Gro许旺细胞在不同孔径丝素蛋白支架上的生长(英文)

背景:细胞在生物支架上的生长行为受到支架表面形貌、润湿性、孔径及孔隙率等多种因素影响。目的:观察许旺细胞在不同孔径丝素蛋白支架上的生长情况。方法:制备大孔径50～60μm、小孔径10～20μm两种多孔丝素材料。选用许旺细胞永生化细胞R3[33-10ras3]为种子细胞,当细胞在培养瓶底形成致密单层时即可消化细胞并进行接种实验,将许旺细胞悬液种于不同形貌的多孔丝素材料表面。复合培养1周后,扫描电镜观察许旺细胞的生长形态及增殖等情况。结果与结论:不同孔径丝素材料的表面可见许旺细胞生长情况不一。在10～20μm孔径材料支架上,细胞浓度较低,细胞表现为特异的双极性形态,细胞与细胞之间或平行排列,或首尾相连成细胞链;细胞与细胞之间或平行排列,或首尾相连成细胞链;在50～60μm孔径丝素材料支架上,细胞浓度较高,细胞多为球形,单个分散在多孔支架表面,或呈现成团成串葡萄样聚集在孔的底部,未延展成双极性形态,只有极少量生长在孔与孔之间嵴上的细胞呈双极样。说明多孔丝素蛋白支架的孔径对许旺细胞的黏附、生长有一定的影响,许旺细胞更适合生长在孔径略大于胞体直径的支架材料上。     

Improving pore interconnectivity in polymeric scaffolds for tissue engineering

A new scaffold fabrication technique aiming to enhance pore interconnectivity for tissue engineering has been developed. Medical grade poly(lactic acid) was utilized to generate scaffolds by a solvent‐evaporating/particulate‐leaching technique, using a new dual‐porogen system. Water‐soluble sodium chloride particles were used to control macro‐pore size in the range 106–255 µm, while organic naphthalene was utilized as a porogen to increase pore interconnections. The three‐dimensional (3D) morphology of the scaffolds manufactured with and without naphthalene was examined by optical coherence tomography and scanning electron microscopy. The mechanical properties of the scaffolds were characterized by compression tests. MG63 osteoblast cells were seeded in the scaffolds to study the cell attachment and viability evaluated by confocal microscopy. It was revealed that introducing naphthalene as the second porogen in the solvent‐evaporating/particulate‐leaching process resulted in improvement of the pore interconnectivity. Cells grew in both scaffolds fabricated with and without naphthalene. They exhibited strong green fluorescence when using a live/dead fluorescent dye kit, indicating that the naphthalene in the scaffold process did not affect cell viability. Copyright © 2009 John Wiley & Sons, Ltd.     

许旺细胞在不同孔径丝素蛋白支架上的生长

背景：细胞在生物支架上的生长行为受到支架表面形貌、润湿性、孔径及孔隙率等多种因素影响。目的：观察许旺细胞在不同孔径丝素蛋白支架上的生长情况。方法：制备大孔径50～60μm、小孔径10～20μm两种多孔丝素材料。选用许旺细胞永生化细胞R3[33-10ras3]为种子细胞,当细胞在培养瓶底形成致密单层时即可消化细胞并进行接种实验,将许旺细胞悬液种于不同形貌的多孔丝素材料表面。复合培养1周后,扫描电镜观察许旺细胞的生长形态及增殖等情况。结果与结论：不同孔径丝素材料的表面可见许旺细胞生长情况不一。在10～20μm孔径材料支架上,细胞浓度较低,细胞表现为特异的双极性形态,细胞与细胞之间或平行排列,或首尾相连成细胞链;细胞与细胞之间或平行排列,或首尾相连成细胞链;在50～60μm孔径丝素材料支架上,细胞浓度较高,细胞多为球形,单个分散在多孔支架表面,或呈现成团成串葡萄样聚集在孔的底部,未延展成双极性形态,只有极少量生长在孔与孔之间嵴上的细胞呈双极样。说明多孔丝素蛋白支架的孔径对许旺细胞的黏附、生长有一定的影响,许旺细胞更适合生长在孔径略大于胞体直径的支架材料上。     

Evaluation of methods for the construction of collagenous scaffolds with a radial pore structure for tissue engineering

Type I collagen is used widely as a biomaterial. The structure of collagenous biomaterials, including pore sizes and general architecture, can be varied by a number of techniques. In this study, we developed a method to construct flat fibrillar type I collagen scaffolds, 6 cm in diameter and with a radially orientated pore structure, by the use of directional freezing. Different methodologies were tested, the optimal one being freezing of a collagen suspension inside-out, using a centrally positioned liquid nitrogen-cooled tube. Pore sizes could be varied by the use of different tube materials. Use of aluminium tubes resulted in radial scaffolds with a pore size of 20-30 μm, whereas use of stainless steel produced radial scaffolds with 70-100 μm pore sizes. Brass- and copper-based tubes produced scaffolds with less homogeneous radial pores, pore sizes being 90-100 and 50-80 μm, respectively. Fibreglass tubes gave even less uniformity (pore size 100-150 μm). Scaffolds were free of cracks, except in case of aluminium. Scaffolds with a radial inner structure may be especially suitable for tissue engineering of organs with a radial scaffold structure, such as the diaphragm.     

Pore size regulates cell and tissue interactions with PLGA-CaP scaffolds used for bone engineering

A common subject in bone tissue engineering is the need for porous scaffolds to support cell and tissue interactions aiming at repairing bone tissue. As poly(lactide-co-glycolide)-calcium phosphate (PLGA-CaP) scaffolds can be manufactured with different pore sizes, the aim of this study was to evaluate the effect of pore diameter on osteoblastic cell responses and bone tissue formation. Scaffolds were prepared with 85% porosity, with pore diameters in the ranges 470-590, 590-850 and 850-1200 μm. Rat bone marrow stem cells differentiated into osteoblasts were cultured on the scaffolds for up to 10 days to evaluate cell growth, alkaline phosphatase (ALP) activity and the gene expression of the osteoblast markers RUNX2, OSX, COL, MSX2, ALP, OC and BSP by real-time PCR. Scaffolds were implanted in critical size rat calvarial defects for 2, 4, and 8 weeks for histomorphometric analysis. Cell growth and ALP activity were not affected by the pore size; however, there was an increase in the gene expression of osteoblastic markers with the increase in the pore sizes. At 2 weeks all scaffolds displayed a similar amount of bone and blood vessels formation. At 4 and 8 weeks much more bone formation and an increased number of blood vessels were observed in scaffolds with pores of 470-590 μm. These results show that PLGA-CaP is a promising biomaterial for bone engineering. However, ideally, combinations of larger (-1000 μm) and smaller (-500 μm) pores in a single scaffold would optimize cellular and tissue responses during bone healing.     

Effect of pore sizes of PLGA scaffolds on mechanical properties and cell behaviour for nucleus pulposus regeneration in vivo

This study investigated the influence of pore sizes of poly(lactic‐co‐glycolic acid) (PLGA) scaffolds on the compressive strength of tissue‐engineered biodiscs and selection of the best suitable pore size for cells to grow in vivo. PLGA scaffolds were fabricated by solvent casting/salt‐leaching with pore sizes of 90–180, 180–250, 250–355 and 355–425 µm. Nucleus pulposus (NP) cells were seeded on PLGA scaffolds with various pore sizes. Each sample was harvested at each time point, after retrieval of PLGA scaffolds seeded with NP cells, which were implanted into subcutaneous spaces in nude mice at 4 and 6 weeks. MTT assay, glycosaminoglycan (GAG) assay, haematoxylin and eosin (H&E) staining, safranin O staining and immunohistochemistry (for collagen type II) were performed at each time point. As the pores became smaller, the value of the compressive strength of the scaffold was increased. The group of scaffolds with pore sizes of 90–250 µm showed better cell proliferation and ECM production. These results demonstrated that the compressive strength of the scaffold was improved while the scaffold had pore sizes in the range 90–250 µm and good cell interconnectivity. Suitable space in the scaffold for cell viability is a key factor for cell metabolism. Copyright © 2014 John Wiley & Sons, Ltd.     

天然及合成尿道重建支架材料的生物相容性及力学性能

背景：目前应用何种尿道组织工程修复重建支架材料更为合适的争论仍不断出现,其生物相容性及力学特性的评估也鲜见报道。目的：评估应用于尿道修复重建多种生物材料的力学特性及生物相容性。方法：脱细胞法制备小肠黏膜下层组织、膀胱脱细胞基质以及脱细胞尿道海绵体基质,同时编织法制备聚乙醇酸支架。单轴拉伸测试测定各类支架生物力学特性,光镜及扫描电镜测定支架表面孔径大小。线粒体代谢活性MTT法检测各种生物材料的细胞毒性。所有支架表面接种海绵体平滑肌细胞,体外培养14d后进一步评估细胞渗透情况。结果与结论：生物力学评估显示脱细胞尿道海绵体基质在弹性模量以及断裂强度方面的检测结果明显优于其余材料（P〈0.05）。MTT结果显示所有支架材料均支持正常的细胞生长代谢,并未发现存在明显的细胞毒性。聚乙醇酸在扫描电镜中显示出最大的孔径（〉200μm）,同时脱细胞尿道海绵体基质的尿道面（〈5μm）和海绵体面（〉10μm）观察到明显不同的孔径大小。细胞接种14d后聚乙醇酸材料的内部可见种子细胞的广泛分布,在膀胱脱细胞基质以及脱细胞尿道海绵体基质的尿道面未发现有明显的细胞渗透迹象,而小肠黏膜下层组织和脱细胞尿道海绵体基质的海绵体面观察到明显的细胞渗透生长表现。提示所有支架材料显示出良好的生物相容性,同时在力学特性方面也与正常尿道组织相仿,但脱细胞尿道海绵体基质在力学和组织学的诸多参数上具有一定的优越性。     

Development of poly(lactide‐co‐glycolide) scaffold‐impregnated small intestinal submucosa with pores that stimulate extracellular matrix production in disc regeneration

The pore size and microstructure of scaffolds influences cell attachment, migration, proliferation and ingrowth, but the optimal pore size of scaffolds for disc tissue formation is not clearly understood. We developed porous poly(lactide‐co‐glycolide) (PLGA) scaffolds with various pore sizes for nucleus pulposus (NP) cell cultures and examined the effects of pore size on cell ingrowth and extracellular matrix (ECM) synthesis. High cell density in the small pores of scaffolds promotes collagen synthesis and cell migration through interconnected pores. Scaffolds with large pores exhibited slower cell proliferation and collagen synthesis. Guided by these results, we investigated a novel, biodegradable, synthetic/natural hybrid scaffold composed of PLGA and small intestinal submucosa (SIS) (PLGA–SIS) with the proper pore size for NP regeneration. We tested the morphological and physical properties of PLGA–SIS scaffolds and initial cell attachment and ECM production of NP in scaffolds. The mechanical and degradable properties of the PLGA–SIS scaffold were superior to those of SIS sponge and were similar to the properties of PLGA scaffold. NP cells grown on PLGA–SIS scaffold exhibited higher initial cell adhesion and ECM production than those grown on pure PLGA scaffold in a biological assay. In conclusion, this study suggests that a proper pore size of scaffolds is critical in NP regeneration, and that PLGA–SIS scaffolds with suitable pores might be useful as substrates for tissue‐engineered biodiscs. Copyright © 2012 John Wiley & Sons, Ltd.     

Breast epithelial cell infiltration in enhanced electrospun silk scaffolds

In the present study, the effects of air‐flow impedance electrospinning and air‐flow rates on silk‐based scaffolds for biological tissues were investigated. First, the properties of scaffolds obtained from 7% and 12% silk concentrations were defined. In addition, cell infiltration and viability of MCF‐10A breast epithelial cells cultured onto these scaffolds were used to determine the biological suitability of these nanostructures. Air‐flow impedance electrospun scaffolds resulted in an overall larger pore size than scaffolds electrospun on a solid mandrel, with the largest pores in 7% silk electrospun with an air pressure of 100 kPa and in 12% silk electrospun with an air pressure of 400 kPa (13.4 ± 0.67 and 26.03 ± 1.19 µm, respectively). After 14 days in culture, the deepest MCF‐10A cell infiltration (36.58 ± 2.28 µm) was observed into 7% silk air‐flow impedance electrospun scaffolds subjected to an air pressure of 100 kPa. In those scaffolds MCF‐10A cell viability was also highest after 14 days in culture. Together, these results strongly support the use of 7% silk‐based scaffolds electrospun with a 100 kPa air‐flow as the most suitable microenvironment for MCF‐10A infiltration and viability. Copyright © 2013 John Wiley & Sons, Ltd.     

Microwave‐sintered 3D printed tricalcium phosphate scaffolds for bone tissue engineering

This study reports the manufacturing process of 3D interconnected macroporous tricalcium phosphate (TCP) scaffolds with controlled internal architecture by direct 3D printing (3DP), and high mechanical strength obtained by microwave sintering. TCP scaffolds with 27%, 35% and 41% designed macroporosity with pore sizes of 500 μm, 750 μm and 1000 μm, respectively, were manufactured by direct 3DP. These scaffolds were then sintered at 1150 °C and 1250 °C in conventional electric muffle and microwave furnaces, respectively. Total open porosity between 42% and 63% was obtained in the sintered scaffolds due to the presence of intrinsic micropores along with designed pores. A significant increase in compressive strength between 46% and 69% was achieved by microwave compared to conventional sintering as a result of efficient densification. Maximum compressive strengths of 10.95 ± 1.28 MPa and 6.62 ± 0.67 MPa were achieved for scaffolds with 500 μm designed pores (~ 400 μm after sintering) sintered in microwave and conventional furnaces, respectively. An increase in cell density with a decrease in macropore size was observed during in vitro cell‐material interactions using human osteoblast cells. Histomorphological analysis revealed that the presence of both micro‐ and macropores facilitated osteoid‐like new bone formation when tested in femoral defects of Sprague–Dawley rats. Our results show that bioresorbable 3D‐printed TCP scaffolds have great potential in tissue engineering applications for bone tissue repair and regeneration. Copyright © 2012 John Wiley & Sons, Ltd.     

应用溶剂浇铸-颗粒滤沥法制备气管软骨组织工程管状泡沫支架

背景:孔径和孔隙率是三维多孔状材料的两个主要评价指标,孔隙率越高越利于软骨细胞的长入及增殖,但是,随着孔隙率的进一步增高支架的抗压强度随之下降,并且一定孔径的有效孔也下降。因此合适的孔径及孔隙率的三维多孔支架材料的制备是气管组织工程成败的重要一环。目的:以溶剂浇铸-颗粒滤沥法制备管状泡沫支架,为气管软骨组织工程寻找实用、理想的支架。设计:观察性实验。单位:郧阳医学院附属太和医院耳鼻咽喉科,四川大学华西医院耳鼻咽喉科。材料:实验于2002-03/05在中科院成都分院化学所完成。外消旋聚乳酸原料,Mr=4.23×104,氯化钠颗粒(直径50～200μm)为致孔剂。方法:在半球形玻璃容器中,将外消旋聚乳酸溶于氯仿中,配制成100g/L的溶液,然后按800,850,900,920,940,960g/L不同质量分数加入氯化钠颗粒(50～200μm)作为致孔剂(1～6号支架),搅拌混匀,制成混悬液,呈糊状。将混悬液浇铸于管柱状模具中加热(90℃)、加压,然后置于通风柜中48h待溶剂挥发,残余溶剂抽真空除去。取出已定型干燥管柱状泡沫支架,放入盛有双蒸水的烧杯中浸泡48h,间隔8h换水1次,去除致孔剂氯化钠。然后将所有管柱状材料放入真空干燥箱中24～48h,即得多孔管柱状外消旋聚乳酸三维支架材料。经过大体及扫描电镜的观察支架形状及强度,同时进行孔隙参数的测定及分析。主要观察指标:①管状泡沫支架大体和扫描电镜观察结果。②孔隙参数的测定。结果:①支架外观为白色、管柱状泡沫,内径8mm,外径12mm。孔径80～250μm、孔隙率90.6%的支架具有一定的强度及韧性。②扫描电镜观察外消旋聚乳酸支架中有大小不等的孔分布,支架内孔与孔之间相互连通,并且大孔中又包含无数小孔。③随着氯化钠质量分数的增加支架的孔隙率也增加,但有效孔并不随着氯化钠质量分数的增加而增加。孔径为80～250μm,各样品有效孔不同,4号样品有效孔为76%,对应的孔隙率为90.6%。4号样品为较理想的支架。结论:通过溶剂浇铸-颗粒滤沥法制备的管状泡沫支架是气管软骨组织工程合适的支架。     

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。     

3D ingrowth of bovine articular chondrocytes in biodegradable cryogel scaffolds for cartilage tissue engineering

A feasibility study was undertaken to examine the potential of biodegradable HEMA-lactate-dextran (HEMA-LLA-D)-based cryogels as scaffolds for cartilage tissue engineering. This was a preliminary in vitro study giving essential information on the biocompatibility of cryogels with cartilage cells. HEMA-lactate (HEMA-LLA) and HEMA-LLA-D were synthesized and characterized by different techniques. Cryogel scaffolds with supermacroporous structures were produced by cryogenic treatment of these macromers. Chondrocytes obtained from bovine articular cartilage were seeded onto cylindrical cryogels and cultured. The samples were examined by several microcopical techniques for cell viability and morphological analyses were performed at two culture points. Histological study of the constructs revealed the cells' growth on the surface and within the scaffolds. Confocal microscopical images demonstrated that the majority of live vs. dead cells had been attached to and integrated with the pores of the scaffold. SEM analysis showed round to oval-shaped chondrocytic cells interconnected with each other by communicating junctions. The chondrocytes rapidly proliferated in the cryogels, manifesting that they fully covered the scaffold surface after 9 days and almost filled the spaces in the pores of the scaffold after 15 days of culture. Chondrocytes secreted significant amount of extracellular matrix in the scaffolds and exhibited highly interconnective morphology. Light and transmission electron microscopy revealed groups of active cartilage cells closely apposed to the cryogel. We concluded that cryogel scaffolds could be excellent candidates for cartilage tissue regeneration with their extraordinary properties, including soft, elastic nature, highly open interconnected pore structure and very rapid, controllable swellability.     

不同孔径的国产PDLLA填充材料修复骨缺损实验研究

目的评价不同孔径三维立体泡沫状外消旋聚乳酸材料（PDLLA）的成骨能力。方法采用溶剂注模并盐结晶颗粒沥滤法制成3种（70～150μm,150～300μm和400～700μm）孔径的外PULLA料,在62例1cm兔桡骨去骨膜缺损模型中分别植入3种孔径材料和空白对照,术后2、4、8、12周行X线、组织学观察及8、12周扫描电镜观察骨缺损区骨生成情况。结果70～150μm与150～300μm材料成骨较好,与400～700μm组和对照组相比差异有显著性P（<0.05）。结论PDLLA具有良好的生物相容性和可吸收性,制成泡沫状具有较好的骨传导性能。     

Greater scaffold permeability promotes growth of osteoblastic cells in a perfused bioreactor

Pore size and porosity have been widely acknowledged as important structural factors in tissue‐engineered scaffolds. In fact, scaffolds with similar pore size and porosity can provide important and varied permeability due to different pore shape, interconnectivity and tortuosity. However, the effects of scaffold permeability on seeded cells remains largely unknown during tissue regeneration in vitro. In this study, we measured the Darcy permeability (K) of tri‐calcium phosphate scaffolds by distributed them into three groups: Low, Medium and High. As a result, the effects of scaffold permeability on cell proliferation, cellular activity and growth in the inner pores were investigated in perfused and static cultures in vitro. Results demonstrated that higher permeable scaffolds exhibited superior performance during bone regeneration in vitro and the advantages of higher scaffold permeability were amplified in perfused culture. Based on these findings, scaffold permeability should be considered in future scaffold fabrications. Copyright © 2013 John Wiley & Sons, Ltd.     

胶原-壳聚糖支架与软骨细胞的生长

背景：目前软骨支架材料的种类比较多,但还没有一种材料能完全符合软骨修复的要求。目的：观察在混合材料胶原-壳聚糖支架中软骨细胞的生长情况。方法：采用冷冻干燥法将质量分数为2%胶原与3%壳聚糖混合制备胶原-壳聚糖多孔支架。将分离培养的第2代兔软骨细胞接种到胶原-壳聚糖支架上,对照组将软骨细胞接种到无支架的培养板中。观察支架的孔隙率、吸水性及内部形态结构,MTT法检测软骨细胞在支架上的增殖情况,组织切片苏木精-伊红染色,扫描电镜观察细胞在支架的生长、贴附情况,RT-PCR检测细胞支架复合物蛋白聚糖和Ⅱ型胶原mRNA表达情况。结果与结论：胶原-壳聚糖支架的吸水性为（80.0±0.55）%,孔隙率为（88.5±1.5）%,孔径为100～150μm,复合细胞培养2周后,细胞增殖活力高,软骨细胞分泌的蛋白聚糖和Ⅱ型胶原mRNA表达明显高于对照组。说明质量分数为2%胶原与3%壳聚糖的混合支架适合软骨细胞生长和快速增殖,是一种良好的修复和重建软骨载体。     

Proliferation and osteogenic differentiation of human bone marrow stromal cells on alginate–gelatine–hydroxyapatite scaffolds with anisotropic pore structure

Porous mineralized scaffolds are required for various applications in bone engineering. In particular, tube‐like pores with controlled orientation inside the scaffold may support homogeneous cell seeding as well as sufficient nutrient supply and may facilitate blood vessel ingrowth. Scaffolds with parallely orientated tube‐like pores were generated by diffusion‐controlled ionotropic gelation of alginate. Incorporation of hydroxyapatite (HA) during the gelation process yielded stable scaffolds with an average pore diameter of approximately 90 µm. To evaluate the potential use of alginate–gelatine–HA scaffolds for bone tissue engineering, in vitro tests with human bone marrow stromal cells (hBMSCs) were carried out. We analysed biocompatibility and cell penetration into the capillary pores by microscopic methods. hBMSCs were also cultivated on alginate–gelatine–HA scaffolds for 3 weeks in the presence and absence of osteogenic supplements. We studied proliferation and osteogenic differentiation in terms of total lactate dehydrogenase (LDH) activity, DNA content and alkaline phosphatase (ALP) activity and found a 10–14‐fold increase of cell number after 2 weeks of cultivation, as well as an increase of specific ALP activity for osteogenic‐induced hBMSCs. Furthermore, the expression of bone‐related genes [ALP, bone sialoprotein II (BSPII)] was analysed. We found an increase of ALP as well as BSPII expression for osteogenic‐induced hBMSCs on alginate–gelatin–HA scaffolds. Copyright © 2008 John Wiley & Sons, Ltd.     

丝素-壳聚糖复合制作的三维可降解多孔支架

背景:单独使用天然材料如胶原、明胶及纤维蛋白制备的支架虽然解决了生物相容性等问题,但由于其降解太快,在作为细胞支架时往往提前塌陷而达不到诱生新组织的目的.目的:探讨应用丝素与壳聚糖复合制作生物可降解三维多孔的支架,并对其特性进行检测.设计、时间及地点:材料学观察实验,于2008-06/2009-06在浙江省医学科学院生物工程研究所完成.材料:春蚕茧由浙江省海宁市马桥镇黄墩庙村蚕农赠送,壳聚糖为上海伯奧生物科技有限公司产品.方法:通过脱胶、溶解、透析这3个主要步骤制成质量浓度为15 g/L的丝素溶液,将壳聚糖溶解在2%的醋酸溶液中制成25g/L.的壳聚糖-乙酸溶液,然后将两者混合,配制成丝素/壳聚糖质量比分别为10:0,5:5,4:6,3:7,2:8,0:10的6种溶液,分别吸入24孔板中,4℃排出气泡后,-20℃预冷冻12 h,再冷冻干燥30 h.取出后梯度乙醇水化,再用NaOH-乙醇溶液中和稳定1h,漂洗后再次冻干.主要观察指标:光镜和扫描电镜观察各种质量比配制的支架孔径、结构;采用改良的液体替代法测定各种支架的孔隙率;测定各种支架在体外4周的降解率.结果:丝素/壳聚糖质量比为10:0制成的支架孔隙粗大,非常蓬松,易碎,溶失率太高;相反仅以壳聚糖制成的支架冻干后较硬,缺乏足够的弹性;以5:5,4:6,3:7,2:8制成的复合支架冻干后,支架较松软,类似海绵,随着壳聚糖浓度增加,支架硬度增加,支架上有分布均匀且细密的小孔.光镜下各个孔形态不规则,每个孔紧密相连并联通,孔径大小均匀,孔径20~100 μm,随着壳聚糖浓度增加,孔径逐渐减小.支架孔隙率测定结果显示,丝素/壳聚糖质量比为4:6组>5:5组>3:7组>2:8组,与丝素/壳聚糖质量比为2:8组比较,5:5组和4:6组的支架孔隙率均明显增大(P<0.05).各种质量配比制成的丝素-壳聚糖复合支架吸水膨胀率无明显差异(P>0.05).第4周时丝素/壳聚糖质量比为2:8组降解最慢,5:5组降解最快.结论:在理化性能方面,将丝素与壳聚糖混合制作的复合支架较单纯用丝素或壳聚糖制作的支架均显示出明显优势,其中用丝素/壳聚糖质量比为5:5及4:6制成的复合支架最符合软骨组织工程的需要.     

Influence of pore size on the redifferentiation potential of human articular chondrocytes in poly(urethane urea) scaffolds

The chemical and physical properties of scaffolds affect cellular behaviour, which ultimately determines the performance and outcome of tissue-engineered cartilage constructs. The objective of this study was to assess whether a degradable porous poly(urethane urea) scaffold could be a suitable material for cartilage tissue engineering. We also investigated whether the post-expansion redifferentiation and cartilage tissue formation of in vitro expanded adult human chondrocytes could be regulated by controlled modifications of the scaffold architecture. Scaffolds with different pore sizes, < 150 μm, 150-300 μm and 300-500 μm, were seeded with chondrocytes and subjected to chondrogenic and osteogenic induction in vitro. The poly(urethane urea) scaffold with the smaller pore size enhanced the hyaline-like extracellular matrix and thus neocartilage formation. Conversely, the chondrocytes differentiated to a greater extent into the osteogenic pathway in the scaffold with the larger pore size. In conclusion, our results demonstrate that poly(urethane urea) may be useful as a scaffold material in cartilage tissue engineering. Furthermore, the chondrogenic and the osteogenic differentiation capacity of in vitro expanded human articular chondrocytes can be influenced by the scaffold architecture. By tailoring the pore sizes, the performance of the tissue-engineered cartilage constructs might be influenced and thus also the clinical outcome in the long run.