rhBMP-2与bFGF序贯缓释对去抗原猪松质骨异体移植成骨效能的影响
发布时间：2018-07-17 01:49
【摘要】：目的 通过组织工程学的方法，利用壳聚糖纳米微粒的缓释作用，构建rhBMP-2与bFGF序贯缓释系统，然后复合于去抗原处理的猪松质骨（Antigen-extractedxenogeneic cancellous bone，AXCB），移植于小鼠腓骨肌袋进行异体成骨效能研究。通过动物实验，研究rhBMP-2与bFGF联合应用时不同缓释顺序对去抗原猪松质骨异体移植的成骨效能的影响，探讨rhBMP-2与bFGF分别在骨形成过程中的作用机制及相互作用的机制。设计并证明具有良好促骨形成效能的rhBMP-2与bFGF序贯缓释系统，为进一步的实验研究及以后的临床应用提供理论基础和实验依据。 方法 实验一：采用表面涂覆/沉积的方法，利用壳聚糖纳米颗粒包裹rhBMP-2小分子与bFGF小分子构建CS/rhBMP-2/bFGF序贯缓释系统，并将该复合纳米颗粒涂覆于去抗原处理后的猪松质骨块的骨小梁结构表面，然后扫描电镜观察形态、比重法检测孔隙率，压缩模量测试检测生物力学性能； 实验二：进行材料载药与体外释放实验，通过包封率和载药量的测定来确定材料的载药能力，并计算药物的缓释时间，描绘缓释曲线，验证复合移植骨材料的成功制备。 实验三：设计动物实验研究方案，进行材料组织相容性及异体成骨效能的研究。选取约30g重的昆明SD小鼠64只，随机分为实验组及对照组两大组，其中实验组按照CS/rhBMP-2/bFGF序贯缓释顺序的不同分为6组：①实验组A：AXCB+rhBMP-2/bFGF，②实验组B：AXCB+CS/rhBMP-2/bFGF，③实验组C：AXCB+rhBMP-2/CS/bFGF，④实验组D：AXCB+bFGF/CS/rhBMP-2，⑤实验组E：AXCB+CS/rhBMP-2，⑥实验组F：AXCB+CS/bFGF；对照组又分为两组：⑦空白对照组G：AXCB+CS，及⑧阴性对照组H：不植入任何材料组。于2w、4w两个时间点分别处死动物后获取标本，进行大体标本观察与称重、μCT观察骨组织显微三维结构、骨参数测量分析、钙含量检测、组织形态学染色（HE染色）以及组织化学染色（ALP免疫组化染色及CD34免疫组化染色）等。对实验得到的所有数据均以x±s表示，应用SPSS13.0软件，采用完全随机设计的单因素方差分析、多个样本均数两两比较的SNK-q检验等方法对数据进行统计分析，以α=0.05为检验水准，规定P0.05为差异具有统计学意义。 结果 实验一：AXCB/CS/rhBMP/bFGF序贯缓释系统的制备与表征 1.1扫描电镜结果显示去抗原猪松质骨块具有疏松多孔状结构，且附载的CS/rhBMP-2/bFGF缓释系统呈约100纳米的颗粒状结构吸附于骨小梁表面； 1.2孔隙率测定结果显示去抗原猪松质骨块具有高达85%的孔隙率，且吸附不同缓释顺序的生长因子纳米颗粒后，其孔隙率均没有发生较大的改变，均保持在85%-86%之间； 1.3力学性能分析结果显示去抗原猪松质骨块的压缩模量为13.44MPa，吸附不同缓释顺序的生长因子纳米颗粒后，其压缩模量均没有发生较大的改变，均保持在12MPa-14MPa之间，且其强度均能达到骨组织工程对于植入材料强度的要求； 实验二：AXCB/CS/rhBMP/bFGF序贯缓释系统的载药及释放研究 2.1各组材料中rhBMP-2及bFGF的包封率均较为接近，分别在55%及60%以上；且各组材料中rhBMP-2及bFGF的载药量也较为接近，分别在22‰及0.012‰左右； 2.2体外释放试验所描绘的释放曲线可知，rhBMP-2与bFGF同时暴释组分别在第9天达到rhBMP80%的累积释放率及第13天达到bFGF70%的累积释放率；rhBMP-2与bFGF同时缓释组则rhBMP-2与bFGF持续释放至第23天；rhBMP-2先暴释，bFGF后缓释组中rhBMP-2第9天就达到80%的累积释放率，而bFGF第8天开始释放，持续释放至第26天累积达70%；bFGF先暴释，rhBMP-2后缓释组中bFGF第13天就达到70%的累积释放率，rhBMP-2第7天开始释放，持续释放至第28天累积达80%。单纯rhBMP-2缓释组中rhBMP-2持续释放至第24天时累积释放率才达到80%；单纯bFGF缓释组中bFGF持续释放至第22天时累积释放率才达到70%。 实验三：AXCB/CS/rhBMP/bFGF序贯缓释系统的异体成骨实验 3.1大体观察及称重结果显示，A-2、B-2、C-2、A-4、B-4、C-4、D-4、E-4材料重量明显高于术前材料重量（P0.05）；2周时A组材料重量高于其他组别（P0.05）；4周时B组材料重量高于其他组别（P0.05）；且B、E组4周时材料重量高于2周时（P0.05）； 3.2钙含量检测结果显示2周时各组材料钙含量浓度值比较为A、C组B组D、E、F、G组(P0.05)，且A组的钙含量浓度最大(348.5±9.66mg/dL)；4周时为B组A、C、D、E组F、G组(P0.05)，且B组的钙含量浓度最大(414.7±12.03mg/dL)。同时B、D、E组4周时的钙含量浓度均高于2周时(P0.05)，分别高出123.1mg/dL、78.2mg/dL、88.3mg/dL; 3.3μCT扫描及骨参数分析结果显示，所有植入的去抗原猪松质骨块的骨体积、骨表面积及骨体积分数均相近(P0.05)；而骨矿物质密度分析发现，2周时各组材料骨密度值比较为A、C、E组B组D、F、G高(P0.05)，且A组的骨密度值最大(307.09±8.27mg/cc)；4周时为B组A、C、D、E组F、G组(P0.05)，且B组的骨密度值最大(367.52±11.64mg/cc)；同时A、B、D、E组4周时的骨密度值均高于2周时(P0.05)，分别高出32.55mg/cc、89.06mg/cc、38.18mg/cc、29.57mg/cc；骨小梁厚度分析发现，2周时各组材料骨小梁厚度比较为A组B、C、E组D、F、G高(P0.05)，且A组的骨小梁厚度最大(95.33±7.28μm)；4周时为B组A、C、D组E、F、G组(P0.05)，且B组的骨小梁厚度最大(126.17±11.36μm)；同时A、B、C、D组4周时的骨小梁厚度均高于2周时(P0.05)，分别高出14.51μm、39.75μm、23.15μm、22.49μm； 3.4H.E.染色结果显示A组、B组、C组、D组及E组在组织学HE染色结果中均可见较大区域的钙化软骨及造血组织的形成，在新生的钙化软骨上可见透亮的软骨细胞及软骨边缘可见的骨母细胞衬里于软骨边缘，同时，在新生的软骨与植入的异种骨小梁及周围软组织之间可见红细胞充盈的造血组织，其中深染的细胞为造血细胞，红染的细胞为血红细胞。 3.5碱性磷酸酶免疫组化染色结果显示，全部植入材料的小鼠部位均可见不同程度程度的ALP阳性表达区域，主要位于新生软骨的外周，表达于软骨细胞的胞浆内。其中A-2组、A-4组、B-4组、C-4组、D-4组及E-4组中ALP的阳性表达要高于其他组； 3.6CD34免疫组化染色结果显示全部植入材料的小鼠部位均可见不同程度程度的CD34阳性表达区域，主要表达于血管内皮细胞的胞膜和胞浆。A-2组、A-4组、B-4组、C-4组、D-4组及F-4组中CD34阳性表达要高于其他组。 结论 1本实验已成功完成对AXCB/CS/rhBMP-2/bFGF序贯缓释系统的制备、表征、体外释放及小鼠体内成骨效能的研究。 2本实验制备的AXCB/CS/rhBMP-2/bFGF序贯缓释系统具有理想的疏松多孔状结构，，生物力学性能满足骨组织工程要求，显微结构形貌观察可见壳聚糖包裹生长因子的纳米颗粒成功地吸附于骨小梁结构表面，且各组材料中rhBMP-2与bFGF的总量及其各自所占移植材料总质量的比例之间无差异； 3体外释放实验证实了AXCB/CS/rhBMP-2/bFGF序贯缓释系统的成功制备，各组材料中rhBMP-2与bFGF均按照实验设计实现先后的缓慢释放顺序。 4动物实验结果均表明2周时，rhBMP-2与bFGF同时暴释组诱导去抗原猪松质骨的成骨效能最高；而4周时，则是rhBMP-2与bFGF同时缓释组诱导去抗原猪松质骨的成骨效能最高；鉴于新骨形成及矿化的周期时间，rhBMP-2与bFGF同时缓释是最为理想的释药方式，对去抗原猪松质骨异体移植具有更好的诱导成骨作用。
[Abstract]:objective
By means of tissue engineering, the slow release of chitosan nanoparticles was used to construct rhBMP-2 and bFGF sequential sustained release system, and then combined with Antigen-extractedxenogeneic cancellous bone (AXCB), which was treated with antigen treatment, and transplanted into the fibula muscle bag of mice to study the osteogenic effect of allogenic bone. The effect of different slow release sequence on the osteogenesis of allogenic porcine cancellous bone allograft in combination with bFGF, and the mechanism of action and interaction between rhBMP-2 and bFGF during bone formation, and the design and demonstration of rhBMP-2 and bFGF sequential sustained release system with good bone formation efficiency for further experimental study and It provides theoretical basis and experimental evidence for future clinical application.
Method
Experiment 1: using the method of surface coating / deposition, chitosan nanoparticles were used to encapsulate rhBMP-2 small molecules and bFGF small molecules to construct CS/rhBMP-2/bFGF sequential slow release system, and the composite nanoparticles were coated on the surface of bone trabecular structure of porcine cancellous bone mass after antigenic treatment, and then scanning electron microscopy was used to observe morphology and specific gravity method to detect holes. The porosity and compression modulus were tested to detect biomechanical properties.
Experiment two: the drug loading and in vitro release experiment were carried out. The drug carrying capacity of the material was determined by the encapsulation rate and the measurement of drug loading, and the release time of the drug was calculated, the sustained release curve was depicted, and the successful preparation of the composite bone graft was verified.
Experiment three: Design animal experimental research program, study the tissue compatibility and allogenic osteogenesis efficiency of materials, select 64 Kunming SD mice with about 30g weight, randomly divide into experimental group and control group two groups, and the experimental group is divided into 6 groups according to the sequence of CS/rhBMP-2/bFGF sequential slow release: (1) the experimental group A:AXCB+rhBMP-2/bFGF, 2 The test group B:AXCB+CS/rhBMP-2/bFGF, the experimental group C:AXCB+rhBMP-2/CS/bFGF, the experimental group D:AXCB+bFGF/CS/rhBMP-2, the experimental group E:AXCB+CS/rhBMP-2, and the experimental group F:AXCB+CS/bFGF; the control group is divided into two groups: the blank control group G:AXCB+CS, and the negative control group H: not in any material group. In 2W, 4W two time points. After the animals were executed, the specimens were collected and the specimen was observed and weighed. The microstructure of the bone tissue was observed by micron CT, the bone parameters were measured and analyzed, the calcium content was detected, the histomorphological staining (HE staining) and histochemical staining (ALP immunohistochemical staining and CD34 immunohistochemical staining) were used. All the data obtained by the experiment were x + s, SPSS13.0 software is used to analyze the data by means of a single factor analysis of variance and SNK-q test of multiple samples, which are compared with two or two, with a test level of alpha =0.05, and the difference between P0.05 and P0.05 has statistical significance.
Result
Experiment 1: preparation and characterization of AXCB/CS/rhBMP/bFGF sequential release system
1.1 the results of scanning electron microscopy showed that the antigenic porcine cancellous bone block had porous and porous structure, and the loaded CS/rhBMP-2/bFGF sustained release system was about 100 nanometers of granular structure adsorbed on the surface of bone trabecula.
The results of 1.2 porosity determination showed that the porcine cancellous bone mass of the antiantigen was up to 85% porosity, and the porosity of the growth factor nanoparticles had not changed greatly after the adsorption of different slow release order growth factor nanoparticles, and all remained at 85%-86%.
1.3 the results of mechanical properties analysis showed that the compression modulus of the antigenic porcine cancellous bone block was 13.44MPa, and the compression modulus was not changed greatly after the adsorption of different slow release growth factor nanoscale nanoparticles, and the strength of the bone fabric could meet the requirement of the strength of the implant.
Experiment two: drug loading and release of AXCB/CS/rhBMP/bFGF sequential release system
2.1 the encapsulation efficiency of rhBMP-2 and bFGF in all kinds of materials were all close to 55% and 60%, respectively, and the drug loading of rhBMP-2 and bFGF in each group were also close, respectively, at 22 per thousand and 0.012 per thousand, respectively.
The release curve depicted in 2.2 in vitro release test showed that the cumulative release rate of rhBMP-2 and bFGF reached rhBMP80% at ninth days and the cumulative release rate of bFGF70% on the 13 day, while rhBMP-2 and bFGF sustained release to twenty-third days in rhBMP-2 and bFGF simultaneous release group, rhBMP-2 first release and rhBMP-2 ninth days in bFGF after release group. The cumulative release rate of 80% was reached, while bFGF eighth days began to release, sustained release to 70% in twenty-sixth days; bFGF was first released, and the cumulative release rate of bFGF thirteenth days in rhBMP-2 release group was reached, rhBMP-2 seventh days began to release, and continued to release to twenty-eighth days to accumulate to accumulate to twenty-fourth days when rhBMP-2 continued to accumulate to twenty-fourth days. The release rate reached 80%, and the sustained release rate of bFGF in the bFGF slow release group reached 70%. only after twenty-second days.
Experiment three: allogeneic osteogenesis of AXCB/CS/rhBMP/bFGF sequential release system.
3.1 gross observation and weighing results showed that the weight of A-2, B-2, C-2, A-4, B-4, C-4, D-4, E-4 was significantly higher than the weight of pre operation material (P0.05), and at the 2 week, the weight of the A group was higher than that of the other groups (P0.05), and the material weight of the B group was higher than that of the other groups at 4 weeks, and the weight of the material was higher than 2 weeks at the week of 4.
The results of 3.2 calcium content test showed that the calcium content of each group was A at 2 weeks, C group B group D, E, F, G group (P0.05), and the concentration of calcium content in A group was maximum (348.5 + 9.66mg/dL), and 4 weeks was B group A, and the concentration of calcium content was the largest (414.7 +). The time (P0.05) is higher than 123.1mg/dL, 78.2mg/dL and 88.3mg/dL respectively.
3.3 CT scanning and bone parameter analysis showed that the bone volume, bone surface area and bone volume fraction of all implanted antigen porcine cancellous bone were similar (P0.05), while bone mineral density analysis showed that the bone mineral density of each group was A, C, B group D, F, G high (P0.05) at the time of 2 weeks, and the BMD value was maximum (307.09 + 8.27mg/cc) in A group (307.09 + 8.27mg/cc); 4 B group A, C, D, E group F, G group (P0.05), and B group (P0.05) in B group (367.52 + 11.64mg/cc). (P0.05), and the thickness of bone trabecula in group A was maximum (95.33 + 7.28 m), and at 4 weeks, B group A, C, D group E, F, G group (P0.05), and B group bone trabecular thickness was maximum (126.17 + 11.36 micron). At the same time, the small bone Liang Houdu of 4 weeks was higher than 2 weeks, 39.75 mu, 23.15 mu, 22.49 micron;
The results of 3.4H.E. staining showed that in group A, group B, group C, group D and group E, the formation of calcified cartilage and hematopoietic tissue in the larger region were found in the histological HE staining results, and the bright chondrocytes and cartilage margins were visible on the edge of cartilage on the newborn calcified cartilage, while the new cartilage and the implanted xenogeneic bone were found. Red blood cells filled with hematopoietic tissue were found between trabecular and surrounding soft tissues. Deep stained cells were hematopoietic cells and red stained cells were erythrocytes.
3.5 the results of immuno histochemical staining of alkaline phosphatase showed that all the implanted materials showed ALP positive expression regions of different degrees, mainly located in the outer circumference of the newborn cartilage, and expressed in the cytoplasm of chondrocytes. The positive expression of ALP in group A-2, group A-4, B-4, C-4, D-4 and E-4 groups was higher than that of other groups.
The results of 3.6CD34 immunohistochemical staining showed that all the implanted materials showed different degrees of CD34 positive expression in different degrees, mainly expressed in the cell membrane and cytoplasm.A-2 group of vascular endothelial cells, and the positive expression of CD34 in group A-4, B-4 group, C-4 group, D-4 group and F-4 group was higher than that of other groups.
conclusion
1 this experiment has successfully completed the preparation, characterization, in vitro release and osteogenic efficiency of AXCB/CS/rhBMP-2/bFGF sequential release system.
2 the AXCB/CS/rhBMP-2/bFGF sequential sustained release system prepared by this experiment has ideal loose porous structure and the biomechanical properties meet the requirements of bone tissue engineering. The microstructure and morphology observe that the chitosan coated growth factor nanoparticles have been successfully adsorbed on the surface of the bone trabecular structure, and the total amount of rhBMP-2 and bFGF in each material and the total amount of the particles in the materials are also observed. There was no difference in the proportion of total mass of transplanted materials.
3 in vitro release test confirmed the successful preparation of AXCB/CS/rhBMP-2/bFGF sequential sustained release system. Both rhBMP-2 and bFGF in all the materials were designed to achieve the slow release sequence according to the experimental design.
The results of 4 animal experiments showed that at 2 weeks, the osteogenic efficacy of rhBMP-2 and bFGF induced antigenic porcine cancellous bone was highest, while at 4 weeks, it was the highest osteogenic efficacy of rhBMP-2 and bFGF simultaneous release group to induce antigenic porcine cancellous bone. In view of the period of new bone formation and mineralization, the simultaneous release of rhBMP-2 and bFGF was the most ideal. The release method has a better osteogenic effect on xenotransplantation of cancellous bone.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R782.2
本文编号：2128496
[Abstract]:objective
By means of tissue engineering, the slow release of chitosan nanoparticles was used to construct rhBMP-2 and bFGF sequential sustained release system, and then combined with Antigen-extractedxenogeneic cancellous bone (AXCB), which was treated with antigen treatment, and transplanted into the fibula muscle bag of mice to study the osteogenic effect of allogenic bone. The effect of different slow release sequence on the osteogenesis of allogenic porcine cancellous bone allograft in combination with bFGF, and the mechanism of action and interaction between rhBMP-2 and bFGF during bone formation, and the design and demonstration of rhBMP-2 and bFGF sequential sustained release system with good bone formation efficiency for further experimental study and It provides theoretical basis and experimental evidence for future clinical application.
Method
Experiment 1: using the method of surface coating / deposition, chitosan nanoparticles were used to encapsulate rhBMP-2 small molecules and bFGF small molecules to construct CS/rhBMP-2/bFGF sequential slow release system, and the composite nanoparticles were coated on the surface of bone trabecular structure of porcine cancellous bone mass after antigenic treatment, and then scanning electron microscopy was used to observe morphology and specific gravity method to detect holes. The porosity and compression modulus were tested to detect biomechanical properties.
Experiment two: the drug loading and in vitro release experiment were carried out. The drug carrying capacity of the material was determined by the encapsulation rate and the measurement of drug loading, and the release time of the drug was calculated, the sustained release curve was depicted, and the successful preparation of the composite bone graft was verified.
Experiment three: Design animal experimental research program, study the tissue compatibility and allogenic osteogenesis efficiency of materials, select 64 Kunming SD mice with about 30g weight, randomly divide into experimental group and control group two groups, and the experimental group is divided into 6 groups according to the sequence of CS/rhBMP-2/bFGF sequential slow release: (1) the experimental group A:AXCB+rhBMP-2/bFGF, 2 The test group B:AXCB+CS/rhBMP-2/bFGF, the experimental group C:AXCB+rhBMP-2/CS/bFGF, the experimental group D:AXCB+bFGF/CS/rhBMP-2, the experimental group E:AXCB+CS/rhBMP-2, and the experimental group F:AXCB+CS/bFGF; the control group is divided into two groups: the blank control group G:AXCB+CS, and the negative control group H: not in any material group. In 2W, 4W two time points. After the animals were executed, the specimens were collected and the specimen was observed and weighed. The microstructure of the bone tissue was observed by micron CT, the bone parameters were measured and analyzed, the calcium content was detected, the histomorphological staining (HE staining) and histochemical staining (ALP immunohistochemical staining and CD34 immunohistochemical staining) were used. All the data obtained by the experiment were x + s, SPSS13.0 software is used to analyze the data by means of a single factor analysis of variance and SNK-q test of multiple samples, which are compared with two or two, with a test level of alpha =0.05, and the difference between P0.05 and P0.05 has statistical significance.
Result
Experiment 1: preparation and characterization of AXCB/CS/rhBMP/bFGF sequential release system
1.1 the results of scanning electron microscopy showed that the antigenic porcine cancellous bone block had porous and porous structure, and the loaded CS/rhBMP-2/bFGF sustained release system was about 100 nanometers of granular structure adsorbed on the surface of bone trabecula.
The results of 1.2 porosity determination showed that the porcine cancellous bone mass of the antiantigen was up to 85% porosity, and the porosity of the growth factor nanoparticles had not changed greatly after the adsorption of different slow release order growth factor nanoparticles, and all remained at 85%-86%.
1.3 the results of mechanical properties analysis showed that the compression modulus of the antigenic porcine cancellous bone block was 13.44MPa, and the compression modulus was not changed greatly after the adsorption of different slow release growth factor nanoscale nanoparticles, and the strength of the bone fabric could meet the requirement of the strength of the implant.
Experiment two: drug loading and release of AXCB/CS/rhBMP/bFGF sequential release system
2.1 the encapsulation efficiency of rhBMP-2 and bFGF in all kinds of materials were all close to 55% and 60%, respectively, and the drug loading of rhBMP-2 and bFGF in each group were also close, respectively, at 22 per thousand and 0.012 per thousand, respectively.
The release curve depicted in 2.2 in vitro release test showed that the cumulative release rate of rhBMP-2 and bFGF reached rhBMP80% at ninth days and the cumulative release rate of bFGF70% on the 13 day, while rhBMP-2 and bFGF sustained release to twenty-third days in rhBMP-2 and bFGF simultaneous release group, rhBMP-2 first release and rhBMP-2 ninth days in bFGF after release group. The cumulative release rate of 80% was reached, while bFGF eighth days began to release, sustained release to 70% in twenty-sixth days; bFGF was first released, and the cumulative release rate of bFGF thirteenth days in rhBMP-2 release group was reached, rhBMP-2 seventh days began to release, and continued to release to twenty-eighth days to accumulate to accumulate to twenty-fourth days when rhBMP-2 continued to accumulate to twenty-fourth days. The release rate reached 80%, and the sustained release rate of bFGF in the bFGF slow release group reached 70%. only after twenty-second days.
Experiment three: allogeneic osteogenesis of AXCB/CS/rhBMP/bFGF sequential release system.
3.1 gross observation and weighing results showed that the weight of A-2, B-2, C-2, A-4, B-4, C-4, D-4, E-4 was significantly higher than the weight of pre operation material (P0.05), and at the 2 week, the weight of the A group was higher than that of the other groups (P0.05), and the material weight of the B group was higher than that of the other groups at 4 weeks, and the weight of the material was higher than 2 weeks at the week of 4.
The results of 3.2 calcium content test showed that the calcium content of each group was A at 2 weeks, C group B group D, E, F, G group (P0.05), and the concentration of calcium content in A group was maximum (348.5 + 9.66mg/dL), and 4 weeks was B group A, and the concentration of calcium content was the largest (414.7 +). The time (P0.05) is higher than 123.1mg/dL, 78.2mg/dL and 88.3mg/dL respectively.
3.3 CT scanning and bone parameter analysis showed that the bone volume, bone surface area and bone volume fraction of all implanted antigen porcine cancellous bone were similar (P0.05), while bone mineral density analysis showed that the bone mineral density of each group was A, C, B group D, F, G high (P0.05) at the time of 2 weeks, and the BMD value was maximum (307.09 + 8.27mg/cc) in A group (307.09 + 8.27mg/cc); 4 B group A, C, D, E group F, G group (P0.05), and B group (P0.05) in B group (367.52 + 11.64mg/cc). (P0.05), and the thickness of bone trabecula in group A was maximum (95.33 + 7.28 m), and at 4 weeks, B group A, C, D group E, F, G group (P0.05), and B group bone trabecular thickness was maximum (126.17 + 11.36 micron). At the same time, the small bone Liang Houdu of 4 weeks was higher than 2 weeks, 39.75 mu, 23.15 mu, 22.49 micron;
The results of 3.4H.E. staining showed that in group A, group B, group C, group D and group E, the formation of calcified cartilage and hematopoietic tissue in the larger region were found in the histological HE staining results, and the bright chondrocytes and cartilage margins were visible on the edge of cartilage on the newborn calcified cartilage, while the new cartilage and the implanted xenogeneic bone were found. Red blood cells filled with hematopoietic tissue were found between trabecular and surrounding soft tissues. Deep stained cells were hematopoietic cells and red stained cells were erythrocytes.
3.5 the results of immuno histochemical staining of alkaline phosphatase showed that all the implanted materials showed ALP positive expression regions of different degrees, mainly located in the outer circumference of the newborn cartilage, and expressed in the cytoplasm of chondrocytes. The positive expression of ALP in group A-2, group A-4, B-4, C-4, D-4 and E-4 groups was higher than that of other groups.
The results of 3.6CD34 immunohistochemical staining showed that all the implanted materials showed different degrees of CD34 positive expression in different degrees, mainly expressed in the cell membrane and cytoplasm.A-2 group of vascular endothelial cells, and the positive expression of CD34 in group A-4, B-4 group, C-4 group, D-4 group and F-4 group was higher than that of other groups.
conclusion
1 this experiment has successfully completed the preparation, characterization, in vitro release and osteogenic efficiency of AXCB/CS/rhBMP-2/bFGF sequential release system.
2 the AXCB/CS/rhBMP-2/bFGF sequential sustained release system prepared by this experiment has ideal loose porous structure and the biomechanical properties meet the requirements of bone tissue engineering. The microstructure and morphology observe that the chitosan coated growth factor nanoparticles have been successfully adsorbed on the surface of the bone trabecular structure, and the total amount of rhBMP-2 and bFGF in each material and the total amount of the particles in the materials are also observed. There was no difference in the proportion of total mass of transplanted materials.
3 in vitro release test confirmed the successful preparation of AXCB/CS/rhBMP-2/bFGF sequential sustained release system. Both rhBMP-2 and bFGF in all the materials were designed to achieve the slow release sequence according to the experimental design.
The results of 4 animal experiments showed that at 2 weeks, the osteogenic efficacy of rhBMP-2 and bFGF induced antigenic porcine cancellous bone was highest, while at 4 weeks, it was the highest osteogenic efficacy of rhBMP-2 and bFGF simultaneous release group to induce antigenic porcine cancellous bone. In view of the period of new bone formation and mineralization, the simultaneous release of rhBMP-2 and bFGF was the most ideal. The release method has a better osteogenic effect on xenotransplantation of cancellous bone.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R782.2
【参考文献】
相关期刊论文 前2条
1 汪炬;肖刚;陈阳;周智优;林永亮;吴昊;查振刚;周天鸿;;自制人骨形态发生蛋白2复合骨修复材料诱导骨再生[J];中国组织工程研究与临床康复;2010年21期
2 周智优;何水连;孙莉;王丁丁;汪炬;;重组人骨形态发生蛋白2-肝素-人工骨复合材料的骨诱导活性[J];中国组织工程研究与临床康复;2011年34期
本文编号：2128496
本文链接：https://www.wllwen.com/yixuelunwen/kouq/2128496.html
最近更新
教材专著
