【作者】 臧俊亭；

【导师】 刘建国；

【作者基本信息】 吉林大学 ， 外科学， 2010， 博士

【摘要】 骨缺损是骨科常见并发症之一,如何有效的修补骨缺损,同时促进骨再生修复一直是骨科工作中的难题。在一些较大的骨缺损修复过程中,单纯依靠骨组织自身的再生和修复能力难以实现骨缺损的自然愈合,而且自身骨移植的骨块一般较小难以满足要求,因此骨移植材料特别是骨组织工程材料的应用在临床工作中将起到越来越大的作用。目前常用的组织工程材料的制作方法有:发泡法;纤维粘结法;自组装法;热凝胶法;颗粒沥滤法;静电纺丝法等。其中静电纺丝技术是一种利用高压电场控制聚合物在接收靶上进行沉积制作组织工程材料的一项前沿技术。通常静电纺丝法所用的溶剂都具有较高的挥发性,通过这项技术可以使聚合物溶液以非常高的速度在电场中螺旋运动,在此过程中溶剂迅速的挥发,剩余的聚合物被拉伸成很细的纤维,并最终以微米-纳米级纤维的形式收集在接收靶上。电纺可以使支架材料具有纳米级结构,制作纳米级三维仿生骨组织工程支架,改变支架的理化性质,促进成骨细胞的粘附、增殖和基因表达,从而达到促进骨再生修复的目的。以往静电纺丝法制备的纤维以单组份或双组分多见。本研究中,我们利用静电纺丝技术制作I型胶原/聚乳酸(poly(lactic acid);PLA)/纳米羟基磷灰石(nano-hydroxyapatite;nHA)三组分混纺支架材料,用于成骨细胞-支架共培养。其中I型胶原取自动物体内,其具有免疫原性低的特点。可以较好的避免免疫排斥;并可以被人体有效利用,形成骨组织。羟基磷灰石(HA)作为天然骨组织的成分之一,能够促进成骨细胞的粘附与钙化。PLA是骨组织工程学中常用的一种聚合物,具有可降解、毒性低的特点,可以在人体内降解、吸收,不会导致毒性反应。本研究的目的是探讨I型胶原、PLA单独电纺以及I型胶原/PLA/nHA混合电纺的条件及最佳参数,对其形态及表征进行分析,并观察I型胶原/PLA/nHA共电纺三维立体支架材料对成骨细胞48小时内黏附率及成骨细胞增殖率的影响。实验部分,我们分5章分别论述了(1)单纯I型胶原静电纺丝纤维的制备;(2)单纯PLA静电纺丝纤维的制备;(3)I型胶原/PLA/nHA静电纺丝支架的制备;(4)骨髓间充质干细胞分离、培养及成骨诱导;(5)细胞与支架共培养及生物学特性研究。通过研究发现:(1)单纯I型胶原和单纯PLA溶液可以通过静电纺丝技术制备纳米级纤维。采用静电纺丝技术构建I型胶原/PLA/nHA纳米纤维骨组织工程支架是可行的,同时混纺纤维在形态上明显优于单组分电纺纤维。(2)I型胶原/PLA/nHA纳米纤维成分组成和形态结构非常接近天然骨基质,在一定程度上达到了成分和结构仿生的目的。其中,I型胶原、PLA能够为细胞粘附提供必要的位点,高孔隙率的支架能够促进细胞的迁徙和增殖,HA通过为成骨细胞提供矿化原料达到调节成骨细胞矿化的作用。(3)通过体外实验发现,I型胶原/PLA/nHA纳米纤维能够促进成骨细胞粘附、增殖,因此其可能成为一种潜在的骨组织工程材料。本研究的创新性在于:(1)解决了I型胶原/聚乳酸/纳米羟基磷灰石共电纺时的相分离问题,成功构建三组份组织工程支架;(2)证实I型胶原/聚乳酸/纳米羟基磷灰石电纺纤维形态优于单组份电纺;(3)解决了以往电纺材料膜厚度增加速度较慢的问题,提高了电纺效率;(4)通过静电纺丝技术基本模拟了天然骨基质微观形态,在一定程度上达到了成分和形态仿生目的;(5)证实该新型材料对成骨细胞粘附、增殖具有促进作用。更多还原

【Abstract】 In clinical work, big bone defects were nard to heal on their own ability of regeneration and restoration, and the bone for autographing was generally small or restricted by its shape, and was difficult to fulfill the requirement. Tissue Engineering provided a new method for born regeneration and restoration, thus more and more researchers focused on the field. At present, various medical materials has been widely utilized in clinic to repair many kinds of tissue defects and worked well to relieve patients’diseases. In the treatment of bone defects, Bone Tissue Engineering Scaffolds were thought to be one of the replacements for bone autographing. An excellent Tissue Engineering Scaffold should have the following features: high quality, reliability, durability and economy; besides it can be applied in the clinic as a medical material. As a bone tissue material, it should possess the feature of porousness, which helped to interact between cells and cells or cells and scaffolds. The 3D scaffolds can support the adhesion and proliferation of the cells, and can be applied as bone grafts. Now plentiful biomaterials have been prepared and come to use in the field of bone tissue engineering. According to their components, these materials can be classified into three as bioactive inorganic material, degradable polymer, and compound or hybrid structure containing the two above.Reviewing the recent studies, we know the component and morphology of the natural bone basically. The main components of the bone were collagen I, hydroxyapatite and water, and in the bone the mineral salts account for 65%, organic matters account for25%, and water 10%. Collagen I presented in the bone matrix, and the diameters of the collagen I fibers were several hundred nanometer. Hydroxyapatite formed the plate like structure and filled the gap among the collage I and the surface of it, and constituted the matrix. The plate like structures overlaid one another and formed the 3D porous structure. The components and morphology of the natural bone have prominent effect on the adhesion, proliferation and differentiation of Osteoblast. Recent years, there are many methods by which the components and morphology of the natural bone were imitated at home and abroad to prepare the bone tissue engineering materials. Beside them, Electrostatic Spinning was a technique utilizing the high voltage field to stretch and deposit on the target. The fibers electrospun had ultrastructure of micro-Nan scale, and can form big hole in materials.The purpose of this study is to prepare a Tissue Engineering Scaffold containing collagen-I/polylactic acid (PLA)/nanohydroxyapatite (nHA) using co-electrospinning methods; evaluate the feasibility of Co-electrospinning single and three components to prepare a novel Tissue Engineering Scaffold; imitate the components and morphostructure of natural bone in vitro; analyze the effect of collagen-I/ PLA/ nHA scaffold and PLA scaffold on Osteoblast and construct a tissue engineering bone. The novelty is that we solved the problem of dissolution of collagen-I/ PLA/ nHA for co-electrospinning, and prepared a tissue engineering scaffold containing three components successfully. We solved the issue that the membrane thickness of the electrospun material was hard to increase. By the technique of Electrostatic Spinning, we imitated the ultrastructure of natural bone basically, and this goal of preparing bionic materials was achieved to a certain extent through imitating the components and morphology. The new material was verified contributing to cell adhesion and proliferation.This study contains five parts. 1. Preparation of Collagen I fibers by electrospinningThis part of experiment was to explore the appropriate concentration of Collagen I solution and the feasibility of electrospinning it. The fittest parameters were analyzed. The morphology of Collagen I fibers were observed. Collagen I was dissolved in HFP in different concentration. Different conditions of dissolving Collagen I by persistent stirring, surfactant, heating were compared. The fittest concentration was analyzed. The homogeneous solution of Collagen I was electrospun. Through regulating voltage, electric field in unitdistance, the fit parameters of electrospinning were analyzed. By SEM, the morphology of Collagen I fibers were observed, and the diameters, density and porosity were measured. The surface features of the fibers were analyzed.2. Preparation of PLA fibers by electrospinningThis part of experiment was to contrast the impacts of Chloroform solvent and HFP solvent on electrospinning PLA respectively; explore the optimum parameters for electrospinning PLA, and analyze the feasibility of Preparation of PLA fibers by electrospinning. The morphologic features of PLA fibers were observed. Different conditions of dissolving PLA in Chloroform and HFP. The concentrations of dissolving PLA for electrospinning were compared and the fit one was chosen at the end. The homogeneous solution of PLA was electrospun. Through regulating voltage, electric field in unitdistance, the fit parameters of electrospinning were analyzed. By SEM, the morphology of PLA fibers were observed, and the diameters, density and porosity were measured. The surface features of the fibers were analyzed.3. Preparation and surface features of Collagen I/PLA/nHA scaffolds by co- electrospinningThe purpose of this part of the experiment was to eliminate the phase separation phenomenon of mix of Collagen I and PLA and prepare the homogenous Collagen I/PLA solution and to prepare the fibers of Collagen I/PLA/nHA by co-electrospinning. Through improving the target substrates, the thickness of the scaffold increased. The morphology and surface features of the fibers were observed. The PLA was dissolved in Chloroform and HFP respectively, and then blended with Collagen I HFP solution. The solutions were electrospun after being stirred 1h and 12h separately. The morphology of fibers and conditions of different solutions were observed and compared. As the reduction of targets area, the increased speed of scaffold’s thickness was observed in different diameters, and the optimal target area was selected. The parameters were optimized. By SEM, the morphology of Collagen I/PLA/nHA fibers were observed, and the diameters, density and porosity were measured. The surface features of the fibers were analyzed. The physicochemical properties of the Collagen I/PLA/nHA fibers were tested by FT-IR and XRD.4. Separation, culture and osteoinductive culture of the Bone Mesenchymal Stem Cells (BMSCs)The objective was to set up a scientific and efficient separating and culturing mode for BMSC. Through osteoinductive culture of the BMSC, the osteogenesis was tested, which provided cells for biological research of scaffolds in vitro. The BMSCs were isolated by density gradient centrifugation and were cultured. The results of morphology, growth cycle, and cell-stain were observed. The osteoinductive culturing cells were tested Alkaline Phosphatase (ALP) and produced calcified nodules.5. Co-culture of the Osteoblast and scaffolds and study of the biological featuresThe objective was to discuss the effect of Collagen I/PLA/nHA 3-D scaffolds on the Osteoblast adherence and proliferation ratio in 48h. After 3h, 6h, 12h, 24h and 48h co-culturing, the cells were digested by trypsin, and the ones alive were accounted by Trypan blue staining. Then the adherence ratio was calculated. On the 1d, 3d, 5d, 7d, 9d, 12d of co-culture, the proliferation ratio of the Osteoblast was tested by means of MTT assay. This research gained following results1. Either continuous stirred 15 mg·ml-1, 20 mg·ml-1 Collagen I or with surfactants could be electrospun and got formed fibers, and gathered on the target substrate. The fibers’diameter of 20 mg·ml-1 Collagen I was 195 nm to 643 nm. The feed liquid was 1 ml·h-1, and the electric field was 2 kV·cm-1. Some connections of few fibers fused. The shape of them was irregular. Plentiful flat or oblate fibers could be seen, but the 3-D pattern was hard to find. The single Collagen I fibers were fragile.2. 40 mg·ml-1 PLA solution could be electrospun and got the best morphology and the largest poriness. The best feed liquid was 1 ml·h-1, and the electric field was 2 kV·cm-1. Compared with the Collagen I fibers, the PLA ones had a better 3-D structure. The fibers were mainly cylinder-shaped, and had more fusions. The fragility decreased obviously.3. PLA and Collagen I were mixed in proportion to 2:1. After being stirred 12 h, they became into homogeneous solution and the fibers electrospun could gathered on the target substrate. Electrospinning after adding 20% nHA, we found that 100nm to 150nm nHA roughness was present on the surface of the fibers due to the deposition of crystals of nHA along the long axis of the fibers. The average diameters of the fibers were 238 nm. The morphology of them improved than single Collagen I or PLA. The poriness could achieve above 90%. The chemical bonds of PLA, Collagen I and nHA could be tested by FT-IR, and it turned out to be that the chain structure of Collagen I did not be destroyed during the dissolving process. The peak of wave of nHA existed obviously when observed by XRD.4. The culture cells were isolated by density gradient centrifugation. They had the common features as human BMSCs in cell morphology, growth cycle and cell staining. After the osteoinductive culture 2w, the ALP positive staining could be observed, and by alizarin red the calcium nodes were apparent.5. The nano 3D collagen-I/PLA/nHA scaffolds fabricated by electrospinning could improve the adherence of osteoblast and raised the proliferation ratio.Conclusions Collagen-I and PLA both could be electrospun into nano-fibers separately. Fabrication of collagen-I/PLA/nHA nano-fibers for bone tissue engineering was feasible. Morphology of collagen-I/PLA/nHA fibers is better than that of collagen-I and PLA fibers. Composition and morphology of collagen-I/PLA/nHA fibers are similar to that of native bone tissue, so we have achieved the biomimetic purpose in some degree. In vitro test, scaffolds of collagen-I/PLA/nHA showed the ability to induce osteoblast adhesion and proliferation, so scaffolds of collagen-I/PLA/nHA are identified as potential bone tissue engineering scaffolds.更多还原