质干细胞的壳聚糖水凝胶和甘露醇晶体）中的粘附、增殖和成骨分化情况。 方法将体外培养、扩增的兔BMSCs与C/GP溶液混合置于培养板底层，放置37℃条件下固化形成水凝胶；将CPC面团置于培养板中水凝胶层的上方，放置37℃条件下固化。通过CCK8法和死/活细胞荧光染色法检测包裹于C/GP水凝胶中的BMSCs通过CPC自凝固化过程后的细胞生存及增殖活性；通过组织化学方法检测碱性磷酸酶(alkaline phosphatase, ALP)活性；茜素红染色检测钙化结节的表达，RT-PCR检测ALP和降钙素(calcitonin, CT)mRNA的表达。通过电子扫描电镜观察BMSCs在新型CPC材料中的生长及粘附情况。 结果包裹于C/GP水凝胶中的BMSCs在CPC材料中培养14天后的死/活细胞荧光染色显示：活细胞比率为(78.77±2.66)%，，同单纯BMSCs细胞悬液组(82.07±4.30)%和C/GP水凝胶组(80.03±3.08)%比较无显著性差异(p0.05)；活细胞密度为(82.54±4.17)%，同单纯BMSCs细胞悬液组(86.37±4.81)%和C/GP水凝胶组(83.63±5.20)%比较亦无显著性差异(p0.05)。包裹于C/GP水凝胶中的BMSCs在CPC材料中使用成骨培养基培养7天和14天后，ALP、茜素红染色均为阳性同对照组无差异(p0.05)；RT-PCR结果亦显示ALP和CT的mRNA基因表达明显增强。BMSCs在新型CPC复合支架材料上培养5天扫描电子显微镜显示BMSCs向CPC材料的孔隙深部长入并且紧紧地附着在类似纳米羟基磷灰石骨矿物质材料上。 结论温敏型C/GP水凝胶对包裹其中的BMSCs在CPC自凝固化过程中可起到保护作用，新型CPC复合支架材料无细胞毒性，其三维孔隙结构及材料特性适合BMSCs粘附、生长、增殖和成骨分化。 目的研究兔桡骨缺损模型的制作以及观察新型CPC材料对兔桡骨缺损模型的修复治疗效果。 方法选用12只成年健康的新西兰大白兔沿其双侧前臂桡侧暴露桡骨中段截取约1cm骨干制造骨缺损模型。其中6只为试验组植入新型CPC，另6只为对照组植入传统CPC。分别于手术当天、术后第4周、8周和第12周行双上肢X线片检查，于术后第12周行双上肢MRI检查、组织切片HE染色观察和生物力学测定。 结果新型CPC材料植入兔桡骨缺损处后未出现炎性、排斥等不良反应。术后第4周、8周和12周X线片检查显示试验组骨折缺损修复情况明显优于对照组。实验组术后12周螺旋CT三维重建可见桡骨缺损区域与周围正常骨质整合完整，骨组织爬行替代理想，骨折缺损间隙模糊消失。对照组桡骨缺损区域植入的传统CPC材料降解吸收，骨折缺损依旧存在，与周围正常骨质无明显连接整合。术后第12周，通过组织形态学观察实验组成骨及塑形等方面明显优于对照组。兔桡骨三点弯曲试验结果显示：术后第12周实验组的最大负荷（Fmax）、抗弯曲强度(Flexural strength)和载荷/位移(F/d)与对照组比较均具有统计学差异(P 0.05)。而实验组弹性模量与对照组比较无统计学差异（P0.05） 结论新型CPC材料具有良好的生物相容性、降解性和细胞活性，对兔桡骨缺损模型的修复具有良好的治疗效果，对于作为理想的骨组织工程支架具有光明的前景。
[Abstract]:Objective To study the preparation of thermosensitive chitosan/beta-glycerol phosphate C/GP hydrogel and its effect on the adhesion, growth and proliferation of rabbit bone marrow mesenchymal stem cells (BMSCs).
Methods Chitosan (CS) was mixed with beta-glycerol phosphate (GP) in a ratio of 3:1 and then placed at 37 C to form a solid-state hydrogel. The growth and proliferation of BMSCs in C/GP hydrogel were detected by CCK8 and dead/living cell fluorescence staining.
Results BMSCs/C/Gp mixed solution could be solidified to form hydrogel in 37 C incubator for 1 0 min. After 14 days of mixed culture with C/GP solution, the cells proliferated in a long spindle shape and exhibited good extension. Compared with the control group, there was no significant difference (P 0.05) on the 4th, 6th and 8th day except for the 2nd day (P 0.05). Fluorescence staining of dead / living cells showed that the majority of BMSCs in the experimental group were stained green and very few dead cells were found after 14 days of culture. The cells were polygonal and well stretched. There was no significant difference in the ratio of living cells and the density of living cells between the two groups (P 0.05).
Conclusion C/Gp thermosensitive hydrogel can keep the state of hydrogel for a long time near human body temperature. C/Gp thermosensitive hydrogel has no cytotoxicity to BMSCs and is suitable for cell adhesion and growth, which lays a foundation for further experiments.
Objective To investigate the cytotoxicity of a new macroporous calcium phosphate cement (CPC) scaffold and its effect on cell adhesion, growth and proliferation.
Methods In the solid phase mixing process of new CPC, the refined tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA) powders were firstly mixed into CPC solid powders at a ratio of 1:1 (molar ratio), and then the water-soluble mannitol crystal with a mass ratio of 50% was added to CPC solid powders for use. Macroporous materials were prepared by mixing CPC solid powder and solidified solution in a 2 g:1 ml ratio in a mortar to obtain a paste mixture, i.e. CPC dough. Three point bending test was used to test the biomechanical properties of the new CPC.
Results By scanning electron microscopy observation and measurement, the pore size of new CPC material was 267.43 6550 The maximum load, bending strength and toughness of the new CPC material were increased about 1 times than those of the traditional CPC material (p0.05). The load bearing capacity of the new CPC material was significantly improved. The optical density value (OD) of the new CPC material was not significantly different from that of the negative control group (p0.0). 5) the cytotoxicity rating of the new CPC material is 1 grade, that is, the material has no obvious toxicity to BMSCs.
Conclusion The new CPC material has strong biomechanical properties, macropore, high porosity and good biocompatibility, and is expected to be an ideal scaffold for bone tissue engineering.
Objective To study the protective effect of temperature-sensitive C/GP hydrogel on the encapsulated BMSCs during CPC autocoagulation and the adhesion, proliferation and osteogenic differentiation of BMSCs in novel CPC mixtures including chitosan hydrogel and mannitol crystals encapsulated bone marrow mesenchymal stem cells.
Methods Rabbit BMSCs were cultured in vitro and mixed with C/GP solution to form a hydrogel. The CPC dough was placed above the hydrogel layer in the culture plate and cured at 37. The BMSCs wrapped in the C/GP hydrogel were detected by CCK8 and dead/living cell fluorescence staining. Alkaline phosphatase (ALP) activity was detected by histochemical method; the expression of calcified nodules was detected by alizarin red staining; the expression of ALP and calcitonin (CT) mRNA was detected by RT-PCR. The growth of BMSCs in new CPC materials was observed by scanning electron microscopy (SEM). And adhesion.
Results BMSCs wrapped in C/GP hydrogel cultured in CPC material for 14 days showed that the ratio of viable cells was (78.77 [2.66]%. There was no significant difference between BMSCs group and BMSCs group (82.07 [4.30]% and C/GP hydrogel group (80.03 [3.08]%) (p0.05). The density of viable cells was (82.54 [4.17]%, and BMSCs group was fine. There was no significant difference (p0.05) between the cell suspension group (86.37 4.81)% and the C/GP hydrogel group (83.63 Scanning electron microscopy showed that BMSCs grew deep into the pores of the CPC scaffolds and adhered tightly to nano-hydroxyapatite-like bone mineral materials.
Conclusion Temperature-sensitive C/GP hydrogel can protect BMSCs encapsulated in CPC during self-coagulation and solidification. The new CPC composite scaffold material has no cytotoxicity. Its three-dimensional pore structure and material characteristics are suitable for BMSCs adhesion, growth, proliferation and osteogenic differentiation.
Objective To study the fabrication of rabbit radius defect model and the effect of new CPC material on the repair of rabbit radius defect model.
Methods Twelve healthy adult New Zealand white rabbits were used to make bone defect models by cutting off about 1 cm of radial shaft along the radial side of their forearms. MRI examination was performed on both upper extremities. Tissue sections were stained with HE and observed by biomechanics.
Results There were no adverse reactions such as inflammation and rejection after implantation of the new CPC material into the radial defect of rabbits.X-ray examination showed that the repair of the fracture defect in the experimental group was better than that in the control group at the 4th, 8th and 12th weeks after implantation.The three-dimensional reconstruction of spiral CT in the experimental group at the 12th week after implantation showed that the radial defect area was integrated with the surrounding normal bone and the bone tissue crawled. In the control group, the traditional CPC material implanted in the radial defect area was degraded and absorbed, the fracture defect still existed, and there was no obvious connection and integration with the surrounding normal bone. The results showed that the maximum load (Fmax), flexural strength (F/d) and load/displacement (F/d) of the experimental group were significantly different from those of the control group at 12 weeks after operation (P 0.05). However, there was no significant difference in the modulus of elasticity between the experimental group and the control group (P 0.05).
Conclusion The new CPC material has good biocompatibility, biodegradability and cell activity, and has a good therapeutic effect on the repair of rabbit radius defect model. It has a bright future as an ideal scaffold for bone tissue engineering.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.08
本文编号：2216734
[Abstract]:Objective To study the preparation of thermosensitive chitosan/beta-glycerol phosphate C/GP hydrogel and its effect on the adhesion, growth and proliferation of rabbit bone marrow mesenchymal stem cells (BMSCs).
Methods Chitosan (CS) was mixed with beta-glycerol phosphate (GP) in a ratio of 3:1 and then placed at 37 C to form a solid-state hydrogel. The growth and proliferation of BMSCs in C/GP hydrogel were detected by CCK8 and dead/living cell fluorescence staining.
Results BMSCs/C/Gp mixed solution could be solidified to form hydrogel in 37 C incubator for 1 0 min. After 14 days of mixed culture with C/GP solution, the cells proliferated in a long spindle shape and exhibited good extension. Compared with the control group, there was no significant difference (P 0.05) on the 4th, 6th and 8th day except for the 2nd day (P 0.05). Fluorescence staining of dead / living cells showed that the majority of BMSCs in the experimental group were stained green and very few dead cells were found after 14 days of culture. The cells were polygonal and well stretched. There was no significant difference in the ratio of living cells and the density of living cells between the two groups (P 0.05).
Conclusion C/Gp thermosensitive hydrogel can keep the state of hydrogel for a long time near human body temperature. C/Gp thermosensitive hydrogel has no cytotoxicity to BMSCs and is suitable for cell adhesion and growth, which lays a foundation for further experiments.
Objective To investigate the cytotoxicity of a new macroporous calcium phosphate cement (CPC) scaffold and its effect on cell adhesion, growth and proliferation.
Methods In the solid phase mixing process of new CPC, the refined tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA) powders were firstly mixed into CPC solid powders at a ratio of 1:1 (molar ratio), and then the water-soluble mannitol crystal with a mass ratio of 50% was added to CPC solid powders for use. Macroporous materials were prepared by mixing CPC solid powder and solidified solution in a 2 g:1 ml ratio in a mortar to obtain a paste mixture, i.e. CPC dough. Three point bending test was used to test the biomechanical properties of the new CPC.
Results By scanning electron microscopy observation and measurement, the pore size of new CPC material was 267.43 6550 The maximum load, bending strength and toughness of the new CPC material were increased about 1 times than those of the traditional CPC material (p0.05). The load bearing capacity of the new CPC material was significantly improved. The optical density value (OD) of the new CPC material was not significantly different from that of the negative control group (p0.0). 5) the cytotoxicity rating of the new CPC material is 1 grade, that is, the material has no obvious toxicity to BMSCs.
Conclusion The new CPC material has strong biomechanical properties, macropore, high porosity and good biocompatibility, and is expected to be an ideal scaffold for bone tissue engineering.
Objective To study the protective effect of temperature-sensitive C/GP hydrogel on the encapsulated BMSCs during CPC autocoagulation and the adhesion, proliferation and osteogenic differentiation of BMSCs in novel CPC mixtures including chitosan hydrogel and mannitol crystals encapsulated bone marrow mesenchymal stem cells.
Methods Rabbit BMSCs were cultured in vitro and mixed with C/GP solution to form a hydrogel. The CPC dough was placed above the hydrogel layer in the culture plate and cured at 37. The BMSCs wrapped in the C/GP hydrogel were detected by CCK8 and dead/living cell fluorescence staining. Alkaline phosphatase (ALP) activity was detected by histochemical method; the expression of calcified nodules was detected by alizarin red staining; the expression of ALP and calcitonin (CT) mRNA was detected by RT-PCR. The growth of BMSCs in new CPC materials was observed by scanning electron microscopy (SEM). And adhesion.
Results BMSCs wrapped in C/GP hydrogel cultured in CPC material for 14 days showed that the ratio of viable cells was (78.77 [2.66]%. There was no significant difference between BMSCs group and BMSCs group (82.07 [4.30]% and C/GP hydrogel group (80.03 [3.08]%) (p0.05). The density of viable cells was (82.54 [4.17]%, and BMSCs group was fine. There was no significant difference (p0.05) between the cell suspension group (86.37 4.81)% and the C/GP hydrogel group (83.63 Scanning electron microscopy showed that BMSCs grew deep into the pores of the CPC scaffolds and adhered tightly to nano-hydroxyapatite-like bone mineral materials.
Conclusion Temperature-sensitive C/GP hydrogel can protect BMSCs encapsulated in CPC during self-coagulation and solidification. The new CPC composite scaffold material has no cytotoxicity. Its three-dimensional pore structure and material characteristics are suitable for BMSCs adhesion, growth, proliferation and osteogenic differentiation.
Objective To study the fabrication of rabbit radius defect model and the effect of new CPC material on the repair of rabbit radius defect model.
Methods Twelve healthy adult New Zealand white rabbits were used to make bone defect models by cutting off about 1 cm of radial shaft along the radial side of their forearms. MRI examination was performed on both upper extremities. Tissue sections were stained with HE and observed by biomechanics.
Results There were no adverse reactions such as inflammation and rejection after implantation of the new CPC material into the radial defect of rabbits.X-ray examination showed that the repair of the fracture defect in the experimental group was better than that in the control group at the 4th, 8th and 12th weeks after implantation.The three-dimensional reconstruction of spiral CT in the experimental group at the 12th week after implantation showed that the radial defect area was integrated with the surrounding normal bone and the bone tissue crawled. In the control group, the traditional CPC material implanted in the radial defect area was degraded and absorbed, the fracture defect still existed, and there was no obvious connection and integration with the surrounding normal bone. The results showed that the maximum load (Fmax), flexural strength (F/d) and load/displacement (F/d) of the experimental group were significantly different from those of the control group at 12 weeks after operation (P 0.05). However, there was no significant difference in the modulus of elasticity between the experimental group and the control group (P 0.05).
Conclusion The new CPC material has good biocompatibility, biodegradability and cell activity, and has a good therapeutic effect on the repair of rabbit radius defect model. It has a bright future as an ideal scaffold for bone tissue engineering.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.08
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