壳聚糖-聚乳酸软骨与软骨下骨复合支架的制备与表征*

目的制备羟乙基壳聚糖-g-左旋聚乳酸（HECS-g-PLLA）和羟乙基壳聚糖-g-左旋聚乳酸＋Ⅱ型胶原蛋白（HECS-g-PLLA＋II型胶原蛋白）复合支架,并进行表征。方法采用热致相分离法制备HECS-g-PLLA、HECS-g-PLLA＋II型胶原蛋白聚合物,再用压片机,设定不同大气压,将聚合物压模成形具有不同性能的复合支架,测定复合支架的微观形貌、红外光谱、压缩模量、孔隙直径及生物相容性。结果复合支架具有纳米微米共存的高孔隙直径的亚微观结构。红外光谱显示壳聚糖成功羟乙基化,羟乙基壳聚糖与左旋聚乳酸聚合成功。随着压片机设定的压模成形的大气压力增大,样品的压缩模量增强,但空隙直径逐渐减少,可以根据需要制作各种不同性能的支架。结论热源实验和全身急性毒性实验提示复合支架浸提液注入动物体内不会引起发热反应和毒性反应,具有良好的生物相容性。     

三种水溶性壳聚糖的制备工艺研究

目的 壳聚糖具有优良的生物相容性和生物可降解性,但其亲水性较差难以满足组织工程的需要.为满足组织工程支架材料的需求,对壳聚糖进行羧甲基化改性,以期改善其溶解性能.方法 通过3种不同的方法来摸索3种不同取代位置的羧甲基壳聚糖(CMC)的制备工艺,分别考察了原料比、反应时间、反应温度等影响因素对产物取代度(DS)的影响.结果 摸索得到3种羧甲基壳聚糖的最佳制备工艺条件,以这种工艺条件制备产物水溶性良好.结论 通过羧甲基化反应可以大大改善壳聚糖的水溶解性能,为以后更深一步的研究提供了科研基础和实验依据.     

三种水溶性壳聚糖的制备工艺研究

目的 壳聚糖具有优良的生物相容性和生物可降解性,但其亲水性较差难以满足组织工程的需要.为满足组织工程支架材料的需求,对壳聚糖进行羧甲基化改性,以期改善其溶解性能.方法 通过3种不同的方法来摸索3种不同取代位置的羧甲基壳聚糖(CMC)的制备工艺,分别考察了原料比、反应时间、反应温度等影响因素对产物取代度(DS)的影响.结...     

聚乳酸/壳聚糖纳米纤维/纳米羟基磷灰石支架与兔软骨细胞的相容性

背景：传统的支架材料存在疏水性强,材料表面缺乏细胞表面受体特异结合的生物活性分子,材料的酸性降解产物易引发无菌性炎性反应等不足。根据仿生原理及软骨真实结构和构成来选择和制备组织工程软骨支架能够获得理想效果。 目的：制备聚乳酸/壳聚糖纳米纤维/纳米羟基磷灰石支架,评价其与兔膝关节软骨细胞的生物相容性,探讨其应用于关节软骨组织工程的可行性。 方法：采用二次相分离技术制备聚乳酸/壳聚糖纳米纤维/纳米羟基磷灰石复合支架,将第3代新西兰兔软骨细胞接种至复合支架材料上复合培养,倒置相差显微镜下观察细胞生长情况。细胞-支架复合物在24孔板中培养5 d以后,将其植入裸鼠皮下8周。 结果与结论：聚乳酸/壳聚糖纳米纤维/纳米羟基磷灰石支架材料经化学合成后,具有合适的三维多孔结构,孔隙率为90%,孔径300~450 μm;植入裸鼠皮下8周后Ⅱ型胶原免疫组织化学染色和甲苯胺蓝染色显示细胞-支架复合物中的软骨细胞可以像天然软骨一样分泌黏多糖和Ⅱ型胶原。提示生物材料聚乳酸/壳聚糖纳米纤维/纳米羟基磷灰石对于兔软骨细胞有良好的生物相容性,可作为生物组织工程支架。     

碳纤维-聚乳酸-聚乙二醇复合支架的制备及性能检测

背景：长期实验发现聚乳酸-聚乙二醇支架的力学性能及细胞相容性能较差,因此多数研究向支架中加入其他材料,以提高其生物活性及力学性能。 目的：制备改性碳纤维-聚乳酸-聚乙二醇支架,并检测其性能。 方法：采用溶液潘注/粒子沥滤法制备改性碳纤维-聚乳酸-聚乙二醇复合支架。对比改性碳纤维-聚乳酸-聚乙二醇复合支架与聚乳酸-聚乙二醇支架的超微结构、孔隙率、吸水性、降解率及力学性能。将改性碳纤维-聚乳酸-聚乙二醇复合支架与聚乳酸-聚乙二醇支架分别与SD大鼠成骨细胞共培养,12 h后采用沉淀法检测细胞黏附率;培养1,3,5,7,9 d后,采用 MTT 法检测细胞增殖。 结果与结论：聚乳酸-聚乙二醇支架材料表面孔结构分布均匀,孔径为(404.0±10.5) µm;改性碳纤维-聚乳酸-聚乙二醇支架碳纤维表面见大量纵向沟槽,表面孔结构分布均匀,孔径为(433.0±3.0) µm,两组支架孔径比较差异有显著性意义(P < 0.05)。改性碳纤维-聚乳酸-聚乙二醇支架的孔隙率、吸水性、弹性模量和抗压强度、降解率、细胞黏附率与增殖率均高于聚乳酸-聚乙二醇支架(P < 0.05)。表明改性碳纤维的加入改善了聚乳酸-聚乙二醇复合支架的力学性能及细胞相容性。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     

丝素蛋白/壳聚糖三维多孔支架的构建及结构表征**

背景：丝素蛋白和壳聚糖均无毒性且具有良好的生物相容性,但是单一成分作为生物支架时都不能满足支架材料的需求。 目的：制备各种不同组分的丝素蛋白及壳聚糖复合支架材料,观察其微观结构及相关性能,筛选出适合成骨细胞生长的理想支架材料。 方法：通过CaCl2∶C2H5OH∶H2O=1∶2∶8(摩尔比)溶解体系溶解、过滤、浓缩提纯,制备出2%的丝素蛋白溶液,壳聚糖溶解于乙酸溶液配制成的3%壳聚糖溶液,将两者以不同的比例相混合,经数次冷冻干燥后,得到成品支架材料。采用电镜观察形貌,计算孔隙率并对支架的结构进行红外、X射线衍射、电子能谱分析观察。 结果与结论：将壳聚糖和丝素蛋白共混后,互为改性,制备出了结构较稳定的支架材料。其中40%丝素蛋白-60%壳聚糖组具有适合成骨细胞生长的较佳孔径,可作为细胞支架的首选配比。 关键词：丝素蛋白;壳聚糖;骨组织工程;支架;生物材料 doi:10.3969/j.issn.1673-8225.2012.12.025     

聚乳酸/壳聚糖复合支架材料的生物相容性研究

为改善聚乳酸作为骨组织工程支架材料降解速率过快、亲水性差和降解产物呈酸性等缺点，本研究制备了一系列高孔隙率的聚乳酸／壳聚糖三维多孔复合支架材料，通过软骨细胞培养、动物皮下和肌肉植入试验对其进行了生物相容性研究。软骨细胞培养试验表明软骨细胞能在复合支架材料贴附增殖，材料无明显毒性；植入试验结果显示纯聚乳酸在体内2个月左右已经降解吸收，失去力学强度，复合材料三个月后仍能保持一定的力学强度和形状，而且组织切片也同时表明复合材料的炎症反应远远低于纯聚乳酸材料。     

聚乳酸-羟基乙酸输尿管支架植入后犬输尿管周围的组织学变化

背景：泌尿系统组织工程支架不仅需要生物相容性良好的生物材料,而且一定要利于组织周围细胞的生长。 目的：制备聚乳酸-羟基乙酸共聚物可降解输尿管支架,观察其植入后犬输尿管周围组织学变化。 方法：制备纳米聚乳酸-羟基乙酸共聚物输尿管支架,并以多聚赖氨酸对支架进行交联、改性,将交联后支架截成长约0.8 cm小段,植入犬损伤输尿管中进行体内观察实验。 结果与结论：①支架制备：支架具有纳米结构,孔隙率约90%,孔径(30±18) µm,多聚赖氨酸交联改性后纤维表面略显粗糙。②支架变化：支架植入30 d时已完全失去原始形态,与周边组织融合,可见裂解小块。③支架植入后输尿管周围组织学变化：植入后15 d炎症表现最为明显,主要是移行上皮脱落,肌层结构被破坏,固有层水肿明显;30 d后,炎症已经明显好转,但组织结构依然不规则;植入后45 d,输尿管全层组织基本恢复正常,组织结构成规则分布。说明聚乳酸-羟基乙酸共聚物输尿管支架具有良好的组织相容性,符合泌尿系统组织工程支架的要求。      

纳米可降解聚乳酸-羟基乙酸尿道支架的制备及性能

背景：聚乳酸-羟基乙酸可作为尿道替代物进行组织缺损的修复。 目的：观察电纺丝法制备聚乳酸-羟基乙酸共聚物可降解尿道支架的可行性,并评价支架管的体外降解性能。 方法：采用电纺丝技术制备纳米聚乳酸-羟基乙酸共聚物(摩尔比80∶20)尿道支架管,并以戊二醛对支架进行交联、改性,将交联后支架截成长约1 cm小段并浸于尿液中进行体外降解实验。 结果与结论：支架管具有纳米结构,孔隙率约89%,孔径(32±19) µm;交联后可见纤维表面变粗糙,但纤维丝直径、孔径及孔隙率与交联前差异无显著性意义(P > 0.05),但交联后支架管力学性能显著提高。支架降解初期速度相对较快,中后期降解速度减慢,至8周时材料质量损失约50%,第10周完全崩解。材料在体内降解过程中相对分子质量的变化趋势与质量损失大体相同,降解早期相对分子质量下降相对较快,后期下降速度减慢并趋于平稳。表明采用电纺丝技术制备的纳米聚乳酸-羟基乙酸共聚物尿道支架可满足尿道组织工程支架的要求。     

壳聚糖及其衍生物在软骨组织工程中的应用

背景：作为生物型支架,壳聚糖因其独特的多孔三维结构、易于改性的特征及良好的生物相容性成为了软骨组织工程支架材料的研究热点。 目的：就壳聚糖及其衍生物的设计、改性及在软骨组织工程中的应用作一综述。 方法：应用计算机检索PubMed数据库和CNKI数据库,中文关键词为“壳聚糖,壳聚糖衍生物,支架材料,组织工程,软骨组织”,英文检索词为“chitosan;chitosan derivatives;scaffold;tissue engineering;cartilage”,检索文献时间范围为1990年1月至2015年1月。 结果与结论：壳聚糖是一种天然的生物多糖,通过化学改性、共混改性等方法可以改变壳聚糖的溶解度、机械强度、生物活性甚至生物降解性等自身特性,从而制成更为合适的生物支架材料。进一步研究表明,将壳聚糖与种子细胞进行共同体外培养可以获得正常形态的软骨细胞并能合成特异性的细胞外基质成分,在动物体内,壳聚糖支架与种子细胞所构建的组织工程软骨能够修复软骨损伤,形成与周围正常软骨相似的组织。壳聚糖及其衍生物支架材料在软骨组织工程中有较为广阔的研究前景。  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     

Peptide surface modification of methacrylamide chitosan for neural tissue engineering applications

Nerve fibres are guided to their targets by the combined actions of chemotactic and haptotactic stimuli; however, translating these stimuli to a scaffold that will promote nerve regeneration is nontrivial. In pursuit of this goal, we synthesized and characterized cell-adhesive, biodegradable chitosan scaffolds. Chitosan amine groups were reacted with methacrylic anhydride resulting in a water soluble methacrylamide chitosan (MC) that was then crosslinked by radical polymerization resulting in a scaffold. Biodegradability by lysozyme and penetrability of the scaffold by rat superior cervical ganglion (SCG) neurons were studied. Maleimide-terminated cell adhesive peptides, mi-GDPGYIGSR and mi-GQASSIKVAV, were coupled to a thiolated form of MC to promote cell adhesion. The MC scaffold was found to be porous, biodegradable, and to allow neurite penetration. Interestingly, all of these properties were found to depend upon the amount of initiator used in crosslinking. Covalent modification of the MC scaffold with cell adhesive peptides significantly improved neuronal adhesion and neurite outgrowth. The MC can be crosslinked to form a novel scaffold, where our results demonstrate its suitability in neural tissue engineering and its potential for other engineered tissues, such as cartilage repair, where chitosan has already demonstrated some utility.     

组织工程神经支架材料的临床应用

背景：神经损伤后没有自我修复的能力,因此,神经组织工程支架材料应用于神经修复、促进神经再生成为研究的热点。 目的：分析目前常用的神经组织工程支架材料的应用范围及效果。 方法：分别对胶原、壳聚糖、凝胶以及透明质酸与人工合成材料形成的聚合物用于神经损伤修复进行动物模型分析,应用免疫染色、生化检测等方法观察评估再生神经的结构和生理功能,确定不同神经组织工程支架材料的应用效果。 结果与结论：神经组织工程支架材料胶原、壳聚糖、凝胶、透明质酸以及人工合成材料均可以通过不同的方式用于损伤神经的再生修复,既可以联合细胞进行生物聚合,也可以联合神经片段移植形成损伤神经桥状联接导管,应用于动物模型实验均显示较好的治疗效果。     

Vascular cell viability on polyvinyl alcohol hydrogels modified with water-soluble and -insoluble chitosan

Polyvinyl alcohol (PVA) hydrogels blended with chitosan or other biological macromolecules have shown promise for cell culture and tissue engineering. This study investigates the attachment and growth of bovine aortic endothelial (BAEC) and smooth muscle cells (BASMC) on the PVA hydrogels modified with water soluble and water insoluble chitosan. Cell adhesion on the surface of the membranes was examined by phase contrast microscopy while cell morphologies were studied using immunocytochemistry staining with EC and SMC specific biomarkers (F-actin and alpha actin respectively). Cells cultured on 6% PVA, 0.4% chitosan (water soluble and insoluble) hydrogel membranes displayed excellent adhesion and spreading characteristics, in addition to negligible cell structural morphological changes in comparison to a polystyrene control. Similar vascular cell adhesion features were apparent on PVA membranes blended with water-soluble and -insoluble chitosan. Fluorescent activated cell sorter (FACS) analysis was used to determine BAEC and BASMC proliferation and cell viability. Apoptotic levels in BAEC after 7 days were 12.8% +/- 2.5% on the PVA- chitosan WS-1 membrane and 10.1% +/- 1.5% on the control well (n = 3) while comparable results were also noted for BASMC. Equivalent proliferative activity was apparent for BAEC on the control and PVA-chitosan membrane after 7 days, while BASMC showed increased proliferative activity on the membranes. These results indicate that the PVA-chitosan blended hydrogel membranes show promise for cell culture and tissue engineering applications.     

Injectable chitosan-based hydrogels for cartilage tissue engineering

Water-soluble chitosan derivatives, chitosan-graft-glycolic acid (GA) and phloretic acid (PA) (CH-GA/PA), were designed to obtain biodegradable injectable chitosan hydrogels through enzymatic crosslinking with horseradish peroxidase (HRP) and H₂O₂. CH-GA/PA polymers were synthesized by first conjugating glycolic acid (GA) to native chitosan to render the polymer soluble at pH 7.4, and subsequent modification with phloretic acid (PA). The CH-GA43/PA10 with a degree of substitution (DS, defined as the number of substituted NH₂ groups per 100 glucopyranose rings of chitosan) of GA of 43 and DS of PA of 10 showed a good solubility at pH values up to 10. Short gelation times (e.g. 10 s at a polymer concentration of 3 wt%), as recorded by the vial tilting method, were observed for the CH-GA43/PA10 hydrogels using HRP and H₂O₂. It was shown that these hydrogels can be readily degraded by lysozyme. In vitro culturing of chondrocytes in CH-GA43/PA10 hydrogels revealed that after 2 weeks the cells were viable and retained their round shape. These features indicate that CH-GA/PA hydrogels are promising as an artificial extracellular matrix for cartilage tissue engineering.     

Osteoblast-like cell attachment to and calcification of novel phosphonate-containing polymeric substrates

In an attempt to interact natural bone and bone cells with biomaterials and to begin to develop modular tissue engineering scaffolds, substrates containing phosphonate groups were identified to mimic mineral-protein and natural polymer-protein interactions. In this study, we investigated poly(vinyl phosphonic acid) copolymer integration with existing materials as a graft-copolymer surface modification. Phosphonate-containing copolymer-modified surfaces were created and shown to have varying phosphate content within different polymeric surfaces. As the phosphonate content in the monomer feed approached 30% vinyl phosphonic acid, increased osteoblast-like cell adhesion (3- to 8-fold increase in adhesion) and proliferation (2- to 10-fold increase in proliferation rate) was observed. Since surfaces modified with 30% vinyl phosphonic acid in the feed exhibited a maximal cell adhesion and proliferation (9.4 x 10(4) cells/cm(2)/day), it was hypothesized that this copolymer composition was optimal for protein-polymer interactions. Osteoblast-like cells formed confluent layers and were able to differentiate on all surfaces that contained vinyl phosphonic acid. Most importantly, cells interacting with these surfaces were able to significantly mineralize the surface. These results suggest that phosphonate-containing polymers can be used to integrate biomaterials with natural bone and could be used for tissue engineering applications.     

Attachment and growth of fibroblasts on poly(L-lactide/epsilon-caprolactone) scaffolds prepared in supercritical CO2 and modified by polyethylene imine grafting with ethylene diamine-plasma in a glow-discharge apparatus

In this study, a copolymer of L-lactide and epsilon-caprolactone (Mn: 73,523, Mw: 127,990 and PI: 1.74) was synthesized by ring-opening polymerization by using stannous octoate as the catalyst. FTIR, 1H-NMR and DSC confirmed the copolymer formation. The copolymer films were prepared and a novel method was developed to produce highly porous sponges for potential use in tissue engineering. Films were subjected to supercritical CO2 at 3300 psi and 70 degrees C to create porous structures for production of possible tissue engineering scaffolds. The pore sizes were in the range of 40-80 microm. The copolymer films were pre-wetted with polyethylene imine (PEI) and then treated with ethylene diamine (EDA)-plasma in glow-discharge apparatus. Gas plasma surface modification of three-dimensional scaffolds fabricated by supercritical carbon dioxide technique was demonstrated to enhance cell adhesion, proliferation, and differentiation over 6 days in culture using L929 fibroblast cell line. Alkaline phosphatase (ALP) activity and glucose uptake in cell culture medium were followed in the cell culture experiments. Fibroblastic cell attachment and growth on the EDA-plasma treated scaffolds were rather low. However, both cell attachment and growth were significantly increased by PEI pre-treatment before EDA-plasma. The changes in ALP activity and glucose uptake also supported the cell growth behavior on these PEI and EDA-plasma treated scaffolds.     

Surface Phosphorylation for Polyelectrolyte Complex of Chitosan and Its Sulfonated Derivative: Surface Analysis,Blood Compatibility and Adipose Derived Stem Cell Contact Properties

Many studies have tried to look for the application of chitosan in tissue engineering since its structure is similar to glycoaminoglycans, the main components of the extracellular matrix. Previous studies had indicated that the incorporation of sulfonic or phosphonic functionalities would be beneficial to the growth of certain cells. However, no study has explored the effect of incorporation of both above-mentioned anionic functionalities onto the chitosan structure. In this study, we have surface-phosphorylated the polyelectrolyte film formed by chitosan and water-soluble sulfonated chitosan with the aim to incorporate phosphonic and sulfonic functionalities onto the film surface. Surface analyses by ESCA and ATR–FT-IR have shown that these two functional groups have been successfully grafted onto the surface, and that the ratio of P/S was dependent upon the weight ratio of phosphorylation agents added. Blood compatibility evaluation indicated that phosphorylated polyelectrolyte complexes extended the plasma recalcification time as compared to non-treated chitosan and direct-phosphorylated chitosan film. In addition, these phosphorylated polyelectrolyte complexes showed similar or slightly less platelet reactivity than the non-phosphorylated counterpart. In contrast, significant platelet activation and adhesion were noted on the direct-phosphorylated chitosan. This implicated the incorporation of sulfonic acid onto the phosphorylated surface can increase the platelet compatibility. An adipose-derived stem cell incubation study has demonstrated that the incorporation of both phosphonic and sulfonic acid functionalities onto the chitosan surface can enhance the stem cell growth. Therefore, the phosphorylated polyelectrolyte complexes were not only blood compatible but also stem cell compatible, and could be a novel biomaterial in tissue-engineering applications.