bFGF基因转染MSCs复合纳米仿生骨治疗骨缺损的实验研究
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摘要
目的
骨缺损是骨科常见病,各种创伤、感染、肿瘤切除、病理发育等因素均可造成骨缺损。较小的骨缺损能自行修复,骨缺损较大时则难以完成自行修复。已有研究表明长管状骨骨干的直径决定了骨缺损能否自行愈合的长度,当骨干骨缺损长度超过于其直径的1.5～2.5倍时即难以自行愈合,造成永久性骨不连。
大的骨缺损需要植骨材料修复,目前临床上用来修复缺损的材料主要有:自体骨,异体骨,异种骨,人工材料,复合材料等,这些材料各有利弊,均难以达到修复骨缺损的理想材料要求。近年来采用骨组织工程技术,将细胞与特定支架材料联合培养,形成有生命活性的组织工程化复合材料,用其修复骨缺损已经取得了初步成效。但对如何构建理想骨组织工程材料有待进一步研究。本课题的目的是,采用骨组织工程技术,以MSCs为种子细胞,转染bFGF基因后,以纳米羟基磷灰石/胶原复合材料(纳米仿生骨nHAC)为支架,形成组织工程化骨,探讨其修复新西兰白兔桡骨节段性骨缺损的可行性。
方法
1.取2～3月龄健康新西兰白兔20只,穿刺抽取双侧股骨大转子部位骨髓约2～3 ml,采用1小时贴壁分离并机械刮除法分离培养骨髓中MSCs细胞,留备下一步实验。其中少量细胞进行形态学观察,并采用成骨细胞诱导条件培养基培养,钙钴法检测碱性磷酸酶表达,茜素红染色观察钙结节形成能力。
2.部分传代培养的MSCs细胞,以质粒为载体,通过电穿孔方法转染bFGF,转染后传代培养备用。少量细胞进行如下鉴定:采用RT-PCR方法检测转染细胞的bFGFmRNA表达水平,采用免疫组化方法检测bFGF蛋白表达。
3.将传代培养的MSCs细胞及转染bFGF的MSCs细胞分别与nHAC支架材料复
ObjectivesBone defects were common conditions caused by trauma, sarcoma or infection. Small bone defects can be repaired automatically by bone induction、 conduction or regeneration. Large bone defects will not heal without bone transplantation.The current bone defects repairing materials include: Autogenous bone, allogenic bone, artificial materials, composite materiel etc. All of these materials were not ideal. It is clinical desirable to find ideal materials to repair larger bone defects. Bone tissue engineering may provide an alternative for bone defects repairing. The aim of this study was to investigate the bone defects repairing ability of the combined material, which with MSCs transduced with bFGF gene as its seeding cells and with nHAC as its scaffolds. Materials and methods1. 20 young New Zealand white rabbits about 2~3 months old were used to harvest bone marrow. The bone marrow was inspired from two femoral trochanter of the rabbits. MSCs were isolated from the bone marrow cells and anchoring at one hour and physically curetle and cultured in vitro. Small samples of MSCs were cultured with conditioned culture medium to assess the alkaline phosphates and the mineralization ability.2. Half of the MSCs were transferred with bFGF gene and cultured. Small samples were used to assess the expression of the bFGF with RT-PCR methods and
immuohistological methods.3. MSCs or MSCs transferred with bFGF gene were combined with nHAC and cultured for 5~7 days respectively. Specimens were obsevered under microscope and electron microscope.4. 62 New Zealand white rabbits which were about 3~4 months old were divided into A、 B、 D group. 10 mm segmental defect were made on the bilateral radius of the rabbits. A group (n=30): the defect areas on the left radius were transplanted with MSCs combined with nHAC. B group (n=30): MSCs transduced with bFGF gene combined with nHAC were transplanted into the defect areas on the left radius. C group (n=60): All the right radiuses of A and B group were divided into this group, the defect areas were implanted with nHAC scaffolds without cells. D group: 2 other rabbits were divided into this group, the same bone defect were made on the bilateral radius without implanting materials. After 2 weeks, 4 weeks, 6 weeks, 12 weeks, 24 weeks from operation, 6 rabbits were killed from A or B group and X ray radio graphical, histological and immuohistological studies were performed. Specimen from C group were harvest and assessed consistent with A and B group, Specimen from D group were harvest at 24 weeks after operation and were assessed with radio graphical and histological method.Results1. The characters of MSCs: the shape of MSC is fusiform or angiogram, with multiple processes, the plasma is pale blue, the nucleus is round with multi-differentiation. MSCs survived and proliferated and differentiated well, the biochemical indexes were stable, MSCs showed the expression of alkaline phosphates, and localized regions of mineralization were found at about 2 weeks with conditioned culture medium.2. bFGF gene transferred MSCs: It didn't change the morphological characteristics of MSCs when MSCs were transferred with bFGF gene. The proliferation and differation of MSCs with bFGF gene transferred were active. The MSCs with bFGF gene transferred can express bFGF at 24h, can express bFGF notably at about 2 weeks and can still express bFGF at 5 weeks after gene transferring.
骨缺损是骨科常见病,各种创伤、感染、肿瘤切除、病理发育等因素均可造成骨缺损。较小的骨缺损能自行修复,骨缺损较大时则难以完成自行修复。已有研究表明长管状骨骨干的直径决定了骨缺损能否自行愈合的长度,当骨干骨缺损长度超过于其直径的1.5～2.5倍时即难以自行愈合,造成永久性骨不连。
大的骨缺损需要植骨材料修复,目前临床上用来修复缺损的材料主要有:自体骨,异体骨,异种骨,人工材料,复合材料等,这些材料各有利弊,均难以达到修复骨缺损的理想材料要求。近年来采用骨组织工程技术,将细胞与特定支架材料联合培养,形成有生命活性的组织工程化复合材料,用其修复骨缺损已经取得了初步成效。但对如何构建理想骨组织工程材料有待进一步研究。本课题的目的是,采用骨组织工程技术,以MSCs为种子细胞,转染bFGF基因后,以纳米羟基磷灰石/胶原复合材料(纳米仿生骨nHAC)为支架,形成组织工程化骨,探讨其修复新西兰白兔桡骨节段性骨缺损的可行性。
方法
1.取2～3月龄健康新西兰白兔20只,穿刺抽取双侧股骨大转子部位骨髓约2～3 ml,采用1小时贴壁分离并机械刮除法分离培养骨髓中MSCs细胞,留备下一步实验。其中少量细胞进行形态学观察,并采用成骨细胞诱导条件培养基培养,钙钴法检测碱性磷酸酶表达,茜素红染色观察钙结节形成能力。
2.部分传代培养的MSCs细胞,以质粒为载体,通过电穿孔方法转染bFGF,转染后传代培养备用。少量细胞进行如下鉴定:采用RT-PCR方法检测转染细胞的bFGFmRNA表达水平,采用免疫组化方法检测bFGF蛋白表达。
3.将传代培养的MSCs细胞及转染bFGF的MSCs细胞分别与nHAC支架材料复
ObjectivesBone defects were common conditions caused by trauma, sarcoma or infection. Small bone defects can be repaired automatically by bone induction、 conduction or regeneration. Large bone defects will not heal without bone transplantation.The current bone defects repairing materials include: Autogenous bone, allogenic bone, artificial materials, composite materiel etc. All of these materials were not ideal. It is clinical desirable to find ideal materials to repair larger bone defects. Bone tissue engineering may provide an alternative for bone defects repairing. The aim of this study was to investigate the bone defects repairing ability of the combined material, which with MSCs transduced with bFGF gene as its seeding cells and with nHAC as its scaffolds. Materials and methods1. 20 young New Zealand white rabbits about 2~3 months old were used to harvest bone marrow. The bone marrow was inspired from two femoral trochanter of the rabbits. MSCs were isolated from the bone marrow cells and anchoring at one hour and physically curetle and cultured in vitro. Small samples of MSCs were cultured with conditioned culture medium to assess the alkaline phosphates and the mineralization ability.2. Half of the MSCs were transferred with bFGF gene and cultured. Small samples were used to assess the expression of the bFGF with RT-PCR methods and
immuohistological methods.3. MSCs or MSCs transferred with bFGF gene were combined with nHAC and cultured for 5~7 days respectively. Specimens were obsevered under microscope and electron microscope.4. 62 New Zealand white rabbits which were about 3~4 months old were divided into A、 B、 D group. 10 mm segmental defect were made on the bilateral radius of the rabbits. A group (n=30): the defect areas on the left radius were transplanted with MSCs combined with nHAC. B group (n=30): MSCs transduced with bFGF gene combined with nHAC were transplanted into the defect areas on the left radius. C group (n=60): All the right radiuses of A and B group were divided into this group, the defect areas were implanted with nHAC scaffolds without cells. D group: 2 other rabbits were divided into this group, the same bone defect were made on the bilateral radius without implanting materials. After 2 weeks, 4 weeks, 6 weeks, 12 weeks, 24 weeks from operation, 6 rabbits were killed from A or B group and X ray radio graphical, histological and immuohistological studies were performed. Specimen from C group were harvest and assessed consistent with A and B group, Specimen from D group were harvest at 24 weeks after operation and were assessed with radio graphical and histological method.Results1. The characters of MSCs: the shape of MSC is fusiform or angiogram, with multiple processes, the plasma is pale blue, the nucleus is round with multi-differentiation. MSCs survived and proliferated and differentiated well, the biochemical indexes were stable, MSCs showed the expression of alkaline phosphates, and localized regions of mineralization were found at about 2 weeks with conditioned culture medium.2. bFGF gene transferred MSCs: It didn't change the morphological characteristics of MSCs when MSCs were transferred with bFGF gene. The proliferation and differation of MSCs with bFGF gene transferred were active. The MSCs with bFGF gene transferred can express bFGF at 24h, can express bFGF notably at about 2 weeks and can still express bFGF at 5 weeks after gene transferring.
引文
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2. Burchardt H. Related Articles, The biology of bone graft repair. Clin Orthop. 1983;(174):28-42.
3. Enneking WF, Eady JL, Burchardt H Autogenous cortical bone grafts in the reconstruction of segmental skeletal defects. J Bone Joint Surg Am. 1980; 62(7): 1039-58.
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