制备软骨组织工程支架的方法

背景：软骨组织工程支架作为软骨细胞外基质的替代物,其外形和孔结构对实现其作用和功能具有非常重要的意义。 目的：回顾目前若干种常用软骨组织工程中三维多孔支架的制备方法。 方法：由第一作者检索2000至2013年PubMed数据库,ELSEVIER SCIENCEDIRECT、万方数据库、中国知网数据库。英文检索词为“Cartilage tissue engineering;scaffolds;fabrication”,中文检索词为“软骨组织工程;制备方法;支架材料;多孔支架”。 结果与结论：制备软骨组织工程支架的方法有相分离/冷冻干燥法、水凝胶技术、快速成型技术、静电纺丝法、溶剂浇铸/粒子沥滤法及气体发泡法等。目前研究发现,支架中孔径的大小对组织的重建有着直接的影响,孔径为100-250 μm的孔有益于骨及软骨组织的再生。通过溶液浇铸/粒子沥滤法、气体发泡法所制备的支架孔径大小在这一范围内,因此比较适合用于骨、软骨组织工程支架的构建。研究人员通常将多种方法结合起来,以期能制备出生物和力学性能方面更加仿生的组织工程多孔支架。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程全文链接：     

明胶/白芨胶多孔材料的细胞相容性研究

目的以明胶、白芨胶及中药黄连、丹参的提取物为原料,制备一种海绵状多孔材料,研究其与细胞的相容性熏为该产品作为创面敷料修复材料和皮肤、肌腱组织工程支架材料提供实验依据。方法将中药提取液复合到明胶和白芨胶支架材料中,通过冷冻干燥法制备成海绵状多孔材料熏以L-929细胞作为对象熏利用浸提液培养及材料表面直接培养法研究该材料的细胞相容性。结果载药材料在第2、4、7天的细胞相对增殖度在100.9%～111.3%范围内,其细胞毒性级别为0级;通过L-929细胞与明胶/白芨胶载药材料复合培养第2、7天的扫描电镜观察,L-929细胞在该材料上能很好地贴附、生长。结论明胶/白芨胶载药多孔材料有良好的细胞相容性,是可以安全使用的创面修复和皮肤、肌腱组织工程用材料。     

组织工程多孔支架材料制备技术的研究进展

组织工程多孔支架材料作为组织工程学的三大要素之一,除本身的性质外,支架材料的形状、孔径大小和孔隙率都直接影响着种子细胞的黏附、增殖和分化,因此如何制备具有高孔隙率、孔径大小合适且内部联通的多孔支架材料.为种子细胞的生长提供良好的微环境是非常重要的.回顾了近年来发展的组织工程多孔支架材料制备技术:纤维粘接法、乳液冷冻干燥法、溶液浇注,沥滤法、气体发泡法、热致相分离法及静电纺丝法.并重点介绍了目前国内外研究较多的快速成形技术;总结分析认为各种基本制备技术的联合应用和具备结构高度可控性、个体化制备特点的快速成形技术将是今后组织工程多孔支架材料制备技术的发展方向.     

制备工艺条件对胶原多孔支架材料结构和性质的影响

目的考察胶原溶胀液浓度、预冻温度等制备工艺条件对胶原多孔支架结构和性质的影响。方法采用冷冻干燥法制备胶原多孔支架,分别以不同浓度(0.2%～0.8%)的胶原溶胀液,经-50℃预冻制备;同时选择0.6%胶原溶胀液,分别经不同预冻温度(-20～-80℃)预冻制备。以甲醛为交联剂对胶原多孔支架进行化学交联。考察不同制备条件下制备的胶原多孔支架孔径结构、力学强度和降解率等性质,同时将兔关节软骨细胞种植在胶原多孔支架上,应用MTT法和扫描电镜观察比较其细胞相容性。结果随着胶原溶胀液浓度增加,多孔支架平均有效孔径逐渐减小,分布更加不均,孔径范围为50～200μm之间,支架的密度和抗拉强度逐渐增加,降解率逐渐减小;随着预冻温度的降低,胶原多孔支架孔径逐渐减小,分布更均一,降解率逐渐减小。细胞培养结果显示,胶原溶胀液浓度的减低和预冻温度的升高,制备的支架有利于软骨细胞黏附和增殖,电镜观察支架上细胞形态呈球形,有大量的丝状伪足相连。结论在不同的胶原溶胀液浓度和预冻温度的制备条件下制备的胶原多孔支架具有不同孔径结构,进而影响到胶原多孔支架的力学性能,降解性能和细胞相容性等。在本实验所选择孔隙尺寸范围内,孔径越大越有利于软骨细胞的黏附和增殖,胶原溶胀液浓度为0.3%～0.6%和预冻温度为-30～-50℃胶原多孔支架适合软骨细胞的黏附和增殖。     

多孔聚乙烯醇/明胶软骨组织工程支架复合材料的制备及其生物相容性

背景：以明胶为基体制备的组织工程支架材料具有良好的生物相容性和生物降解性能,但存在力学性能低,降解速率难以控制的缺陷。 目的：制备一种软骨组织工程支架材料多孔聚乙烯醇/明胶复合物,并检测其理化性能和生物相容性。 方法：采用乳化发泡法制备聚乙烯醇/明胶多孔支架,并通过电镜分析、力学测试、皮下植入实验,检测材料孔径和孔隙率、IR光谱、力学性能和生物相容性。 结果与结论：多孔材料内部呈三维网状多孔结构,孔径均匀,有相似的孔隙率61.8%,含水率44.6%,抗拉强度为(5.01±0.03) MPa,抗压强度为(1.47±0.36) MPa,有较好的力学性能,IR光谱分析表明材料内部结构均匀。皮下植入后,炎症反应逐渐减轻,囊壁逐渐变薄,并趋于稳定,提示多孔聚乙烯醇/明胶支架材料具有较好的生物相容性和力学性能。     

丝素蛋白/明胶/壳聚糖三维多孔软骨组织支架的制备及体外评价

背景：软骨缺损是骨科医生面临的主要临床挑战之一，组织工程是一种结合了工程学和细胞生物学知识的跨学科方法，为软骨缺损的修复提供了新思路与途径。目的：基于丝素蛋白、明胶和壳聚糖制备多组分复合支架，通过评估其理化性质和生物学性能，筛选能够适合软骨再生的三维多孔支架。方法：以丝素蛋白、明胶和壳聚糖为基础材料，通过真空冷冻干燥法制备4组多孔支架，分别为明胶/壳聚糖支架、丝素蛋白/壳聚糖支架、丝素蛋白/明胶支架和丝素蛋白/明胶/壳聚糖支架，通过扫描电镜、X射线衍射、孔隙率、吸水膨胀率和生物降解率及力学性能等检测筛选出合适的软骨支架。然后将软骨支架与骨关节炎患者软骨细胞共培养，通过细胞黏附率、活死染色和增殖活性等检测体外评估多孔支架用于软骨损伤修复的可行性。结果与结论：(1)4组支架均具有多孔结构，综合物理性能检测结果得出丝素蛋白/明胶/壳聚糖支架更符合软骨缺损修复的要求，该支架的孔径为(176.00±53.68)μm，孔隙率为(80.15±2.57)%，吸水溶胀率为(3 712±358)%，体外浸泡于含溶菌酶的PBS中28 d后的生物降解速率为(46.87±3.25)%，且具有良好的机械性能；(2...     

钙磷生物陶瓷多孔骨修复材料的制备*

以适量的Mg(H2PO4)2-(NaPO3)6为粘结剂,HA和-TCP粉末为原料,用有机泡沫浸渍法制备钙磷多孔生物陶瓷坯体,并在850℃烧成,探索在较低烧结温度下制备钙磷多孔生物陶瓷的工艺。采用X射线衍射(XRD)、扫描电镜(SEM)、能谱(EDS)等方法对多孔生物陶瓷的物相组成、显微结构、物理性能进行了分析。烧成后的钙磷生物陶瓷多孔支架主要由-TCP、-Ca2P2O7和CaO-MgO-Na2O-P2O5磷酸盐玻璃组成。烧结过程中,HA发生了向-TCP的转化,部分-TCP转化为-Ca2P2O7。多孔支架具有良好三维连通性的孔隙结构,孔径为200～500m,孔隙率达81%,抗压强度为1.1～1.5MPa。     

超临界二氧化碳流体发泡技术制备组织工程支架及其泡孔形貌控制研究进展

组织工程支架作为细胞生长的载体,在组织工程再生组织的研究中具有非常重要的地位。传统方法在三维支架的理论研究与制备技术方面均已趋于成熟,但制备过程仍存在工艺复杂,有机溶剂难以去除,制备条件不利于保持生物分子活性等问题。近年来,超临界二氧化碳流体技术利用优越的传质性与环境友好性,已广泛用于制备各种三维结构的组织工程支架,其中以超临界发泡技术最为经典。三维多孔支架在超临界发泡技术制备过程中,泡孔形貌受材料性质、致孔剂种类及工艺参数等方面的影响。本文就采用该技术制备的多孔支架研究进展及存在的问题进行综述,同时对该技术控制泡孔形貌的发展方向提出展望。     

聚乳酸多孔支架制备及细胞实验

以冰粒子作为致孔剂，采用冷冻干燥-粒子滤出复合法制备了块状聚乳酸多孔支架。将聚乳酸溶于氯仿溶液后加入冰粒子，在液氮中冷冻后冷冻干燥获得多孔支架。对支架孔隙结构分析表明，该工艺制备的多孔支架无致孔剂残留，其孔隙大小由加入的冰粒子大小决定。细胞实验表明该多孔支架具有较好的生物相容性并且无细胞毒性。     

肝素化胶原/壳聚糖多孔支架的制备及其血管化的研究

本研究旨在构建一种能快速血管化的人工真皮替代物。用冻干法制备了胶原／壳聚糖多孔支架，并对其进行肝素化，观察此支架的结构特征、亲水性、体外降解性和组织相容性，同时将血管生成素引入到此支架，对复合有血管生成素的肝素化支架的体内血管化进行了初步研究。结果表明，肝素化胶原／壳聚糖多孔支架具有合适的三维多孔结构、良好的吸水性和较理想的酶解稳定性，体内实验表明，此支架具有良好的组织相容性，血管生成素可加快支架的血管化。     

The design of scaffolds for use in tissue engineering. Part I. Traditional factors.

In tissue engineering, a highly porous artificial extracellular matrix or scaffold is required to accommodate mammalian cells and guide their growth and tissue regeneration in three dimensions. However, existing three-dimensional scaffolds for tissue engineering proved less than ideal for actual applications, not only because they lack mechanical strength, but they also do not guarantee interconnected channels. In this paper, the authors analyze the factors necessary to enhance the design and manufacture of scaffolds for use in tissue engineering in terms of materials, structure, and mechanical properties and review the traditional scaffold fabrication methods. Advantages and limitations of these traditional methods are also discussed.     

Cytocompatibility and blood compatibility of multifunctional fibroin/collagen/heparin scaffolds

An applicable matrix used in tissue engineering should not only have suitable mechanical properties, porous structures and biocompatibility that facilitate the adhesion, growth and proliferation of tissue cells, but also have the ability to release bioactive factors to provide a more conducive and inductive environment for tissue growth. Because of the harsh preparation conditions and deficiency of mechanical properties, it is still difficult for fibroin and collagen matrices to possess these multifunctional properties. In this research, we successfully prepared fibroin/collagen hybrid scaffolds containing heparin that possess multifunctional properties under mild conditions. These scaffolds maintain outstanding mechanical properties and porous structures of fibroin-based scaffolds. Furthermore, the scaffolds keep the bioactivity of collagen, becoming delivering systems that release heparin slowly to make the scaffolds blood compatible. Compared with fibroin/collagen scaffolds, the scaffolds containing heparin further facilitate the growth of HepG2 cells since a more complex, dynamic environment was formed to promote the cell growth. Considering the mild aqueous preparation environment without crosslinking reaction, besides promoting the progress in blood contacting tissue engineering, our research has also opened a door to prepare various multifunctional fibroin/collagen hybrid matrices that combine the advantages of fibroin and collagen.     

Effect of sterilization on structural and material properties of 3-D silk fibroin scaffolds

The development of porous scaffolds for tissue engineering applications requires the careful choice of properties, as these influence cell adhesion, proliferation and differentiation. Sterilization of scaffolds is a prerequisite for in vitro culture as well as for subsequent in vivo implantation. The variety of methods used to provide sterility is as diverse as the possible effects they can have on the structural and material properties of the three-dimensional (3-D) porous structure, especially in polymeric or proteinous scaffold materials. Silk fibroin (SF) has previously been demonstrated to offer exceptional benefits over conventional synthetic and natural biomaterials in generating scaffolds for tissue replacements. This study sought to determine the effect of sterilization methods, such as autoclaving, heat-, ethylene oxide-, ethanol- or antibiotic–antimycotic treatment, on porous 3-D SF scaffolds. In terms of scaffold morphology, topography, crystallinity and short-term cell viability, the different sterilization methods showed only few effects. Nevertheless, mechanical properties were significantly decreased by a factor of two by all methods except for dry autoclaving, which seemed not to affect mechanical properties compared to the native control group. These data suggest that SF scaffolds are in general highly resistant to various sterilization treatments. Nevertheless, care should be taken if initial mechanical properties are of interest.     

Fabrication of PLGA scaffolds using soft lithography and microsyringe deposition

Construction of biodegradable, three-dimensional scaffolds for tissue engineering has been previously described using a variety of molding and rapid prototyping techniques. In this study, we report and compare two methods for fabricating poly(DL-lactide-co-glycolide) (PLGA) scaffolds with feature sizes of approximately 10-30 microm. The first technique, the pressure assisted microsyringe, is based on the use of a microsyringe that utilizes a computer-controlled, three-axis micropositioner, which allows the control of motor speeds and position. A PLGA solution is deposited from the needle of a syringe by the application of a constant pressure of 20-300 mm Hg, resulting in a controlled polymer deposition. The second technique is based on 'soft lithographic' approaches that utilize a poly(dimethylsiloxane) mold. Three variations of the second technique are presented: polymer casting, microfluidic perfusion, and spin coating. Polymer concentration, solvent composition, and mold dimensions influenced the resulting scaffolds as evaluated by light and electron microscopy. As a proof-of-concept for scaffold utility in tissue engineering applications, multilayer structures were formed by thermal lamination, and scaffolds were rendered porous by particulate leaching. These simple methods for forming PLGA scaffolds with microscale features may serve as useful tools to explore structure/function relationships in tissue engineering.     

纤维基组织工程支架结构对细胞行为的影响

有效引导细胞的生长对于组织工程的发展至关重要,而目前研究表明细胞与支架的相互作用受到材料表面结构的影响,这为设计细胞诱导生长的新型支架提供理论依据。通过静电纺丝技术制备的纤维基支架可以模拟天然细胞外基质的纤维网络结构,对于细胞的生长和组织修复有促进作用,因此成为组织工程支架设计的研究热点。从纤维直径、空间排列、孔径等方面综述支架结构对细胞增殖、迁移、分化等行为的影响,并进一步讨论利用静电纺和静电纺复合技术制备不同纤维结构的常用方法,并展望纤维基支架的未来发展方向。     

Fabrication and in vitro degradation of porous fumarate-based polymer/alumoxane nanocomposite scaffolds for bone tissue engineering

In this work, the fabrication and in vitro degradation of porous fumarate-based/alumoxane nanocomposites were evaluated for their potential as bone tissue engineering scaffolds. The biodegradable polymer poly (propylene fumarate)/propylene fumarate-diacrylate (PPF/PF-DA), a macrocomposite composed of PPF/PF-DA and boehmite microparticles, and a nanocomposite composed of PPF/PF-DA and surface-modified alumoxane nanoparticles were used to fabricate porous scaffolds by photo-crosslinking and salt-leaching. Scaffolds then underwent 12 weeks of in vitro degradation in phosphate buffered saline at 37 degrees C. The presence of boehmite microparticles and alumoxane nanoparticles in the polymer inhibited scaffold shrinkage during crosslinking. Furthermore, the incorporation of alumoxane nanoparticles into the polymer limited salt-leaching, perhaps due to tighter crosslinking within the nanocomposite. Analysis of crosslinking revealed that the acrylate and overall double bond conversions in the nanocomposite were higher than in the PPF/PF-DA polymer alone, though these differences were not significant. During 12 weeks of in vitro degradation, the nanocomposite lost 5.3% +/- 2.4% of its mass but maintained its compressive mechanical properties and porous architecture. The addition of alumoxane nanoparticles into the fumarate-based polymer did not significantly affect the degradation of the nanocomposite compared with the other materials in terms of mass loss, compressive properties, and porous structure. These results demonstrate the feasibility of fabricating degradable nanocomposite scaffolds for bone tissue engineering by photo-crosslinking and salt-leaching mixtures of fumarate-based polymers, alumoxane nanoparticles, and salt microparticles.     

Ice-template-induced silk fibroin-chitosan scaffolds with predefined microfluidic channels and fully porous structures

Scaffold-based tissue engineering has made great progress in fabricating relatively simple tissues. One of the major challenges in creating thick complex organs is to achieve sufficient nutrient supply as well as uniform cell distribution in a three-dimensional (3D) scaffold. Here we employed microstructured ice templates to fabricate silk fibroin-chitosan (SF-CS) scaffolds with predefined microfluidic channels, open-pore surface and oriented porous structures. The effects of these structural organizations in ice-template-induced (ITI) scaffolds on nutrient delivery, cell seeding as well as cell growth were well investigated in comparison with that of polydimethylsiloxane-template-induced scaffolds. The ITI scaffolds exhibited better structural properties in promoting mass transport, facilitating uniform cell distribution and growth. The ITI scaffolds uniformly seeded with living cells could be further rolled up to form a thick tissue-engineered construct with predefined microfluidic channels. We envision that our ITI scaffolds can be potentially used to engineer thick prevascularized organs when the oriented porous structures are uniformly seeded with primary cells and the predefined microfluidic channels are incorporated with endothelial cells.     

纳米羟基磷灰石/Ⅰ型胶原/壳聚糖复合支架材料的制备与优化

目的制备适合于骨组织工程的高强度纳米羟基磷灰石/Ⅰ型胶原/壳聚糖复合支架材料。方法用原位合成法代替传统的直接分散法,以胶原和壳聚糖为模板原位合成羟基磷灰石,再用冷冻干燥法使材料成型,制成可用于骨组织工程的多孔支架材料。结果制备的材料孔隙率高,孔的连通性好,材料中羟基磷灰石结晶度更小,表面能大,与有机物基底结合紧密,也能为成骨细胞的粘附提供更多的活性位点。结论用紫外辐照对材料进行处理,能使其抗压性能得到提高。制备的支架材料适用于骨组织工程。     

Low-pressure foaming: a novel method for the fabrication of porous scaffolds for tissue engineering

Scaffolds for tissue engineering applications must incorporate porosity for optimal cell seeding, tissue ingrowth, and vascularization, but common fabrication methods for achieving porosity are incompatible with a variety of polymers, limiting widespread use. In this study, porous scaffolds consisting of poly(1,8-octanediol-co-citrate) (POC) containing hydroxyapatite nanocrystals (HA) were fabricated using low-pressure foaming (LPF). LPF is a novel method of fabricating an interconnected, porous scaffold with relative ease. LPF takes advantage of air bubbles that act as pore nucleation sites during a polymer mixing step. Vacuum is applied to expand the nucleation sites into interconnected pores that are stabilized through cross-linking. POC was combined with 20%, 40%, and 60% by weight HA, and the effect of increasing HA particle content on porosity, mechanical properties, and alkaline phosphatase (ALP) activity of human mesenchymal stem cells (hMSC) was evaluated. The effect of the prepolymer viscosity on porosity and the mechanical properties of POC with 40% by weight HA (POC-40HA) were also assessed. POC-40HA scaffolds were also implanted in an osteochondral defect of a rabbit model, and the explants were assessed at 6 weeks using histology. With increasing HA content, the pore size of POC-HA scaffolds can be varied (85 to 1,003 μm) and controlled to mimic the pore size of native trabecular bone. The compression modulus increased with greater HA content under dry conditions and were retained to a greater extent than with porous scaffolds fabricated using salt-leaching under wet conditions. Furthermore, all POC-HA scaffolds prepared using LPF supported hMSC attachment, and an increase in ALP activity correlated with an increase in HA content. An increase in the prepolymer viscosity resulted in increased compression modulus, greater distance between pores, and less porosity. After 6 weeks in vivo, cell and tissue infiltration was present throughout the scaffold. This study describes a novel method of creating porous osteoconductive POC scaffolds without the need for porogen leaching and provides the groundwork for applying LPF to other elastomers and composites.