【作者】 王富友；

【导师】 杨柳；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 由创伤或骨病所致关节骨软骨缺损临床常见,主要表现为顽固性疼痛、关节运动功能受限,严重影响患者生活质量,已成为目前肢体残障的主要原因之一。美国发病率为1.5‰～3‰,我国约为美国的5～6倍,且随着中老年人群比例的增加呈逐年上升趋势。由于软骨没有神经、血管及淋巴系统,自身修复能力非常有限,直径>2mm的软骨缺损几乎不能完全修复,如果合并软骨下骨缺损治疗更为困难。现有临床治疗措施均存在明显缺陷,其中保守治疗与关节清理术只能暂时缓解疼痛,不能阻止病程的发展;自体骨软骨移植术人为造成供区缺损,且来源有限,难以修复面积较大的缺损;异体骨软骨移植术存在免疫排斥反应及传播疾病的可能;关节融合术改变了关节原有的解剖结构,丧失了关节的运动功能,患者难以接受;人工关节置换术费用昂贵、并发症较多、翻修率较高,且经济负担较重,特别是对年轻患者的身心健康影响极大。快速发展的组织工程技术,尤其是单一组织工程骨和组织工程软骨构建技术的日趋成熟,为关节骨软骨缺损的再生修复提供了新思路和新技术。国内外对组织工程骨软骨复合组织的构建研究比较深入,从“分层构建”的可行性初探到“一体构建”的动物实验研究取得了阶段性成果,然而目前尚存在缺损区修复组织质量缺陷、与宿主界面整合欠佳及缺乏相应力学功能等主要问题。组织工程学是应用生命科学和工程学的原理与技术,在正确认识哺乳动物正常及病理两种状态下组织结构与功能关系的基础上,研究、开发用于修复、维持和促进人体各种组织或器官损伤后功能和形态生物替代物的科学。与单一的软骨组织和骨组织比,骨软骨复合组织是成分与结构较为复杂的功能器官。因此,我们推测目前制备的组织工程骨软骨之所以存在上述问题,主要原因是:(1)缺乏对人体关节部位正常骨软骨复合组织的基本成分、形态结构、生理功能及其生理微环境深入认识,忽视了对关节软骨与软骨下骨之间的界面连接结构――钙化软骨层的构建;(2)所选用的生物材料与宿主成分存在较大的差异,植入体内容易产生免疫排斥反应,或在降解过程中造成生理微环境的改变,不利于植入细胞的增殖与分化;(3)理想的构建策略,不是将再造的软骨与骨组织简单地缝合或粘合在一起,而应以其正常形态结构为依据,采用仿生生物材料构建与宿主结构相似的一体化组织工程骨软骨。针对上述原因,本研究首先采用组织形态学相关技术与方法,对人体正常膝关节骨软骨复合组织,尤其是其功能界面――钙化软骨层的形态结构、主要组分进行了深入研究;然后以所获得的研究结果为依据,利用仿生学原理,选用胶原蛋白、壳聚糖和羟基磷灰石为组织工程骨软骨支架材料;应用壳聚糖――胶原蛋白嫁接技术、羟基磷灰石――胶原蛋白矿化技术、以及喷雾干燥技术等方法对仿生原材料进行调控与修饰;通过体外细胞材料共培养实验、兔背部皮下植入实验,证实所研制的三种仿生生物材料均具有良好生物相容性与安全性;最后建立兔膝关节股骨滑车面骨软骨缺损模型,采用体内一体化构建策略,将体外制备好的细胞材料复合体分层植入体内,研究发现由于软骨层材料固定不佳,导致修复结果不理想。因此,又采用体外一体化构建策略,应用计算机辅助下的三维打印技术对组织工程骨软骨一体仿生支架进行了初步研制。第一部分人体正常关节骨软骨形态结构观察与组分分析本部分研究,以人体膝关节正常骨软骨复合组织为实验材料,采用组织形态学相关技术与方法,对骨软骨复合组织的形态结构、组成成分以及各层组织间的界面连接方式进行了探索,为组织工程骨软骨复合组织的仿生构建提供理论依据。一、实验方法1.人体正常关节骨软骨形态结构观察(1)采用组织切片技术,番红O/固绿染色、冯库萨染色等方法观察骨软骨复合组织的形态结构。(2)采用扫描电镜技术、连续切片组织三维重建技术观察骨软骨各层组织间界面连接方式。2.人体正常关节骨软骨组分分析(1)采用免疫组织化学与氨基酸分析技术对骨软骨复合组织中胶原蛋白进行定性、定量分析。(2)采用X-射线衍射技术对骨软骨复合组织中羟基磷灰石进行定性、定量分析。二、实验结果1.成人关节骨软骨自关节面向深部依次分为透明软骨层、钙化软骨层和软骨下骨三层结构。透明软骨层厚约3mm左右,钙化层厚度仅为几微米至300多微米,其上界面以波浪状的潮线结构与透明软骨层紧密连接,其下界面以凸凹不平的梳齿状结构与软骨下骨相互锚合。2.透明软骨主要由Ⅱ型胶原蛋白组成,占组织干重的61.39%±0.38%,余为蛋白多糖;钙化软骨层主要由Ⅱ型胶原蛋白和羟基磷灰石组成,分别占组织干重的20.16±0.96%和65.09%±2.31%;软骨下骨主要由Ⅰ型胶原蛋白和羟基磷灰石组成,分别占组织干重的13.69%±0.45%和85.78%±3.42%。三、结论成人关节骨软骨自关节面向深部可分为透明软骨层、钙化软骨层和软骨下骨三层结构。透明软骨主要由软骨细胞和Ⅱ型胶原蛋白与蛋白多糖等细胞外基质组成;钙化软骨层主要由少量肥大软骨细胞和Ⅱ型胶原蛋白与羟基磷灰石等细胞外基质组成;软骨下骨主要由成骨细胞和Ⅰ型胶原蛋白与羟基磷灰石等细胞外基质组成。钙化层上界面以波浪状潮线结构与透明软骨层紧密连接,下界面以凸凹不平梳齿状粘合线结构与软骨下骨相互锚合。这种特殊界面连接方式,即增加了软骨与骨界面之间的连接面积,同时也增加了连接强度。钙化层的结构致密,像隔离带一样将骨软骨复合组织分为两个理化因素不同的生理微环境,透明软骨氧和营养缺乏,其主要来源于关节腔内的滑液;软骨下骨氧和营养丰富,主要来源于骨髓和干骺端血管。关节骨软骨的这种特殊结构组成及其界面连接方式,不但有利于各层组织利用各自的生理微环境进行新陈代谢,而且有利于其生物力学功能的发挥。第二部分组织工程骨软骨仿生基质材料研制本部分研究以第一部分的结果为依据,利用仿生学原理,选用胶原蛋白、壳聚糖和羟基磷灰石为组织工程骨软骨基质材料,应用壳聚糖――胶原蛋白嫁接技术、羟基磷灰石――胶原蛋白矿化技术、以及喷雾干燥技术等方法对仿生原材料进行调控与修饰;采用傅利叶变换红外光谱分析技术、细胞――材料共培养以及兔背部皮下植入等方法,对仿生基质材料的理化性能、生物相容性及安全性进行检测。一、实验方法1.组织工程骨软骨仿生基质材料研制(1)采用壳聚糖――胶原蛋白嫁接技术制备软骨层仿生基质材料;羟基磷灰石――胶原蛋白矿化技术制备钙化层和软骨下骨仿生基质材料。(2)采用扫描电镜技术、傅利叶变换红外光谱分析技术对各层基质材料的性能表征进行检测。2.组织工程骨软骨仿生基质材料生物相容性及安全性检测(1)将各层基质材料与兔骨髓间充质干细胞进行共培养检测基质材料的细胞相容性。(2)将各层基质材料植入兔背部皮下检测基质材料的组织相容性和生物安全性。二、实验结果1.按照正常骨软骨复合组织的分层结构,采用相关技术成功研制出三种仿生基质材料:透明软骨层基质材料――Ⅱ型胶原蛋白/壳聚糖聚合体干粉;钙化层基质材料――Ⅱ型胶原蛋白/羟基磷灰石聚合体干粉;软骨下骨基质材料――Ⅰ型胶原蛋白/羟基磷灰石聚合体干粉。扫描电镜分析,各层材料干粉颗粒直径在2μm～5μm之间;傅利叶变换红外光谱分析表明,与单纯混合的材料比较,经过调控、修饰的材料光普峰数明显减少,证实构成聚合体的材料分子之间形成稳固的键合。2.细胞――材料共培养7天,细胞形态正常、生长旺盛,与单纯细胞培养组相比无统计学差异;细胞粘附于材料之上,结果证实所研制材料具有良好的细胞相容性。细胞材料混合体植入兔背部皮下1.5月,大体观察植入材料被纤维组织包裹,软骨材料具有一定的弹性,骨材料较硬;组织学观察植入细胞分布均匀、形态正常;主要脏器心、肝、肺、肾的形态、结构正常,结果证实所研制材料具有良好的生物安全性。三、结论采用壳聚糖――胶原蛋白嫁接技术、羟基磷灰石――胶原蛋白矿化技术、及喷雾干燥技术,成功研制出用于构建工程化骨软骨的三种基质材料;各层材料颗粒直径在2μm～5μm之间,构成聚合体的材料分子之间形成稳固的键合。细胞――材料共培养实验、兔背部皮下植入实验表明,所研制的仿生材料具有良好的细胞相容性、组织相容性和生物安全性。第三部分组织工程骨软骨仿生设计与初步构建本部分研究,以人体正常关节骨软骨的形态、结构为依据,利用仿生学原理确定组织工程骨软骨主要参数;然后应用第二部分研制的生物材料,采用体内一体构建策略制备细胞材料复合体,植入兔膝关节骨软骨缺损并检测修复情况;采用体外一体化构建策略,应用三维打印快速成型技术初步制备组织工程一体化仿生支架,并对其主要结构参数进行验证。一、实验方法1.组织工程骨软骨体内一体化构建(1)建立兔膝关节股骨滑车面骨软骨缺损模型。(2)体外制备组织工程骨软骨各层基质材料――细胞复合体,一次性分层植入并观察缺损修复情况。2.组织工程骨软骨一体化支架仿生制备(1)应用计算机辅助设计软件,建立组织工程骨软骨一体化支架模型。(2)采用三维打印快速成型技术制备组织工程骨软骨仿生支架。二、实验结果1.兔膝关节骨软骨缺损修复实验结果表明,术后1.5月,空白对照组未被修复;单纯支架组缺损边缘少部分修复;无钙化层细胞材料复合组可见缺损深度变浅,面积缩小,但表面有开裂;有钙化层细胞材料复合组修复组织表面呈白色,深度变浅,面积缩小,触之具有一定的弹性,修复组织与界面整合良好,但缺损中央仍存在未修复区域,约为原缺损的1/3,可能是因为软骨材料固定不牢所致。2.采用三维打印快速成型技术成功制备出组织工程骨软骨仿生支架模型,经检测模型的各项参数与计算机设计的模型完全一致,证实三维打印技术可以实现具有复杂结构的组织工程骨软骨仿生支架的一次性制备;所研制的仿生材料能够满足三维打印技术要求,但因为目前现有的三维打印机主要应用于工业模型制备,尚不能制备出结构精细组织工程支架。三维打印技术具有操作规范、重复性好及可以产业化制备等优点,可望成为未来组织工程支架制备的先进方法,因此尚需对其进行深入探索。三、结论体内一体化构建的骨软骨复合组织修复情况较好,缺损深度变浅、体积缩小,修复组织表面呈白色,触之具有一定的弹性,修复组织与宿主界面整合良好,组织学检测可见少量新生软骨形成;但由于软骨层材料固定不牢,部分脱落至关节腔,影响修复效果,因此尚需对软骨层材料的固定技术进行深入探索。采用三维打印技术可以一次性制备出与计算机设计完全一致的组织工程骨软骨模型,但因为目前现有的三维打印机主要应用于工业模型制备,尚不能制备出结构精细组织工程支架。三维打印技术具有操作规范、重复性好及可以产业化制备等优点,对其进行深入探索,可望成为未来组织工程支架制备的先进方法。更多还原

【Abstract】 The articular osteochondral defects caused by trauma or bone disease are common and become one of the main reasons of physically handicap in clinic. Those patients with osteochondral defects will frequently be with intractable pain, restrict joint activities and seriously impact on their quality of life. The incidence rate of osteochondral defects in the United States is 1.5‰to 3‰. In our country, it is about 5 to 6 times of that in the United States. With the increase in the proportion of elderly population, the incidence is increasing. Because cartilage have not nerves, blood vessels, lymphatic system and a enough self-repair capacity, in almost cartilage defects whose diameters is large than 2mm can not be completely restored, and the treatment of cartilage defects merging subchondral bone defect is more difficult. The current clinical treatment measures have obvious flaws, conservative treatment and joint clean-up operation could only temporarily alleviate the pain, can prevent the development of disease; autogenous bone graft is difficult to repair large defect for its limited source and may cause human-induced; osteochondral allograft transplantation may transmitted diseases; arthrodesis is possible to change joint anatomy of the original structure and loss of joint function and is difficult to accept by patients; joint replacement is costly, complications leaded to a high rate of revision and especially have a great physically and mentally impact to the younger patients. The rapid development of tissue engineering technology, especially single bone tissue engineering and tissue-engineered cartilage construction technology gradually get success, it provide new ideas and new technologies for the regeneration and repair of the osteochondral defects.The tissue engineering of osteochondral composite tissue has been deeply studied both in China and aboard. Preliminary results have been achieved from the feasibility of "construction by layers" to the experimental study of "integration construction". But the poor quality of the repair tissue in the area of osteochondral defects, poor integration with the host interface and the lack of corresponding mechanical functions are the major problems. On the base of correct understanding of normal and pathological state of the structure-function relationship ,tissue engineering apply life sciences and engineering principles and technology to research and develop biological substitutes for repairing, maintaining and promoting the human body in various injury or morphology state. Osteochondral composition is a more complex organ than a single cartilage and bone tissue. Therefore, we speculate the main problems of the current bone and cartilage tissue engineering are: (1) the lack of basic knowledge about the components, organizational structure, physiological functions of human osteochondral composite, and in-depth understanding of its micro-environment; disregard the important functional structure between the articular cartilage and subchondral bone - calcified cartilage zone; (2) there is a big difference between the biological materials and body composition, it is easily lead to immune rejection reaction in vivo, induce an acid environment during the degradation process (PLGA) to the detriment of cell proliferation; (3) the ideal strategy of construction will be build and host similar structure and function of the integration of osteochondral tissue engineering composite tissue in its normal structure, composition and foundation, rather than simply paste cartilage and bone tissue together.For the above reasons, this study first use the technology and methods related to tissue morphology, to study the normal human osteochondral composition, in particular its interface structure - calcified layer structure and the major component; and then based on the results of the study, with the use of collagen, and chitosan bionic raw materials, such as hydroxyapatite, using the technology of the mineralization of collagen and the methods such as spray-drying techniques to modify the regulation and make it with good plasticity and test the compatibility of their cell, tissue compatibility and biological safety; finally with the construction of the integration strategy, a preliminary tissue engineered osteochondral composite and animal experimental study was carried out with bionic structure and composition.PRIMARY PART: Normal Human Osteochondral Morphology and Component AnalysisUsing tissue morphology related technology and methods, to explore the morphology and component of osteochondral tissue and interface between bone and cartilage in normal human knee. Provide relevant theory for building bionic osteochondral tissue engineering composite. Materials and methods1. Normal human articular osteochondral morphology(1) To observe osteochondral composite tissue morphology in Fan-O / solid green staining and the Feng Savimbi staining.(2) To observe interface connections by scanning electron microscope and three-dimensional reconstruction technology.2. Normal human articular cartilage and bone component analysis(1) To analyse collagen protein of osteochondral composite by immunohistochemistry and amino acid detection technology qualitatively and quantitatively.(2) To analyse hydroxyapatite of osteochondral composite by X-ray diffraction technique qualitatively and quantitatively.Results1. Adult articular cartilage composite can be divided into three-layer structure: hyaline cartilage, calcified cartilage and subchondral bone zone. Hyaline cartilage is about 3 mm in thickness; alcified cartilage zone is only a few microns to 300 microns in thickness and its up interface showed a wave-like structure, the bottom interface of the calcified cartilage zone showed different sizes of comb teeth to anchor with subchondral bone.2. The main component of hyaline cartilage is collagen typeⅡ, which account 61.39%±0.38% of the dry weight, The rest component is proteoglycan; calcified cartilage zone mainly composited by typeⅡcollagen protein and hydroxyapatite, they account 20.16±0.96% and 65.09%±2.31% of the dry weight respectively; the main component of subchondral bone is typeⅠcollagen protein and hydroxyapatite, they account 13.69%±0.45% and 85.78%±3.42 % of the dry weight respectively.ConclusionAdult articular cartilage composite can be divided into three layer structure: hyaline cartilage, calcified cartilage and subchondral bone zone. the main component of hyaline cartilage is collagen type , calcified cartilage zone mainly composited by typeⅡcollagen protein and hydroxyapatiteⅡand the main component of subchondral bone is typeⅠcollagen protein and hydroxyapatite and the bottom interface of the calcified cartilage zone showed different sizes of comb teeth to anchor with subchondral bone. SECOND PART: Production of bionic materials of osteochondral matrixThis part the study was based on the results of the first part study, the first the composition and ratio of bionic materials of osteochondral matrix composite were identified, then modified the matrix composite by the way of grafting technique, collagen mineralization technology, as well as spray-drying technology, the last detected the physical and chemical properties, compatibility of cells and organizations and biological security of the matrix composite.Methods1. Production of matrix materials of tissue engineered bionic osteochondral composite(1) Cartilage matrix material was produced by collagen and chitosan graft technology; calcified layer and subchondral bone material was produced by collagen protein and mineralized hydroxyapatite, then micron powder was prepared by spray-dried technique.(2) Test the matrix material by Fourier Transform Infrared Spectroscopy.2. Biocompatibility of bionic materials of osteochondral matrix(1) Co-cultured matrix material with rabbit bone marrow-derived mesenchymal stem cells.(2) Implant matrix materials subcutaneously to the rabbit back to test the tissue compatibility and bio-safetyResults1. Successfully production three osteochondral matrix material: cartilage matrix materials -Ⅱcollagen protein / chitosan polymer powder; matrix material of the calcified zone -Ⅱcollagen protein / hydroxyapatite polymer powder; Matrix materials of subchondral bone.Ⅰcollagen protein / hydroxyapatite polymer powder. Fourier transforms infrared spectroscopy shows that the composition of the polymer material link in the molecular bonding.2. Cytocompatibility experiment showed that cocultured cells grow well, illustrate normal morphology and adhere on materials. Tissue compatibility experiment showed that implant material was wrapped by fibrous tissue, and did not happen inflammatory response, the organ pathological biopsy showed normal structure; which showed good cell, tissue compatibility and good bio-security. ConclusionCollagen grafting techniques, mineralization and spray drying technology can successfully produce the three osteochondral matrix material, physical and chemical properties of the structure showed that the biological material have good cells and tissues compatibility and biological security.THIRD PART: Design and Preparation of Tissue Engineered Bionic Osteochondral compositeThe design of tissue engineered bionic osteochondral composite is based on the first part of the research. Then use the second part of the development of the biological material to construct tissue engineered bionic osteochondral composite in vivo. The preliminary preparation of the tissue engineered bionic osteochondral composite by the method of rapid prototyping 3D printing technology.Methods1. Preparation of Engineered osteochondral composite tissue in vivo(1) Establish osteochondral defect model on the rabbit knee femoral trochlea(2) Prepare composite of materials and MSCs, implant the composite in defect model layerly then observe it.2. Preparation of bionic integrated engineering osteochondral scaffolds(1) Establish CAD model of bionic integrated engineering osteochondral scaffolds(2) Produce bionic integrated engineering osteochondral scaffolds by 3D printing technologyResults1. Control group :defect had not been repaired; scaffold group : a small portion of the edge repaired; material group without calcified zone:defect is shallow, area is narrow, but the surface is cracking; material group without calcified zone : repair tissue was white and elastic, defect is shallow, area is narrow, interface integration are continuous, but there is still a central defect.2. Bone cartilage bionic scaffold model produced by rapid prototyping 3D printing technology, the parameters of the model conform to that of the computer model. At present rapid prototyping 3D printing technology mainly used in industrial model and can not be used for the biomedical field now. Binder should improve to meet the requirements of 3D printing technology and have good biocompatibility.ConclusionUsing osteochondral composite to repair bone cartilage defect in vivo, we observed repair tissue was white and elastic, defect is shallow, defect is narrow, interface integration are continuous, but there is still a central defect.A small number of new cartilage were observed in tissue section, but most of that exfoliated to joint cavity for unfast fixation, therefore this technology require an advance study. Bone cartilage bionic scaffold model was produced by rapid prototyping 3D printing technology, the parameters of the model conformed to that of the computer model. At present rapid prototyping 3D printing technology mainly used in industrial model and cannot be used for the biomedical field now. Binder should improve to meet the requirements of 3D printing technology and have good biocompatibility.更多还原