【作者】 王冠聪；

【导师】 刘宏；

【作者基本信息】 山东大学 ， 材料学， 2012， 博士

【摘要】 目前,因创伤、先天性畸形、骨关节炎、骨质疏松和肿瘤等原因造成的骨缺损疾病对骨移植材料的需求日趋增加。当前常用的骨修复材料包括自体骨移植物、异种或同种异体骨移植物以及人工合成的骨替代材料,然而这些材料均存在一定的缺陷或局限性。自体骨移植一直被视为治疗骨缺损的“黄金法则”,但它受限于来源,并且存在着引起供区正常骨结构破坏、感染和疼痛等潜在问题。异种或同种异体骨移植物具有免疫原性,会引发机体的免疫排斥反应,还可能导致病原体传染。传统的人工合成的骨替代材料,如羟基磷灰石或磷酸钙陶瓷,虽然可以避免生物源性移植物的缺陷,但其仅具有填充、支持和骨传导作用,不具有高的生物活性,尤其骨诱导能力弱,不能形成“活”的具有生物功能的骨组织。如何理想地实现骨缺损的修复,一直是临床研究的热点和难点。如今,骨组织工程为骨缺损的修复提供了重要的选择性策略。支架材料作为人工细胞外基质材料是骨组织工程的核心。根据天然骨的结构和组成,天然聚合物/羟基磷灰石复合支架材料成为了研究热点。生物可降解的天然衍生高分子聚合物壳聚糖(chitosan),由于其良好的生物相容性,生物可降解性,正电性,易化学改性性而成为仅次于胶原的最具有潜力的用于骨组织工程支架材料的天然聚合物。目前,壳聚糖/羟基磷灰石骨组织工程支架材料的制备方法主要基于混合共沉淀法和原位共沉淀法。以上制备方法虽然工艺简单,却很难在一个具有复杂结构的三维支架内实现表面化学组成和微观结构的可控调节,且使用的交联剂戊二醛有毒,其在体内降解过程中很容易释放出来。此外,为了获得与骨组织结构类似来达到理想的力学性能和生物活性的骨支架材料,生物矿化方法已成为实现形貌和结晶度与天然骨相似的磷灰石在天然聚合物自组装的重要策略。然而,目前很难利用类似在胶原表面进行生物矿化的方法实现羟基磷灰石在壳聚糖基的支架材料表面组装。因此,通过一种无毒且可控的方法实现具有生物活性的羟基磷灰石在壳聚糖基的支架材料表面精确组装仍然是骨组织工程的重要挑战。骨髓来源的间充质干细胞(BMSCs),作为一种成体干细胞,因具有低的免疫原性、易获取性和多向分化潜能性已成为骨组织工程中最为理想的种子细胞之一。近来研究表明生物材料的表面特征,包括化学组成、粗糙度、微结构,特别是纳米级微结构,可以直接影响干细胞的命运。因此,了解骨髓间充质干细胞在支架微环境的生理反应,可以在分子水平明确支架微环境对细胞行为的影响,这为骨髓间充质干细胞在骨组织工程的应用提供了理论依据并有助于研发具有特定生物活性的组织诱导性支架材料。而且,目前随着干细胞与生物材料表面微环境相互作用的逐步广泛认识,组织工程逐步朝向设计和工程化具有促进细胞特殊表型表达功能的支架材料表面的方向发展。此外,骨支架材料的表面特征,特别是考虑到对生长因子(例如骨形态发生蛋白-2,BMP-2)的持续释放,或许能够提供一种新型而有效的药物释放系统来达到促进成骨的目的。根据上述背景,本论文旨在通过一种无毒且可控的方法实现具有生物活性的羟基磷灰石在壳聚糖基的支架材料表面精确组装,并探讨该复合支架材料的表面微环境对接种的干细胞体外成骨分化潜能以及对成骨生长因子(如BMP-2)缓释能力的影响,提供一种具有潜在应用价值的骨组织工程支架材料。本论文主要包括以下几个方面的工作：1.二级三维多孔网络结构壳聚糖/羟基磷灰石骨组织工程支架材料(HGCCS)的构建及细胞生物相容性表征使用无毒的交联剂京尼平(Gneipin),根据纳米籽晶诱导的仿生矿化方法,利用水热合成、原位复合和冷冻干燥制备技术,实现了羟基磷灰石在壳聚糖支架表面的组装,并成功地制备了具有二级三维网状结构的壳聚糖网络/HAp骨组织工程支架材料(HGCCS):第一级网络结构为壳聚糖支架构成的三维连通的多孔结构(约150μm),利于细胞迁移和物质传输;第二级网络结构是由羟基磷灰石在孔道表面白组装形成的纳米网络结构(约150nm),为细胞的生长和生理功能提供了特殊的微环境。X射线衍射(XRD)、高分辨投射电镜(HRTEM)和傅里叶变化红外光谱(FTIR)分析证实了在HGCCS孔道表面上连续的纳米结构是由结晶性的羟基磷灰石组成。使用的无毒交联剂京尼平,赋予了复合支架材料特有的荧光特性和高的力学强度。羟基磷灰石纳米籽晶的掺入促进了羟基磷灰石纳米网络结构在支架孔道表面的快速组装,且共同起到了增强支架材料力学性能的作用。HGCCS的弹性模量(EM)高达50.49±2.23MPa。利用京尼平与壳聚糖共聚物的荧光特性,开发了其在支架材料成像与示踪以及支架材料降解过程中结构观察的潜在应用。此外,其荧光特性或许为研究细胞与支架材料之间的相互作用,以及观察细胞在支架表面的黏附、定位和迁移提供了一个有效手段。将前成骨细胞MC3T3-E1接种于京尼平交联的壳聚糖支架(GCF)和HGCCS上体外培养3天后,通过扫描电镜(SEM)对细胞形貌的观察以及通过激光扫描共聚焦显微镜(CLSM)对细胞核和细胞骨架染色后进行观察,证实了GCF和HGCCS具有良好的细胞生物相容性。将提取的大鼠BMSCs接种于支架材料内,通过细胞形貌和细胞骨架组装观察以及细胞增殖实验,进一步证实了HGCCS具有良好的细胞生物相容性。此外,胎牛血清蛋白(fetal calf serum,FBS)作为模式蛋白被用于评价了HGCCS的蛋白吸附性能。相比于GCF和京尼平交联的壳聚糖／纳米羟基磷灰石复合物(GCGF), HGCCS特有的孔道表面特征,包括高的比表面积、纳米尺寸的结晶度以及微孔隙度促进了FBS在支架材料表面的吸附,并为BMSCs的生长获得了更多的营养。FBS在三种支架材料上的吸附结果也进一步支持了细胞增殖结果。2.体外评价BMSCs在HGCCS上的成骨分化潜能体外评价了BMSCs在HGCCS上的成骨分化潜能。在相同的体外培养条件下,培养7天后,BMSCs在GCF和HGCCS上的细胞形状和细胞骨架组装均呈现出显著的差异。BMSCs在GCF上呈现出成纤维形貌,具有BMSCs的典型表型,而BMSCs在HGCCS上的多角的形状类似成骨细胞的表型。碱性磷酸酶(ALP)作为成骨重要标志物之一,其活性检测结果表明HGCCS诱导了更高的ALP活性。基于实时定量PCR(RT-PCR)检测,HGCCS诱导了成骨分化标志物在mRNA水平的最高表达,分别是第7天的成骨特异性转录因子(Runx2),第7天的骨桥蛋白(OPN)和第14天的骨钙素(OCN)。分别培养7天和14天后,茜素红染色结果证实了BMSCs在HGCCS上具有促进矿化基质和钙结节形成的能力。在相同的体外培养条件下,相比于GCF, HGCCS表面的羟基磷灰石纳米网络结构作为重要的信号因素促进了BMSCs的体外成骨分化。3.基于具有二级三维多孔结构的壳聚糖／羟基磷灰石复合支架的表面微结构调控BMP-2缓释及促进BMSCs体外成骨分化体外评价了HGCCS表面特殊的羟基磷灰石纳米网络结构对BMP-2的吸附和长期释放作用以及对大鼠BMSCs体外成骨分化潜能的影响。酶联免疫吸附实验(ELISA)结果表明,与对照组GCF的28%的BMP-2载药率相比,HGCCS具有65%的有效载药率。ELISA结果还表明BMP-2从HGCCS可持续释放到第14天,而在第1和第3天BMP-2在GCF上呈现出爆发式地释放。进而,BMP-2从HGCCS的缓释能力促进了体外BMSCs的ALP活性增加。基于实时定量PCR检测,BMP-2荷载的HGCCS也诱导了成骨分化标志物在mRNA水平的最高表达,分别是第14天的Runx2,第3天的OPN和第14天的OCN。本次研究结果证实具有二级三维多孔结构的HGCCS的表面羟基磷灰石纳米网络结构作为BMP-2的荷载系统可以促进BMSCs的体外成骨分化,表明HGCCS是具有潜在应用价值的骨组织工程支架材料。4.猪脱细胞真皮基质(PADM)-羟基磷灰石复合支架材料对大鼠颅骨临界骨缺损修复性能的初步研究课题组前期首次利用PADM作为基础材料体外构建了具有二级三维多孔结构的天然胶原-羟基磷灰石复合支架材料,并研究了其对大鼠下颌骨临界骨缺损的修复性能。在前期研究基础上,进一步构建了8mm大鼠颅骨临界骨缺损模型用于评价PADM和PADM-HAp的成骨生物活性。修复5周后,Micro-CT分析和组织化学染色结果表明相比于PADM, PADM-HAp具有更好的骨修复能力。更多还原

【Abstract】 In the modern world there are increasingly urgent demands for various biomedical bone implants to repair bone defects caused by bone fractures, congenital malformation, osteoarthritis, osteoporosis or bone cancers. However, bone substitute including autografts, allografts, and an assortment of synthetic or biomimetic materials and device, all have some drawbacks. Although bone autografts are recognized as gold standard, their supply is limited and there are many potential drawbacks associated with their use including donor site morbidity, danger of infection, and pain. Allografts have the risk of introducing infectious agents or immune rejection. Though the traditional synthetic bone substitute including hydroxyapatite or calcium phosphate ceramics do not have the drawbacks from autografts and allografts, they only possess the functions of filling, supporting and osteoconduction, and do not have high bioactivity, especially osteoinduction. Therefore, it is difficult to achieve the functions of natural bone. Thus, the requirement for complete regeneration of bone and restoration of its function is a major clinical need.Bone tissue engineering has been regarded as an important strategy to repair and regenerate bone tissue. Bone scaffolds as synthetic extracellular matrix materials are significant for bone tissue engineering. According to the structure and composition of natural bone, biodegradable polymer/hydroxyapatite (HAp) composites have been widely studied. Now, chitosan as an important biodegradable natural polymer, due to its biocompatibility, biodegradability, cationic nature, ready availability, and anti-bacteria, has been the most promising natural polymer next to collagen for bone tissue engineering.Presently, the fabrication techniques for porous HAp/chitosan composite bone scaffolds are based on either mixing or in situ co-precipitation methods. Though the mixing and in situ co-precipitation approaches are simple processes with low cost, it is difficult to control the surface chemistry and geometry within a large and complex structure. In addition, these methods for chitosan/HAp scaffold preparation normally use glutaraldehyde (GTA) as a crosslinker, a substance that is toxic when it is released in the host during the biodegradation process. In order to develop ideal bone-like composites with enhanced mechanical properties and improved bioactivity, biomimetic mineralization has become an effective strategy to assemble bone-like apatite that is close to natural bone with low crystallinity and nanoscale size. However, it is difficult to achieve the controlled nucleation and growth of HAp nano-crystals on a chitosan-based framework. Thus, it is critical to control and attain desirable bioactive mineral surface structures within a chitosan-based framework through a nontoxic and controllable preparation method, and it is still a great challenge for bone tissue engineering.Bone marrow-derived mesenchymal stem cells (BMSCs) as one of adult stem cells have attracted particular attention in bone tissue engineering, because they are readily accessible, with low immunogenicity and multilineage differentiation potential. Recent studies have indicated the surface characteristics of biomaterials including chemical composition, roughness, and topography, especially nanotopography, can profoundly affect cell functions. Thus, understanding BMSCs response to the microenvironment in scaffold can identify the effect of the microenvironment in scaffold on BMSCs behaviors at the molecular level. It can provide the basis of the theory for the application of BMSCs in bone tissue engineering and exploring tissue-inducing biomaterials that have special bioacitivity. Recently, with increased knowledge of the interactions of stem cells with biomaterial surfaces, tissue engineering is becoming increasingly oriented toward designing and engineering biomaterial surfaces that promote specific cellular phenotypes. Moreover, the surface characteristics of bone scaffold, especially regarding the sustained delivery of growth factors (such as bone morphogenetic protein-2,BMP-2), can possibly provide a novel and effective drug delivery system that can enhance osteogenesis.Based on the backgrounds mentioned above, the objective of this study was to control and attain desirable bioactive hydroxyapatite structures within a chitosan-based scaffold through a nontoxic and controllable preparation method, and investigate the effect of surface microenvironment of the obtained composite scaffolds on the osteogenic differentiation potential of seeded BMSCs in vitro and sustained delivery of bioactive molecules (such as BMP-2). The main topics of this dissertation are as follows:1. Construction of two-level three-dimensional networked chitosan/hydroxyapatite composite scaffold for bone tissue engineering and its cell biocompatibilityUsing a nontoxic cross-linker (genipin), a nano-crystallon induced biomimetic mineralization method, and hydrothermal synthesis, in situ co-precipitation and lyophilization technology, we have demonstrated the construction of a2-level3D networked chitosan/hydroxyapatite (HGCCS) by assembling HAp nanostructures on the chitosan framework for bone tissue engineering. The first level of the network is the chitosan3D interconnected macroporous framework (-150μm) that is favorable for cell immigration and mass transport; the second level is the nano-network and nanostructure of HAp self-assembled on the channel surface of the chitosan framework (-150nm) that provides the special microenvironment for cell growth and function.X-ray diffraction (XRD), high resolution transmission electron microscopy (HRTEM) and fourier transform infrared spectroscopy (FTIR) analysis confirm that the continuous network-like nanostructure on the channel surface of the HGCCS is composed of crystalline HAp. Non-toxic genipin is used as a crosslinker and it endows high strength and intrinsic fluorescence to the chitosan framework. The nano-crystalline HAp seeds play a determining role in the construction of HAp nanostructure on the channel surface, and they reinforce the produced scaffold.The compressive elastic modulus (EM) of the HGCCS obtained increases to50.49±2.23MPa. We further explore the novel fluorescence properties of genipin-cross-linked chitosan that suggest a promising application for3D scaffold imaging and tracking, and structural observation of the degradation process. In addition, the intrinsic fluorescence may provide an effective way to image the cell-scaffold interaction and effectively monitor the adhesion, localization and migration of cells on the surface of the scaffolds.The results of SEM observation of cell morphology, and CLSM observation of MC3T3-E1pre-osteroblasts cultured for3days on GCF and HGCCS after nuclei and F-actin staining confirmed their good cell compatibility. Rat BMSCs were extracted and seeded in scaffolds, the results of SEM observation of cell morphology, and CLSM observation of cytoskeleton organization further confirmed that HGCCS possess good cell compatibility.Fetal bovine serum (FBS) as a model protein was used to evaluate the protein adsorption capacity of HGCCS. Compared to GCF and GCGF, the special surface properties of HGCCS, including increased surface area, nanocrystallinity and microporosity with nanosized pores enhanced increased adsorption of FBS for easier access to nutrients by BMSCs. The results of adsorption experiments of FBS further confirmed the results of cell proliferation assay.2. In vitro assessment of the osteogenic differentiation potential of BMSCs on HGCCSWe examined the osteogenic differentiation potential of BMSCs on HGCCS in vitro. Given the same culture conditions, cell shape and cytoskeleton organization showed significant differences between cells cultured on GCF and those cultured on HGCCS after7days of incubation. The result of specific alkaline phosphatase (ALP) activity as an indicator of osteogenic differentiation showed that the ALP activity of rat BMSCs was higher on HGCCS. Based on quantitative real time PCR (RT-PCR), HGCCS induced highest mRNA expression of osteogenic differentiation markers, runt-related transcription factor2(Runx2) by7days, osteopontin (OPN) by7days and osteocalcin (OCN) by14days, respectively. The enhanced ability of BMSCs on HGCCS to produce mineralized extracellular matrix and nodules was also assessed on day14with Alizarin red staining. The results of this study suggest that in the same culture conditions, compared to GCF, the surface HAp nano-network structure of HGCCS acts as a critical signal cue promoting osteogenic differentiation in vitro.3. Enhanced osteogenic differentiation of BMSCs in vitro on two-level three-dimensional networked HGCCS based on surface microstructure for sustained delivery of BMP-2We evaluated the effect of the surface special HAp nano-network structure of HGCCS on the BMP-2adsorption and release ability, and the resulting osteogenic differentiation of rat BMSCs in vitro. HGCCS exhibited a loading efficiency of65%, which is significantly higher than28%for GCF, as quantified by an enzyme-linked immunosorbent assay (ELISA). We also found that the release of BMP-2from HGCCS was sustained for at least14days in vitro, compared to that from GCF, which consisted of burst releases on days1and3. Moreover, the BMP-2release from HGCCS induced an increase in alkaline phosphatase activity of BMSCs for14days in vitro. Based on quantitative real time PCR, HGCCS also stimulated the highest mRNA expression of osteogenic differentiation markers, Runx2for14days, OPN for3days and OCN for14days. The results of this study suggest that the surface HAp nano-network structure of two-level3D HGCCS used as a delivery system for BMP-2is capable of promoting osteogenic differentiation in vitro. As a result, HGCCS is a promising scaffold for bone tissue engineering.4. Study of repairing of rat cranial critical size defect with porcine accellular dermal matrix (PADM)-HAp composite scaffold in a cranial modelIn previous studies, we first demonstrated the construction of a2-level3D PADM-HAp composite scaffold. Further, critical bone defects of rat cranial about4mm in diameter were used for investigation of the bioactivity of PADM and PADM-HAp. After15weeks, the results of histochemical stainings and Micro-CT showed that compared to PADM, the defect was well repaired with use of PADM-HAp.更多还原