组织工程纤维环—髓核双相支架的制备与性能评估
发布时间：2018-07-31 05:40
【摘要】：目的：以天然猪骨脱钙基质明胶(BMG,主要成分含Ⅰ型胶原)和软骨细胞外基质(CECM,主要成分为Ⅱ型胶原/GAG)为材料,利用仿生学原理研制新型纤维环-髓核双相支架,并探讨其作为组织工程椎间盘支架的可行性。 方法： 1.纤维环支架：取猪股骨近端松质骨,环钻钻取外径10mm、内径5mm、厚3mm的中空环状骨环,高压水冲洗,依次进行脱脂、脱钙、脱细胞等处理,制成中空环状脱钙骨基质明胶为材料的椎间盘纤维环支架。评估支架材料的孔径和孔隙率、吸水率,采用微型力学测试机行力学测试,采用分离培养的兔脂肪干细胞和MTT法分析支架浸提液毒性。 2.纤维环-髓核双相支架：将猪关节软骨粉碎采用梯度离心法选取5μm～100μm软骨微丝,脱细胞等处理后制备成质量体积比为3.5%的悬液,将软骨浆料悬液注入中空环状脱钙骨基质纤维环支架中央冷冻冻干,采用紫外线以及化学交联剂碳化二亚胺等对支架进行交联。行组织学及扫描电镜观察,测定支架孔径和孔隙率、吸水率。将兔脂肪干细胞种植在支架上,倒置显微镜以及扫描电镜观察细胞在支架上的粘附以及生长情况。 3.双相纤维环支架：取猪掌骨中央圆形区域,锯取厚5mm的皮质骨-松质骨环,冲洗、脱钙、脱细胞制成皮质骨-松质骨环双相纤维环支架。观察支架的孔径及孔隙,测定吸水率及孔隙率,用微型力学测试机行力学测试。结果： 1.纤维环支架：以松质骨脱钙骨基质明胶制备的纤维环支架为乳白色的环形多孔结构,孔径大致均匀,孔隙相通。湿性状态下质地柔软。HE染色显示组织内无细胞结构残留。光镜及扫描电镜示支架孔隙均匀,支架纤维环部孔径为401.44±13.1μm,孔隙率为62.12±1.52%,吸水率为409.774±11.34%。生物力学结果示支架的弹性模量为47.75±6.32MPa。细胞增殖曲线显示不同浓度支架浸提液与对照DMEM培养液吸光度值比较,差异无统计学意义(P0.05)。 2.纤维环-髓核双相支架：以松质骨脱钙骨基质明胶与软骨细胞外基质构建的纤维环-髓核双相支架为淡黄白色的圆形多孔结构,孔隙相通,湿性状态下质地柔软。孔径大致均匀,纤维环部孔径大于髓核部,支架髓核部孔径为101.3±22.5μm,孔隙率为83.24±4.31%,吸水率为421.45±8.43%,支架总体弹性模量为53.45±3.21MPa。HE染色显示组织内无细胞结构残留。光镜及扫描电镜示支架孔隙均匀。倒置显微镜下细胞在支架上粘附良好。扫描电镜下细胞在支架上有良好的粘附,分布均匀,生长良好。 3.纤维环双相支架：以皮质骨-松质骨环制备的的纤维环双相支架为淡黄色类圆形多孔结构,孔径、孔隙率以及吸水率比松质骨纤维环支架小,孔隙率为51.41±2.71%,吸水率为350.15±3.21%。 结论： 1.以松质骨脱钙骨基质明胶制备的纤维环支架具有良好的孔隙率、孔径、吸水率、生物相容性,弹性模量与纤维环在同一数量级。 2.以松质骨脱钙基质明胶和软骨细胞外基质制备的纤维环-髓核双相支架具有良好的孔径、孔隙率、生物力学强度和物相容性,在基质成分上与正常椎间盘细胞外基质类似,可作为组织工程椎间盘的支架材料。 3.以皮质骨-松质骨环制备的双相纤维环支架具有一定的发展前景。
[Abstract]:Objective: to use natural pig bone decalcification matrix gelatin (BMG, mainly composed of type I collagen) and cartilage extracellular matrix (CECM, the main component of type II collagen /GAG) as material, a new type of fibrous ring nucleus pulposus biphasic stent was developed by bionics, and the feasibility of it as a scaffold for tissue engineering intervertebral disc was discussed.
Method:
1. fibrous ring scaffold: Taking the proximal cancellous bone of the pig femur, taking the outer diameter 10mm, the inner diameter 5mm, the hollow ring bone ring of the thick 3mm, and rinsing with high pressure water. The hollow ring decalcified bone matrix gelatin was made in order to make the hollow ring decalcified bone matrix gelatin as the intervertebral disc scaffold. Mechanical tests were carried out using a micro mechanical testing machine. The isolation and culture of rabbit adipose derived stem cells and MTT method were used to analyze the toxicity of the scaffold extract.
2. fibrous ring and nucleus pulposus biphasic stent: the porcine articular cartilage was crushed by gradient centrifugation to select 5 m ~ 100 m cartilage microfilament, and the mass volume ratio was 3.5%. The suspension was injected into the central cold freeze dry of the hollow ring decalcified bone matrix fibrous ring scaffold, and the UV and chemical crosslinking agent carbon was used. The scaffolds were crosslinked with two imines. The pore size, porosity and water absorption of the scaffold were measured by histological and scanning electron microscopy. The rabbit adipose stem cells were planted on the scaffold, inverted microscope and scanning electron microscope were used to observe the adhesion and growth of the cells on the scaffold.
3. double phase fibrous ring scaffold: Taking the central round area of the pig metacarpal bone, sawing the thick 5mm cortical bone - cancellous bone ring, rinsing, decalcification and deactivating the cortical bone - the double phase fibrous ring scaffold of the cancellous bone ring. The pore size and pore size of the scaffold were observed and the water absorption and porosity were measured. The results of mechanical test were measured by micromechanical test machine.
1. fibrous ring scaffold: the fibrous ring scaffold prepared with cancellous bone decalcified bone matrix gelatin is a milk white annular porous structure. The pore size is roughly uniform and the pores are interconnected. Under wet condition, the texture of soft.HE shows no cell structure residue in the tissue. The aperture of the scaffold is 401.44 + 13.1 with the light and scanning electron microscope. With the porosity of 62.12 + 1.52% and the water absorption rate of 409.774 + 11.34%., the biomechanical results showed that the elastic modulus of the scaffold was 47.75 + 6.32MPa. cell proliferation curve and showed that there was no significant difference in the absorbance between the different concentrations of the scaffold and the control DMEM culture (P0.05).
2. fiber ring and nucleus pulposus biphasic stent: the fibrous ring nucleus pulposus double phase scaffold constructed with cancellous bone decalcified bone matrix gelatin and cartilage extracellular matrix is yellowish white circular porous structure, pore is interconnected, and the texture is soft under wet condition. The pore size is roughly uniform, the diameter of the ring is larger than that of nucleus pulposus, and the diameter of the nucleus pulposus is 101.3 + 22.5 m. The porosity was 83.24 + 4.31%, the water absorption rate was 421.45 + 8.43%. The overall elastic modulus of the scaffold was 53.45 + 3.21MPa.HE. The cell structure was not residual in the tissue. The light and scanning electron microscope showed that the scaffold pores were uniform. The cells adhered well on the scaffold under the inverted microscope. Good long.
3. fiber ring biphasic stent: the fibrous ring biphasic stent made with cortical bone cancellous bone ring is a light yellow circular porous structure. The pore size, porosity and water absorption are smaller than that of the cancellous bone fibrous ring scaffold, the porosity is 51.41 + 2.71%, and the water absorption rate is 350.15 + 3.21%.
Conclusion:
1. the fibrous ring scaffolds prepared with cancellous bone decalcified bone matrix gelatin have good porosity, pore size, water absorption, biocompatibility, and modulus of elasticity with the fiber ring at the same order of magnitude.
2. the fibrous ring nucleus pulposus biphasic scaffold prepared with cancellous bone decalcification matrix gelatin and cartilage extracellular matrix has good pore size, porosity, biomechanical strength and substance compatibility. It is similar to the extracellular matrix of normal intervertebral disc on the matrix component. It can be used as a scaffold material for tissue engineering intervertebral disc.
3. the biphasic fiber ring scaffolds made from cortical bone cancellous bone rings have certain development prospects.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08
本文编号：2154598
[Abstract]:Objective: to use natural pig bone decalcification matrix gelatin (BMG, mainly composed of type I collagen) and cartilage extracellular matrix (CECM, the main component of type II collagen /GAG) as material, a new type of fibrous ring nucleus pulposus biphasic stent was developed by bionics, and the feasibility of it as a scaffold for tissue engineering intervertebral disc was discussed.
Method:
1. fibrous ring scaffold: Taking the proximal cancellous bone of the pig femur, taking the outer diameter 10mm, the inner diameter 5mm, the hollow ring bone ring of the thick 3mm, and rinsing with high pressure water. The hollow ring decalcified bone matrix gelatin was made in order to make the hollow ring decalcified bone matrix gelatin as the intervertebral disc scaffold. Mechanical tests were carried out using a micro mechanical testing machine. The isolation and culture of rabbit adipose derived stem cells and MTT method were used to analyze the toxicity of the scaffold extract.
2. fibrous ring and nucleus pulposus biphasic stent: the porcine articular cartilage was crushed by gradient centrifugation to select 5 m ~ 100 m cartilage microfilament, and the mass volume ratio was 3.5%. The suspension was injected into the central cold freeze dry of the hollow ring decalcified bone matrix fibrous ring scaffold, and the UV and chemical crosslinking agent carbon was used. The scaffolds were crosslinked with two imines. The pore size, porosity and water absorption of the scaffold were measured by histological and scanning electron microscopy. The rabbit adipose stem cells were planted on the scaffold, inverted microscope and scanning electron microscope were used to observe the adhesion and growth of the cells on the scaffold.
3. double phase fibrous ring scaffold: Taking the central round area of the pig metacarpal bone, sawing the thick 5mm cortical bone - cancellous bone ring, rinsing, decalcification and deactivating the cortical bone - the double phase fibrous ring scaffold of the cancellous bone ring. The pore size and pore size of the scaffold were observed and the water absorption and porosity were measured. The results of mechanical test were measured by micromechanical test machine.
1. fibrous ring scaffold: the fibrous ring scaffold prepared with cancellous bone decalcified bone matrix gelatin is a milk white annular porous structure. The pore size is roughly uniform and the pores are interconnected. Under wet condition, the texture of soft.HE shows no cell structure residue in the tissue. The aperture of the scaffold is 401.44 + 13.1 with the light and scanning electron microscope. With the porosity of 62.12 + 1.52% and the water absorption rate of 409.774 + 11.34%., the biomechanical results showed that the elastic modulus of the scaffold was 47.75 + 6.32MPa. cell proliferation curve and showed that there was no significant difference in the absorbance between the different concentrations of the scaffold and the control DMEM culture (P0.05).
2. fiber ring and nucleus pulposus biphasic stent: the fibrous ring nucleus pulposus double phase scaffold constructed with cancellous bone decalcified bone matrix gelatin and cartilage extracellular matrix is yellowish white circular porous structure, pore is interconnected, and the texture is soft under wet condition. The pore size is roughly uniform, the diameter of the ring is larger than that of nucleus pulposus, and the diameter of the nucleus pulposus is 101.3 + 22.5 m. The porosity was 83.24 + 4.31%, the water absorption rate was 421.45 + 8.43%. The overall elastic modulus of the scaffold was 53.45 + 3.21MPa.HE. The cell structure was not residual in the tissue. The light and scanning electron microscope showed that the scaffold pores were uniform. The cells adhered well on the scaffold under the inverted microscope. Good long.
3. fiber ring biphasic stent: the fibrous ring biphasic stent made with cortical bone cancellous bone ring is a light yellow circular porous structure. The pore size, porosity and water absorption are smaller than that of the cancellous bone fibrous ring scaffold, the porosity is 51.41 + 2.71%, and the water absorption rate is 350.15 + 3.21%.
Conclusion:
1. the fibrous ring scaffolds prepared with cancellous bone decalcified bone matrix gelatin have good porosity, pore size, water absorption, biocompatibility, and modulus of elasticity with the fiber ring at the same order of magnitude.
2. the fibrous ring nucleus pulposus biphasic scaffold prepared with cancellous bone decalcification matrix gelatin and cartilage extracellular matrix has good pore size, porosity, biomechanical strength and substance compatibility. It is similar to the extracellular matrix of normal intervertebral disc on the matrix component. It can be used as a scaffold material for tissue engineering intervertebral disc.
3. the biphasic fiber ring scaffolds made from cortical bone cancellous bone rings have certain development prospects.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08
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