【作者】 张瑗；

【导师】 周跃；

【作者基本信息】 第三军医大学 ， 外科学， 2009， 博士

【摘要】 当前,组织工程学的突破进展为研发具有应用价值的骨修复材料赋予了极大启示,而多样化的骨替代材料在系列研究中亦表现出极高的潜在价值,但在应用中往往并不具备实际意义,即其修复能力仍难达到临床需求。从表象上来讲,主要归因于材料本身(如金属、多聚物、生物陶瓷)特性单一,不足以提供骨传导和骨诱导双重信号。然而,本课题组认为：只有充分认识到材料-组织整合过程中的分子机制和细胞作用,才能从根本上解决如上科学问题。骨整合(osteointegration)信号传递到材料-组织界面有两个关键因素：1.要有足够数量的细胞与生物分子充分作用以启动胞内信号；2.生物信号的活性／强度必须高于细胞活性的刺激阂值。从这一要求出发,本课题旨在通过双嗜性分子重组纤维连接蛋白／钙粘附蛋白(recombinant fibronectin/cadherin chimera, rFN/CDH)仿生修饰骨支架材料,构建具有优良生物理化特征的仿生学界面和适宜骨种子细胞粘附、增殖和分化的微环境。通过提高种子细胞在界面的粘附效率和生长信号的强度进而增强组织工程化材料在体内外的骨修复能力,从而有效解决传统组织工程材料粘附效率低和成骨信号弱的问题。方法：1.rFN/CDH融合蛋白的构建设计、基因克隆和蛋白表达纯化以FN和CDH11作为前体分子,生物信息学分析以了解其结构序列与功能基团。采用“同源模建”作为分子模拟策略确定FN和CDH11功能片段的接合方式。常规PCR扩增FNⅢ7-10和CDH11 EC1-2基因片段后应用重叠延伸PCR(Splice-over-extension PCR, SOE-PCR)进行基因拼接。将此融合基因插入原核表达载体pET-22b进而构建重组载体。重组载体转化表达菌株Rsoetta-gami(DE3),优化参数实现rFN/CDH的诱导表达,金属离子层析纯化蛋白。免疫印迹和生物质谱分析确定融合蛋白种属。离心促粘附实验和成骨诱导验证rFN/CDH体外生物活性。2. rFN/CDH-BCP仿生界面的组装策略与表征评价DTBP同双官交联法将rFN/CDH以共价方式接合在双相钙磷陶瓷(Biphasiccalcium phosphate ceramic, BCP)表面构建仿生界面。扫描电镜法(Scanning electro-microscopy, SEM)、X光电子能谱法(X ray photoelectron spectroscopy, XPS)、接触角分析和蛋白吸附实验分别了解仿生修饰对材料表面微观结构、化学元素组成和分布、亲／疏水性、rFN/CDH分布密度的影响,初步评价rFN/CDH-BCP仿生界面的组织相容性。3.体外功能学与机制研究：rFN/CDH-BCP仿生界面对骨种子细胞增殖、粘附和分化的影响以人骨髓间充质干细胞(human Mesenchymal stem cells, hMSCs)为骨种子细胞,采用离心促粘附实验、MTT法和SEM观察rFN/CDH-BCP仿生界面对细胞粘附、增殖、形态的影响。同时,以碱性磷酸酶(Alkaline phosphatase, ALP)和骨钙素(osteocalcin,OCN)作为成骨分化的早、晚期指标,分光比色法观察成骨诱导后hMSCs的ALP酶活性,实时荧光定量PCR(real-time RT PCR)和免疫印迹(western blotting)法检测OCN基因和蛋白表达,茜素红染色法鉴定钙结节形成情况。以此综合判断rFN/CDH-BCP仿生界面对hMSCs成骨分化的影响。进一步采用粘附斑激酶(Focal adhesion kinase,FAK)397酪氨酸磷酸化(phosphorylation of Tyrosine 397, pY397)特异性抗体封闭hMSCs表面作用位点,初步探讨FAK pY397与rFN/CDH-BCP介导的细胞粘附和分化间的关系。4.体内功能研究：复合bMSCs的rFN/CDH-BCP组织工程骨的体内成骨效应以兔MSCs(bMSCs)为种子细胞构建rFN/CDH-BCP组织工程化人工骨,将其植入兔腰椎横突5-6间行融合术。影像学手段(X片和CT三维重建)观察骨愈合情况；三点抗弯实验检测腰椎融合部位力学强度；HE、Masson和甲苯胺蓝染色法观察材料-组织间的骨整合状况。结果：1. rFN/CDH融合蛋白的构建设计、基因克隆和蛋白表达纯化确定CDH11 EC1-2-(Ser)3-FNⅢ7-10是适宜融合蛋白构建的接合方式,生物信息学分析提示改良大肠杆菌系统适合融合蛋白优化表达。采用SOE-PCR将0.8kbp的CDH11 EC1-2和1.1kbp的FNⅢ7-10片段成功拼接为1.9kbp的融合基因,双酶切实验和DNA测序证实重组载体构建成功、准确。转化Rosetta-gami(DE3)后于IPTG终浓度0.4mM,重组菌对数生长值0.5-0.6,诱导温度30℃,诱导时间4-6hr的条件下实现对rFN/CDH(75 kDa)的高效、可溶性表达。6xHis标签行镍柱(Ni-NTA)吸附纯化后纯度大于96%。质谱分析确认rFN/CDH种属完全正确,属于一种FN前体分子和一种CDH11前体分子。离心促粘附实验表明rFN/CDH涂层的TCPS表面对成骨细胞株MC3T3-E1的吸附作用明显强于单纯FN和CDH(1.4倍和2.9倍,p<0.05)。成骨诱导实验显示rFN/CDH促进ALP活性升高和OCN基因表达上调,显著优于阳性对照(p<0.05)。2. rFN/CDH-BCP仿生界面的组装策略与表征评价XPS数据显示仿生修饰后BCP表面分别独立出现164.0eV和401.0eV的峰谱,即硫和氮元素的分布,提示界面构建成功、有效。SEM显示BCP表面呈巨孔／微孔不规律分布,且仿生修饰对微结构无明显影响。XPS表明BCP表面分布有C、O、P、Ca四种元素,而仿生修饰后N元素出现,并发生了C元素的价态变化(CC-N)。且元素含量变化为C、N含量增加而P、Ca含量降低(p<0.05)。接触角测定表明θ角显著减小(45°→31°,p<0.05),提示rFN/CDH-BCP界面亲水性增加。蛋白吸附实验提示BCP对rFN/CDH的吸附在一定范围内呈浓度依赖性,且rFN/CDH的饱和工作浓度为5μg/ml,分布密度为1700fmol/cm2。3.体外功能学与机制研究：rFN/CDH-BCP仿生界面对骨种子细胞增殖、粘附和分化的影响hMSCs在rFN/CDH-BCP界面上培养1-9天,其增殖率在前期(1-5天)明显高于阴性和阳性对照组(p<0.01,p<0.05)。SEM发现观察rFN/CDH修饰BCP界面更利于细胞的贴壁生长,表现为hMSCs充分伸展为多边形状或圆盘状,伪足样结构减少。离心粘附实验发现hMSCs在rFN/CDH-BCP表面的粘附数量与生物配基密度表现为正相关,在1500 fmol/cm2的表面密度下是阳性对照的的4.9倍和1.52倍(p<0.05)。此结果提示：rFN/CDH-BCP界面具有极佳的生物相容性,hMSCs在其表面表现出优越的粘附、增殖和生长状态。hMSCs成骨诱导10天后,在rFN/CDH-BCP表面的ALP活性表现最高(约57μmol nitrophenol/min/mg protein, p<0.05)。成骨诱导14天后,在rFN/CDH-BCP表面的OCN基因和蛋白表达水平亦为最高(p<0.05,p<0.05)。成骨诱导21天后,rFN/CDH-BCP表面的细胞间形成的椭圆形橘红色结节在数量和面积上分布最多。此结果表明：仿生修饰界面可更高效地促进hMSCs的成骨分化。。利用FAK pY397特异性抗体来封闭hMSCs表面的信号通路,发现如上指标均受一定程度的抑制,但并非完全性阻断,此结果提示该仿生界面发挥功能不完全依赖于FAK酪氨酸397的磷酸化,尚有其他磷酸化位点或信号分子参与该过程。4.体内功能研究：复合hMSCs的rFN/CDH-BCP组织工程骨的体内成骨效应将bMSCs复合的rFN/CDH-BCP植入兔腰椎5、6横突间成功建立腰椎融合模型,术后3月,采用X片结合CT三维重建的影像学手段定性分析发现rFN/CDH-BCP材料与骨床间隙相延续、模糊不可辨,定量分析显示横突间隙的骨痂填充率以rFN/CDH-BCP为最高(p<0.05)。建立应力-应变曲线分析力学载荷、弯曲强度、弹性模量的数据变化,发现bMSCs复合rFN/CDH-BCP组行腰椎融合后的刚性(0.044±0.0059GPa)显著强于单纯BCP组(0.026±0.0058 GPa,p<0.05),略高于bMSCs复合BCP组(0.041±0.0093),但不及自体髂骨移植组(0.071±0.0119)。组织学水平上,以HE法、Masson法和甲苯胺蓝法染色评价材料-组织间的骨融合状况,发现bMSCs复合rFN/CDH-BCP组植入3月后的组织学表现与自体髂骨移植组相似,表现为融合区域出现成熟度较高的骨小梁连续性、交错性桥接,其在骨量上甚至略高于后者。该成骨能力显著强于bMSCs复合BCP组和单纯BCP植入组(p<0.01)。综合数据提示：rFN/CDH-BCP复合bMSCs后表现出优越的骨传导和骨诱导特性,可显著提高BCP在体内的骨修复和融合能力,具有一定的替代意义。但与自体骨移植标准相比,需进一步提高其力学性能。结论：1.本研究设计、制备的rFN/CDH嵌合式融合蛋白实现了对FN和CDH11功能学的叠加,体外具有良好的促粘和成骨活性。2. rFN/CDH生物配基共价交联生物陶瓷BCP界面后具备优良的粗糙度、微结构、亲水性、化学组成和可控的配基密度等生物理化特征,是一种生物相容性界面。3. rFN/CDH-BCP仿生界面在体外有效增强hMSCs的粘附、增殖效率和生长活力,显著上调了干细胞成骨诱导后ALP、OCN等成骨标志物的表达,明显促进了钙结节形成和基质矿化能力。4.特异性ITG-FAK pY397通路在rFN/CDH-BCP介导的干细胞粘附和分化过程中发挥了重要作用,FAK pY397可能是rFN/CDH-BCP发挥生物效应的调节位点。5. rFN/CDH-BCP用以兔腰5-6横突间融合具有优良的促进骨愈合的能力,具有临床效果的影像学征象、力学整合意义的抗压强度和生物学整合意义的组织学表现,具有一定的替代意义。6. rFN/CDH-BCP具备良好的骨传导和骨诱导特性,是一种有光明应用前景的新型组织工程来源的骨替代材料,可用于非负重部位的骨修复／融合。但与自体骨移植标准相比,需进一步提高其力学性能。更多还原

【Abstract】 The current approaches in tissue engineering have presented tremendous inspiration for developing applicable biomaterial for bone repair, and diversified substitutions have been proved to be magnificently potential in a series of studies. Even though, they didn’t possess enough significance in application because of the difficulty in meeting clinical requirements. Apparently, this is due to the infertile property of materials (metal, polymer, bioceramic, etc.), and the incompetence in osteoconduction and osteoinduction. As far as we know, only the full insight into the molecular mechanism and cell interaction on the material-tissue interface, could we settle this scientific issue.Two prerequisites are necessary for delivering the osteointegration signal to the interface:(1) local cell populations must interact with the biomolecules for a period of time to initiate cellular events and (2) concentrations of bio-molecules must exceed threshold levels for cellular activity. Proceeding from this requirement, this study aimed at fabricating a scaffold with a novel recombinant fibronectin/cadherin chimera (rFN/CDH) and constructing superior microenvironment for osteoblastic adhesion, proliferation and differentiation with excellent bio-physico-chemical properties. Therefore, it can be expected to improve the repair ability of tissue engineered scaffold in vitro and in vivo by up-regulating the adhesion effect of bone seeding cells and the intensity of growth factors on the biomaterial surface, and to find an efficient solution to this traditional problem.Methods1. Engineering, cloning, expression and purification of rFN/CDH In this study, FN and CDH11, as two pre-molecules of the recombinant protein, were analysed bioinformatically to acquired the structural sequences and functional groups. The fragments of FN and CDH11 were integrated using a homology modeling strategy. The gene fragments of FNⅢ7-10 and CDH11 EC1-2 were spliced by SOE-PCR and then inserted into the expression plasmid of pET-22b for a reconstructed vector. After transforming the vector into Rsoetta-gami(DE3) strain and optimizing the experimental parameters, rFN/CDH was induced to express using IPTG and purified via nickel ion metal affinity chromatography. Further, immuno-blotting and mass spectrometry were employed to verify the identity of the protein. While centrifugal cell adhesive assay and osteoblastic induction were used to determine the bioactivity in vitro.2. Fabrication and characterization of rFN/CDH bio-inspired BCP biomimetic surface.The biomimetic surface was achieved by immobilizing rFN/CDH onto biphasic calcium phosphate ceramic (BCP) covalently, using a DTBP crosslinking method. Scanning electro-microscopy (SEM), X-ray photoelectron spectroscopy (XPS), contact angle and protein adsorption assay were introduced to determine the microstructure, chemical distribution and composition, hydrophilicity and ligand density of the surface after modification. Hereby the biocompatibility of the biomaterial was preliminarily evaluated.3. The effects of rFN/CDH-BCP biomimetic surface on cell proliferation, adhesion and differentiation:an functional and mechanism study in vitrohuman mesenchymal stem cells (hMSCs) were employed as the bone seed cells to observe the responses of adhesion, proliferation and morphology using cell centrifugal adhesive assay, MTT method and SEM, respectively. Meanwhile, the effect of rFN/CDH-BCP on differentiation were evaluated comprehensively as follows. Alkaline phosphatase (ALP) activity and osteocalcin (OCN) gene/protein expression, as the early and late markers for osteoblastic differentiation, were determined by colorimetric quantitation and real-time RT PCR/western blotting. Additionally, calcium nodules formed in extracellular matrix were visualized by alizarin red staining. Furthermore, a specific antagonist of phosphorylation of tyrosine 397 in focal adhesion kinase (FAK pY397) was adopted to block the reaction site of hMSCs, so as to explore the relationship between FAK pY397 and adhesion/differentiation of hMSCs which were mediated by rFN/CDH-BCP.4. The healing response of a tissue engineered bone of bMSCs combined rFN/CDH-BCP in a rabbit model:a functional study in vivoA tissue engineered bone was prepared with biomaterial of bMSCs loaded rFN/CDH-BCP, then implanted into the intertransverse process space of lumbar 5 to 6, to establish a rabbit lumbar fusion model for observing the healing response. Radiological methods (X plain and 3-dimensional CT remodeling) were employed to clinically evaluate the overall healing status, and 3 point bending test was introduced to determine the mechanical strength. Hematoxylin eosin (HE), Masson and Toluidine blue stainings were adopted to detect material-tissue interfacial integration.Results1. Engineering, cloning, expression and purification of rFN/CDHMolecular simulation indicated that the framework of CDH11 ECl-2-(Ser)3-FNⅢ7-10 was suitable for gene splicing. Bioinformatics suggested that a refined E. coli. system would be superior for rFN/CDH expression. Experimentally, a combined gene of 1.9 kbp was acquired by splicing the FNIII7-10 (1.1 kbp) with CDH11 EC 1-2 (0.8 kbp), which was verified as valid by double digestion and DNA sequencing. rFN/CDH was expressed high-efficiently and solubly at 30℃, with an OD value of 0.5-0.6, an final IPTG concentration of 0.4 mM, and an induction time of 4-6 hours. The purity reached 96% after chromatography. Data from MS showed that the protein corresponded to precursors of human fibronectin and cadherin. Additionally, cell centrifugal adhesive assay demonstrated that rFN/CDH coating TCPS adhered more MC3T3-E1 osteoblasts, as compared to single FN and CDH (1.4-fold and 2.9-fold, p<0.05). While results from osteoblastic induction indicated that rFN/CDH further promoted ALP activity and OCN gene expression (p<0.05).2. Fabrication and characterization of rFN/CDH bio-inspired BCP biomimetic surface.Data from XPS displayed that two spectra of 164.0 eV and 401.0 eV appeared on the biomimetic surface, representing the existence of sulfur and nitrogen elements and suggesting the validity in surface fabrication. SEM observations revealed an irregular distribution of macro/micro-pores among the surface and no obvious influence after modification. XPS results also showed that additional N spectrum and C-N peak for C appeared on modified surface. Besides, the chemical composition exhibited increase for C and N contents while decrease for P and Ca contents. For contact angle measurement, the 0 angle was significantly decreased after modification (45°→31°, p<0.05), indicating increased hydrophilicity. As for protein adsorption assay, adsorption of rFN/CDH onto BCP was concentration-dependent within a certain range, and the saturation concentration of rFN/CDH was 5μg/ml while ligand density was 1700 fmol/cm2.3. The effects of rFN/CDH-BCP biomimetic surface on cell proliferation, adhesion and differentiation:an functional and mechanic study in vitroThe proliferation rate were significantly higher within the first 5 days, as compared to negative and positive controls (p<0.01, p<0.05). SEM observations found that the hMSCs of rFN/CDH-BCP evenly dispersed on the surface as a rounded or polygonal morphology, exhibiting better status for cell ingrowth. Cell centrifugal adhesive assay indicated the adherent hMSCs on rFN/CDH-BCP was 4.9-fold and 1.52-fold higher at the ligand density of 1500 fmol/cm2 when compared to positive controls (p<0.05). The results unveils biomimetic surface possesses excellent biocompatibility, which is convenient for cell adhesion, proliferation and growth.As for differentiation, the ALP activity on rFN/CDH-BCP surface was the highest among all samples at the 10th day after induction (p<0.05). Correspondingly, the expression level of OCN gene and protein on rFN/CDH-BCP surface also exhibited the highest manifestation at the 14th day (p<0.05, p<0.05). At the 21st day, alizarin red staining showed that the oval-shaped and orange-red nodules, either the number or the area, distributed wider on rFN/CDH-BCP surface. These results suggest that the biomimetic surface could substantially promoted hMSCs’differentiation towards osteo-lineage.After blocking the signal transduction with specific FAK pY397 antibody, the above indices were suppressed to some extent, but not completely inhibited. This implies that the the mechanism by which the rFN/CDH-BCP promotes adhesion and ossification is not fully dependent on the phosphorylation of tyrosine 397 of FAK, but other unknown phosphorylated sites or signal molecules might be involved in this process.4. The healing response of a tissue engineered bone of bMSCs combined rFN/CDH-BCP in a rabbit model:a functional study in vivoA rabbit lumbar fusion model was established by implanting bMSCs combined rFN/CDH-BCP into intertransverse process space of L5-L6. Radiologically, the results from X plain and 3-dimensional reconstruction after 3 months revealed a fuzzy gap between material and bone bed, and higher cover rate of intertransverse process space with new bone deposition on rFN/CDH-BCP surface (p<0.05). Mechanically, a stress-strain curve was set up to illustrate the changes of mechanic load, flexural strength, and elastic modulus. The results demonstrated that the bending stiffness at the fusion site of rFN/CDH-BCP group was significantly higher than that in BCP group (0.044±0.0059 GPa vs 0.026±0.0058 GPa, p<0.01), while slightly improved as compared with bMSCs combined BCP group (0.044±0.0059 GPa vs 0.041±0.0093 GPa, p>0.05). However, no comparability can be found when compared to autogenous iliac implanting group (0.044±0.0059 GPa vs 0.071±0.0119, p<0.01). Histologically, rFN/CDH-BCP revealed incorporating bone islands consistent with autogenous iliac group, which exhibited as interlacing bone trabecula bridging biomaterial suface and cortical bone of transverse process continously. The bone mass was even more as compared with autograft, while the maturation was significantly higher than the groups of BCP and bMSCs loaded BCP groups (p<0.01).The comprehensive data reveal that when loaded with MSCs, rFN/CDH-BCP demonstrates superior characteristics of osteoconduction and osteoinduction, and substantially enhances healing capacity in vivo.Conclusions:1. The rFN/CDH chimera prepared in this study possesses excellent capacity in promoting osteoblastic adhesion and ossification, which achieves the functional integration of FN and CDH11.2. The rFN/CDH bio-functionalized BCP ceramic demonstrates superior characteristics of bio-physico-chemstry such as roughness, micro-topography, hydrophilicity, chemical composition and controllable surface density, indicating an excellent biocompatibility.3. rFN/CDH-BCP exhibits superior characteristics in promoting hMSCs’adhesion, proliferation and viability, up-regulating ALP and OCN expression, and advancing calcium nodule formation and matrix mineralization.4. Specific ITG-FAK pY397 pathway plays an important role in mediating cell adhesion and differentiation on rFN/CDH-BCP surface, and may be a regulatory and controllable target in the process.5. rFN/CDH-BCP significantly promotes bone repair efficiency in a rabbit lumbar fusion model, as evidence by the enhanced healing manifestation in radiological, mechanical and histological integration, and may be an applicable candidate for autograft.6. rFN/CDH-BCP is a novel and promising bone substitution derived from tissue engineering strategy, and can be greatly expected for repair/fusion in non-load bearing regions. However, its mechanical property should be improved as an ideal substiution for autograft.更多还原