生物材料表面生物功能化及可控微结构陶瓷支架的研究
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摘要
随着人口老龄化和各种创伤的增加,生物材料尤其是骨修复材料的需求日益增长。临床上骨缺损现象主要是由于外伤、肿瘤以及先天性缺陷等原因引起,是骨科中最常见的疾病之一。对于较大尺寸的骨缺损病例其骨缺损不能自体愈合,很大程度上依靠骨移植体进行修复。一般考虑骨移植体来源是自体骨移植和异体骨移植或异种骨移植。但存在诸多问题,如自体骨供应量有限、异种骨存在免疫反应或炎症反应。人工合成的生物材料也是一类重要的骨修复材料,但是,能承力的金属材料缺乏生物活性,而非金属材料又不能满足骨修复的力学性能要求。因此,骨组织工程构建骨组织修复材料显得尤为重要。骨组织工程中支架材料需满足以下要求：(1)具有良好的生物相容性;(2)具有良好的三维多孔结构；(3)具有良好的生物降解性；(4)良好的材料/细胞界面；(5)具有良好的力学性能和可塑性。体内构件组织工程骨,在选择良好生物相容性及良好三维贯通多孔结构的支架基础上,需重点研究生物材料表面性质(如化学成分,支架微结构)对材料功能化的影响。
本研究中,首先涉及热化学法在常用生物金属材料(钛合金)表面制备生物活性钛酸钙涂层,该制备方法简单、有效。接着重点研究溶胶-凝胶法制备可控微结构生物无机陶瓷(羟基磷灰石,HA)球粒,并将其堆积成支架,通过体外细胞、仿生矿化实验和体内非骨部位植入实验,考察支架微结构对其生物功能化的影响,并进行了异位杂化支架原位骨修复实验,取得了较为满意结果。本研究主要结论如下：
1.通过在钛合金基底上覆盖无水硝酸钙,并升温使其发生热化学反应,能制备均匀分布的钛酸钙生物涂层。该方法相比于其他方法,更简单、稳定,制备的涂层同样具有生物活性,可实现对医用钛及其合金的表面改性。
2.通过系统优化溶胶-凝胶体系中HA/Chitin比例,制备了可控微结构的HA球粒,并对形貌、结构和性能进行表征。结果显示：该体系能制备多孔表面和致密表面的HA球粒；在体外细胞和仿生矿化实验中,控制支架宏观大孔尺寸和贯通性一致,发现球粒表面微结构会影响堆积支架整体的生物功能性；多孔表面HA球粒堆积支架比致密表面HA球粒堆积支架具有更好的生物活性,表现为更好的成骨细胞黏附、增殖活性以及早期更迅速地钙磷盐沉积。
3.通过动物狗体内非骨部位(腹腔)植入多孔表面的球粒堆积支架和致密表面的球粒堆积支架异位杂化实验,主要考察两种支架异位骨诱导性和改善支架力学性能两方面。结果显示：两种支架异位杂化后均能改善陶瓷支架的力学性能；多孔表面HA球粒支架其异位骨诱导能力要优于致密表面HA球粒支架,说明球粒微结构对支架生物功能化具有十分重要的作用。
4.通过在动物狗体内非骨部位(腹腔)预先植入多孔表面HA球粒堆积支架进行杂化,然后采用杂化支架对原位骨缺损进行修复。结果显示：异位杂化支架具有优越的原位修复能力,组织学切片染色图可观察到杂化支架进行原位修复时出现大量新骨形成。通过本研究,说明球粒堆积支架体内构建组织工程骨的可行性,为临床中获取骨修复体提供了新途径,具有良好的应用前景。
With the aging of the population, the demand for biomaterials has been increasing, especially bone repair materials. Bone defect is commonly seen in clinics, mainly due to infection, trauma, tumor and congenital disorders. Currently, critical-size bone defects remain difficult to repair. Various approaches have been developed for the repair of large defects, including the use of autografts, allografts and synthetic materials. Autografts are currently the gold standard but are limited in availability. Allografts, on the other hand, involve the risks of immune reaction and inflammatory reaction. Synthetic materials have been regarded as important alternatives to allografts and autografts. However, metallic materials generally lack bioactivity and non-metallic materials do not meet the requirement of mechanical properties for load-bearing applications.
Tissue engineering has been introduced as a promising approach to repair large bone defects. 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, firstly, calcium titanate coatings were prepared on Ti6A14V substrates by a thermochemical surface transformation technique. This technique was simple and efficient. Secondly, HA spheres with controllable microstructure were prepared by a method combining sol-gel and water/oil emulsification techniques and then acculmulated in a porous tube as a porous scaffold. The effects of scaffold microstructure and the bio-functionalization were studied by in vitro cell culture, in vitro biomimetic mineralization and in vivo implantation in non-osseous sites. Then, ectopic hybrid scaffolds were implanted in experimentally created bone defects to evaluate their bone repair capability. The following conclusions were obtained:
1. Reaction between the molten calcium nitrate and Ti6A14V formed a uniform layer of calcium nitrate. This technique was simple and efficient, and the coating was bioactive. This technique could be used as a way of surface modification for titanium and its alloys.
2. HA spheres with controllable microstructure were successfully prepared by combining sol-gel and water/oil emulsification. Spheres with porous or dense surface could be prepared by adjusting the processing conditions. The surface microstructures of spheres could influence the bio-functionalization of scaffolds. The scaffolds constructed from spheres with a porous surface were found to have superior bioactivities, including a better osteoblasts adhesion, more active proliferation, and a faster deposition of calcium phosphate salts during in vitro biomimetic mineralization.
3. Scaffolds constructed from spheres with porous or dense surfaces were implanted in the abdominal cavities of dogs, and the ectopic bone formation and the improvement of mechanical properties were evaluated. Results showed that:the mechanical properties of both scaffolds were improved. The scaffolds consisted of porous-surfaced spheres showed a better capability of inducing ectopic bone formation compared with those consisted of dense-surfaced spheres.. These findings suggest that the surface microstructure of spheres is an important factor for the in vivo functionalization of scaffolds.
4. Repair of experimental bone defects with ectopically hybridized scaffolds showed that the hybrid scaffolds had a superior ability of bone repair. The histological analyses revealed that new bone tissues were formed when using hybrid scaffolds for bone repair. These results demonstrate the feasibility of in vivo construction of bone tissues. This approach may provide new possibilities of creating clinically viable bone grafts for the repair of large bone defects.
本研究中,首先涉及热化学法在常用生物金属材料(钛合金)表面制备生物活性钛酸钙涂层,该制备方法简单、有效。接着重点研究溶胶-凝胶法制备可控微结构生物无机陶瓷(羟基磷灰石,HA)球粒,并将其堆积成支架,通过体外细胞、仿生矿化实验和体内非骨部位植入实验,考察支架微结构对其生物功能化的影响,并进行了异位杂化支架原位骨修复实验,取得了较为满意结果。本研究主要结论如下：
1.通过在钛合金基底上覆盖无水硝酸钙,并升温使其发生热化学反应,能制备均匀分布的钛酸钙生物涂层。该方法相比于其他方法,更简单、稳定,制备的涂层同样具有生物活性,可实现对医用钛及其合金的表面改性。
2.通过系统优化溶胶-凝胶体系中HA/Chitin比例,制备了可控微结构的HA球粒,并对形貌、结构和性能进行表征。结果显示：该体系能制备多孔表面和致密表面的HA球粒；在体外细胞和仿生矿化实验中,控制支架宏观大孔尺寸和贯通性一致,发现球粒表面微结构会影响堆积支架整体的生物功能性；多孔表面HA球粒堆积支架比致密表面HA球粒堆积支架具有更好的生物活性,表现为更好的成骨细胞黏附、增殖活性以及早期更迅速地钙磷盐沉积。
3.通过动物狗体内非骨部位(腹腔)植入多孔表面的球粒堆积支架和致密表面的球粒堆积支架异位杂化实验,主要考察两种支架异位骨诱导性和改善支架力学性能两方面。结果显示：两种支架异位杂化后均能改善陶瓷支架的力学性能；多孔表面HA球粒支架其异位骨诱导能力要优于致密表面HA球粒支架,说明球粒微结构对支架生物功能化具有十分重要的作用。
4.通过在动物狗体内非骨部位(腹腔)预先植入多孔表面HA球粒堆积支架进行杂化,然后采用杂化支架对原位骨缺损进行修复。结果显示：异位杂化支架具有优越的原位修复能力,组织学切片染色图可观察到杂化支架进行原位修复时出现大量新骨形成。通过本研究,说明球粒堆积支架体内构建组织工程骨的可行性,为临床中获取骨修复体提供了新途径,具有良好的应用前景。
With the aging of the population, the demand for biomaterials has been increasing, especially bone repair materials. Bone defect is commonly seen in clinics, mainly due to infection, trauma, tumor and congenital disorders. Currently, critical-size bone defects remain difficult to repair. Various approaches have been developed for the repair of large defects, including the use of autografts, allografts and synthetic materials. Autografts are currently the gold standard but are limited in availability. Allografts, on the other hand, involve the risks of immune reaction and inflammatory reaction. Synthetic materials have been regarded as important alternatives to allografts and autografts. However, metallic materials generally lack bioactivity and non-metallic materials do not meet the requirement of mechanical properties for load-bearing applications.
Tissue engineering has been introduced as a promising approach to repair large bone defects. 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, firstly, calcium titanate coatings were prepared on Ti6A14V substrates by a thermochemical surface transformation technique. This technique was simple and efficient. Secondly, HA spheres with controllable microstructure were prepared by a method combining sol-gel and water/oil emulsification techniques and then acculmulated in a porous tube as a porous scaffold. The effects of scaffold microstructure and the bio-functionalization were studied by in vitro cell culture, in vitro biomimetic mineralization and in vivo implantation in non-osseous sites. Then, ectopic hybrid scaffolds were implanted in experimentally created bone defects to evaluate their bone repair capability. The following conclusions were obtained:
1. Reaction between the molten calcium nitrate and Ti6A14V formed a uniform layer of calcium nitrate. This technique was simple and efficient, and the coating was bioactive. This technique could be used as a way of surface modification for titanium and its alloys.
2. HA spheres with controllable microstructure were successfully prepared by combining sol-gel and water/oil emulsification. Spheres with porous or dense surface could be prepared by adjusting the processing conditions. The surface microstructures of spheres could influence the bio-functionalization of scaffolds. The scaffolds constructed from spheres with a porous surface were found to have superior bioactivities, including a better osteoblasts adhesion, more active proliferation, and a faster deposition of calcium phosphate salts during in vitro biomimetic mineralization.
3. Scaffolds constructed from spheres with porous or dense surfaces were implanted in the abdominal cavities of dogs, and the ectopic bone formation and the improvement of mechanical properties were evaluated. Results showed that:the mechanical properties of both scaffolds were improved. The scaffolds consisted of porous-surfaced spheres showed a better capability of inducing ectopic bone formation compared with those consisted of dense-surfaced spheres.. These findings suggest that the surface microstructure of spheres is an important factor for the in vivo functionalization of scaffolds.
4. Repair of experimental bone defects with ectopically hybridized scaffolds showed that the hybrid scaffolds had a superior ability of bone repair. The histological analyses revealed that new bone tissues were formed when using hybrid scaffolds for bone repair. These results demonstrate the feasibility of in vivo construction of bone tissues. This approach may provide new possibilities of creating clinically viable bone grafts for the repair of large bone defects.
引文
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