多孔生物陶瓷支架的组织学性能和力学性能表征
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摘要
较大尺寸的骨缺损不能自体愈合,往往在缺损处移植骨代替物以帮助骨愈合。针对这一难题,骨组织工程应运而生。目前,骨组织工程研究主要集中在三个方面：种子细胞、支架、生物反应器。对于支架的探索又分为两个方面：一是支撑和诱导骨组织生长的支架,即使用一种多孔结构材料支架来充填缺损,这种支架具有骨传导和骨诱导能力,能引发成骨细胞及该区域其他细胞长入并吸附在支架上；二是支架材料与细胞的相互作用,在具骨传导性能的支架上成骨细胞迁移有助于骨组织长入和细胞外基质形成,这对于骨缺损的愈合具有重要的功能。骨组织工程支架的基本要求包括：良好的生物相容性,三维贯通的孔隙结构,良好的材料／细胞界面,一定的生物降解性能,并且具有良好的力学性能。因此羟基磷灰石(HA)多孔生物陶瓷支架优良的生物相容性决定了其发展的必然性。
实验对多孔羟基磷灰石颗粒堆积支架、泡沫浸渍法制备的多孔网状羟基磷灰石支架和石蜡造孔剂造孔的羟基磷灰石支架进行体式显微镜和SEM表征,从宏观和微观角度观察支架的孔隙结构,对比其孔隙率和孔隙结构特点。然后选取多孔HA颗粒堆积支架在体内构建组织工程骨,再将构建时间点不同的组织工程骨置于骨缺损部位进行修复实验。通过组织学染色分析,观察支架材料与组织的相互作用以及构建的组织工程骨骨缺损的修复能力。同时,我们改良了硬组织切片制备技术,得到了较为理想的薄层组织学染色标本。另外,骨的力学完整性得到恢复是判断组织工程骨修复骨缺损是否成功的重要标志。本文对构建的组织工程骨以及组织工程骨原位修复的修复骨进行了系统的力学性能表征。本实验获得的主要结果如下：
1.三种HA支架均具有均匀的孔隙分布,较高的孔隙率,且力学性能良好。选用多孔HA颗粒堆积支架和致密HA颗粒堆积支架考察其在体内构建组织工程骨的情况。
2.针对传统硬组织切片技术的缺陷,对超薄硬组织切片技术进行了改进,大大提高了超薄硬组织切片的完整性。传统硬组织切片技术体内构建的组织工程骨标本在切的很薄的情况无法保持其完整性,同时还存在染料吸附和容易脱片的缺陷。通过大量的探索和尝试,我们队超薄硬组织切片技术进行了优化,克服了组织学检测技术处理生物陶瓷材料时易于破坏组织和材料结构的缺点,从根本上解决了标本脱片及染色吸附的问题,从而保证超薄硬组织切片形态完整和后续组织学标本染色层次清晰。
3.通过对腹腔和背肌内构建的组织工程骨组织学染色分析,证明了HA颗粒堆积支架在动物模型体内具有良好的骨诱导性,有利于细胞粘附、组织的长入和血管化。材料在体内构建组织工程骨的能力与所处的生理环境和材料的孔隙结构关系密切。对修复骨的组织学染色显示,组织工程骨的构建时间与原位修复时间共同影响着骨修复能力。
4.通过对体内构建的组织工程骨抗压强度测试表明,多孔颗粒堆积支架相较于致密颗粒堆积支架具有更高的抗压强度,且其抗压强度随体内构建时间的延长而增加,6个月后抗压强度达到自然松质骨的92%左右。利用体内构建的组织工程骨对胫骨长段骨缺失进行修复,构建6个月修复3个月(6M-3M)的修复骨标本的抗弯强度达到正常骨的94%左右。
综和以上结果,得出结论：多孔颗粒堆积支架的三维空间结构利于细胞的粘附、组织的生长；组织学染色分析表明,支架孔隙结构和所处的生物环境对组织工程骨的构建有很大的影响,构建时间与原位修复时间共同影响着骨修复能力：通过对硬组织切片技术的改良,制得了较为理想的HA支架材料类骨修复体硬组织切片标本；影响修复骨力学性能的因素包括组织工程骨构建时间,体内原位修复的时间。研究发现,随体内修复时间的延长,修复骨的抗弯强度能够达接近正常骨的力学强度,力学实验结果与组织学分析相一致。
Since the large bone defect cases can not self heal, the bone graft is needed to auxiliary healing. Bone tissue engineering research is mainly concentrated in two aspects. One is bone induction, a porous biodegradable scaffold was used to fill the defect, which has the ability of bone conduction and bone induction, can cause the osteoblast and otther cells move into and spread on the scaffold. The other is cell migration, the autologous osteoblast and osteoblastoma in the sacffold play a important role in defect healing, the migration of osteoblast is contribute to tissue ingrowth and the formation of the extracellular matrix. The scaffold materials for bone tissue engineering need to fulfill a few basic requirements, including biocompatibility, three-dimensional (3D) porous structure, biodegradability, favorable material/cell interface and mechanical properties. Furthermore, for in vivo bone tissue engineering, the surface properties of scaffolds (chemical composition, surface microstructure) also play critical roles.
In this study, HA porous scaffolds were prepared by three methods, then the macro and micro porous structures of the scaffold were characterized by stereomicroscope and SEM. Used dog as an experimental mode, the HA spherule scaffolds were implanted in enterocoelia to study the biological properties of the material. Observation of material deformation, vaseularized and ossification were characterized by histotomy, and the mechanical properties were also characterized, then the implant were used to repair the bone defect. We improved the method of hard tissue slicing for research of bone tissue engineering of bioceramic materials, prepared the better thin layer histologic dyeing specimens. The mechanical properties of HA scaffolds is the main rejection for applied in bone repair.
The main conclusions were obtained as follows:
· The HA scaffold has regular pore structure, suitable porosity and good mechanical properties, and HA spherule scaffolds were implanted in enterocoelia to study the biological properties of tissue engineered bone.
· We improved the hard biopsy technology to make a better analysis of the interaction between materials and tissues. Traditional hard biopsy technology had the following defects: when the ceramic specimens were cut to very thin, it can hardly keep the integrity, and were easy to take off when dyeing. The improved technology overcame the defect that it may destroy organization, when we use the histologic detection technology to deal with the biological ceramic materials. It fundamentally solved the defects such as dyeing of take off and dye absorption, so as to got the integral and clear histologic dyeing specimens.
· The compressive property of the tissue engineered bone built by scaffolds was increased significantly along with the increase of the implant time. The porous scaffolds had the better compressive property compared with the density ones. That may be due to the porous surface was more advantageous to the protein absorption and cell proliferation, and induct the tissue extended into the scaffolds, induct the formation of the bone eventually.
· The mechanical property of defect bone healed with the tissue engineered bone was increased along with the increase of the healing time, the influence factors of the healed bone mechanical property included the implant time and the in suit repair time.We found that the mechanical property of the healed bone were almost close to normal bone along with the increase of the healing time. The materials and the bone were combined well, bone tissue conduction were well done, the interface of material-bone were indistinct, and the materials were degradation transformation unceasingly.
实验对多孔羟基磷灰石颗粒堆积支架、泡沫浸渍法制备的多孔网状羟基磷灰石支架和石蜡造孔剂造孔的羟基磷灰石支架进行体式显微镜和SEM表征,从宏观和微观角度观察支架的孔隙结构,对比其孔隙率和孔隙结构特点。然后选取多孔HA颗粒堆积支架在体内构建组织工程骨,再将构建时间点不同的组织工程骨置于骨缺损部位进行修复实验。通过组织学染色分析,观察支架材料与组织的相互作用以及构建的组织工程骨骨缺损的修复能力。同时,我们改良了硬组织切片制备技术,得到了较为理想的薄层组织学染色标本。另外,骨的力学完整性得到恢复是判断组织工程骨修复骨缺损是否成功的重要标志。本文对构建的组织工程骨以及组织工程骨原位修复的修复骨进行了系统的力学性能表征。本实验获得的主要结果如下：
1.三种HA支架均具有均匀的孔隙分布,较高的孔隙率,且力学性能良好。选用多孔HA颗粒堆积支架和致密HA颗粒堆积支架考察其在体内构建组织工程骨的情况。
2.针对传统硬组织切片技术的缺陷,对超薄硬组织切片技术进行了改进,大大提高了超薄硬组织切片的完整性。传统硬组织切片技术体内构建的组织工程骨标本在切的很薄的情况无法保持其完整性,同时还存在染料吸附和容易脱片的缺陷。通过大量的探索和尝试,我们队超薄硬组织切片技术进行了优化,克服了组织学检测技术处理生物陶瓷材料时易于破坏组织和材料结构的缺点,从根本上解决了标本脱片及染色吸附的问题,从而保证超薄硬组织切片形态完整和后续组织学标本染色层次清晰。
3.通过对腹腔和背肌内构建的组织工程骨组织学染色分析,证明了HA颗粒堆积支架在动物模型体内具有良好的骨诱导性,有利于细胞粘附、组织的长入和血管化。材料在体内构建组织工程骨的能力与所处的生理环境和材料的孔隙结构关系密切。对修复骨的组织学染色显示,组织工程骨的构建时间与原位修复时间共同影响着骨修复能力。
4.通过对体内构建的组织工程骨抗压强度测试表明,多孔颗粒堆积支架相较于致密颗粒堆积支架具有更高的抗压强度,且其抗压强度随体内构建时间的延长而增加,6个月后抗压强度达到自然松质骨的92%左右。利用体内构建的组织工程骨对胫骨长段骨缺失进行修复,构建6个月修复3个月(6M-3M)的修复骨标本的抗弯强度达到正常骨的94%左右。
综和以上结果,得出结论：多孔颗粒堆积支架的三维空间结构利于细胞的粘附、组织的生长；组织学染色分析表明,支架孔隙结构和所处的生物环境对组织工程骨的构建有很大的影响,构建时间与原位修复时间共同影响着骨修复能力：通过对硬组织切片技术的改良,制得了较为理想的HA支架材料类骨修复体硬组织切片标本；影响修复骨力学性能的因素包括组织工程骨构建时间,体内原位修复的时间。研究发现,随体内修复时间的延长,修复骨的抗弯强度能够达接近正常骨的力学强度,力学实验结果与组织学分析相一致。
Since the large bone defect cases can not self heal, the bone graft is needed to auxiliary healing. Bone tissue engineering research is mainly concentrated in two aspects. One is bone induction, a porous biodegradable scaffold was used to fill the defect, which has the ability of bone conduction and bone induction, can cause the osteoblast and otther cells move into and spread on the scaffold. The other is cell migration, the autologous osteoblast and osteoblastoma in the sacffold play a important role in defect healing, the migration of osteoblast is contribute to tissue ingrowth and the formation of the extracellular matrix. The scaffold materials for bone tissue engineering need to fulfill a few basic requirements, including biocompatibility, three-dimensional (3D) porous structure, biodegradability, favorable material/cell interface and mechanical properties. Furthermore, for in vivo bone tissue engineering, the surface properties of scaffolds (chemical composition, surface microstructure) also play critical roles.
In this study, HA porous scaffolds were prepared by three methods, then the macro and micro porous structures of the scaffold were characterized by stereomicroscope and SEM. Used dog as an experimental mode, the HA spherule scaffolds were implanted in enterocoelia to study the biological properties of the material. Observation of material deformation, vaseularized and ossification were characterized by histotomy, and the mechanical properties were also characterized, then the implant were used to repair the bone defect. We improved the method of hard tissue slicing for research of bone tissue engineering of bioceramic materials, prepared the better thin layer histologic dyeing specimens. The mechanical properties of HA scaffolds is the main rejection for applied in bone repair.
The main conclusions were obtained as follows:
· The HA scaffold has regular pore structure, suitable porosity and good mechanical properties, and HA spherule scaffolds were implanted in enterocoelia to study the biological properties of tissue engineered bone.
· We improved the hard biopsy technology to make a better analysis of the interaction between materials and tissues. Traditional hard biopsy technology had the following defects: when the ceramic specimens were cut to very thin, it can hardly keep the integrity, and were easy to take off when dyeing. The improved technology overcame the defect that it may destroy organization, when we use the histologic detection technology to deal with the biological ceramic materials. It fundamentally solved the defects such as dyeing of take off and dye absorption, so as to got the integral and clear histologic dyeing specimens.
· The compressive property of the tissue engineered bone built by scaffolds was increased significantly along with the increase of the implant time. The porous scaffolds had the better compressive property compared with the density ones. That may be due to the porous surface was more advantageous to the protein absorption and cell proliferation, and induct the tissue extended into the scaffolds, induct the formation of the bone eventually.
· The mechanical property of defect bone healed with the tissue engineered bone was increased along with the increase of the healing time, the influence factors of the healed bone mechanical property included the implant time and the in suit repair time.We found that the mechanical property of the healed bone were almost close to normal bone along with the increase of the healing time. The materials and the bone were combined well, bone tissue conduction were well done, the interface of material-bone were indistinct, and the materials were degradation transformation unceasingly.
引文
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