壳聚糖及其改性材料对骨髓基质细胞的作用

壳聚糖是一种广泛应用的生物可降解材料,该论文研究了几种与壳聚糖相关的材料对骨髓基质细胞生长和分化的作用,主要实验方法是在材料表面培养骨髓基质细胞并对其进行诱导促使其向成骨细胞方向分化。通过对细胞生长和分化情况的观察和测定,对几种材料与骨髓基质细胞的亲和性作出了评价。另外,通过ELISA法测定了细胞外基质分子在材料上的吸附量,测量了各材料的表面接触角以研究细胞在材料表面的铺展和增殖。结果表明尽管壳聚糖本身与骨髓基质细胞并不具有很好的亲和性,但通过与明胶混合,壳聚糖的生物相容性得到了明显提高,是很有应用前景的骨修复材料。     

几种神经支架原材料的生物力学性质评价

组织工程支架都需要有一定的力学性能以满足实际使用时的要求。本文考察了9种候选神经支架原材料的力学性能,为神经支架的设计制作的选材提供力学依据。本研究以PLGA、胶原、海藻酸钠、脱细胞猪皮、脱细胞血管、脱细胞猪皮-PLGA的复合体、脱细胞血管-PLGA的复合体以及经改性的壳聚糖和明胶作为考察材料,用拉伸实验机对其进行拉伸试验。本试验获得了这9种材料的最大应变、最大应力厦杨氏模量。其结果可以为神经支架的选材、设计和制作提供力学参考依据。     

动脉损伤后早期中膜原位明胶酶活性变化

动脉平滑肌细胞迁移到内膜和内膜细胞增生是经皮腔内血管成型术后再狭窄的关键。迁移时平滑肌细胞必须将包绕其周围的基底膜消化溶解。基底膜由ＩＶ型胶原蛋白和层粘连蛋白构成 ,明胶酶能够消化这两种蛋白。血管损伤后明胶酶原位活性变化并不清楚。本研究目的是探明损伤后动脉壁明胶酶活性的变化规律 ,为在早期抑制血管再狭窄提供基础。1　材料和方法(1)动脉壁损伤　Ｗｉｓｔａｒ系雄性大鼠 (12周 ,2 2 0～ 2 5 0ｇ)苯巴比妥钠 (0 .8ｍｌ ｋｇ)腹腔麻醉下 ,显露右颈内、外动脉 ,将 2Ｆｒ球囊导管从颈外动脉向心方向插入右颈总动脉近端 ,将球…     

通过研究改性壳聚糖与细胞的相互作用评价其生物相容性

利用细胞生物学的方法, 研究了四种不同的细胞在经过改性的壳聚糖（ＣＨＩＴＯＳＡＮ） 膜上的生长,测定了细胞相对黏附力、细胞初始黏附率, 并利用ＦＤＡ 实验测定了细胞活力,从而从多个方面评价了这几种不同材料的生物相容性。实验结果表明,与明胶交联的壳聚糖膜明显比其它两种膜有利于细胞的黏附和生长,为进一步对材料进行筛选奠定了基础。     

快速膨胀片层多孔壳聚糖止血海绵的制备及生物相容性研究

目的:探索快速膨胀片层多孔壳聚糖止血海绵的制备工艺,评价止血海绵的理化性能及生物相容性,并探讨原料脱乙酰度对止血海绵性能的影响。方法:考察止血海绵的理化性质,包括扫描电子显微镜(SEM)观察表观形貌,检测力学性能、吸水率、快速吸水膨胀时间和膨胀率,研究其体内外的生物相容性,包括体外细胞毒性实验、动物皮内刺激实验和皮下植入实验。结果:确定了止血海绵的制备工艺,采用该工艺制备的止血海绵均具有片层多孔结构,且具有较高的力学强度和快速膨胀的特点。证实高脱乙酰度原料(DD=95.14%)制备的止血海绵力学性能、吸水率、膨胀率均优于低脱乙酰度原料(DD=69.70%)制备的止血海绵。脱乙酰度69.70%和脱乙酰度95.14%的壳聚糖止血海绵,拉伸强度分别为10.1 N和15.4 N,吸水率分别为1904%和2131%,吸水膨胀时间分别为13.4 s和14.0 s,膨胀率分别为8.4倍和10.8倍。体外细胞毒性实验表明脱乙酰度为95.14%的壳聚糖止血海绵更有利于细胞的增殖,皮内刺激和皮下植入实验结果表明脱乙酰度为95.14%的壳聚糖海止血海绵表现出更小的组织炎性反应。结论:脱乙酰度为95.14%的壳聚糖止血海绵具有优良的力学性能、优异的吸水膨胀能力以及良好的生物相容性,在临床止血特别是腔隙止血方面具有广阔的应用前景。     

壳聚糖基角膜细胞载体的制备及其细胞相容性

为探讨羟丙基壳聚糖基共混膜作为组织工程技术中角膜细胞培养载体的可行性, 分别制备了羟丙基壳聚糖/硫酸软骨素、羟丙基壳聚糖/明胶/硫酸软骨素以及羟丙基壳聚糖/氧化透明质酸/硫酸软骨素三种共混膜。测定其透光率、含水量和蛋白吸附性能; 在共混膜上培养兔角膜上皮细胞, 通过观察角膜上皮细胞在不同载体膜上的生长状态、贴附情况, 测定细胞活性以及上清液中乳酸脱氢酶的活性, 研究三种壳聚糖基载体膜片与角膜上皮细胞的相容性。膜片理化性质测定结果表明三种共混膜片具有良好的透明度, 适宜的含水量和较强的蛋白吸附性能; 细胞相容性实验结果表明羟丙基壳聚糖/明胶/硫酸软骨素共混膜对细胞的损伤最小, 有利于细胞在膜上的贴附和生长, 表现出良好的细胞相容性, 有望作为角膜细胞载体体外构建组织工程化角膜。     

不同脱乙酰度和分子量壳聚糖的神经亲和性研究

研究了四种不同脱乙酰度和分子量的壳聚糖与神经细胞的亲和性。利用酶联免疫技术（ELISA）测定了人血清白蛋白和纤粘蛋白在四种壳聚糖膜上的吸附量,并利用圆二色柱（CD）测定了人血清白蛋白吸附在四种壳矣糖膜上的二极结构。通过研究它们对胎鼠大脑皮层细胞（FMCC）生长的影响,发现在这四种壳聚糖中,分子量较大的两种壳聚糖更有利于神经细胞的生长,许多轴突聚集成树干状。     

多聚赖氨酸改性壳聚糖对神经细胞的作用

壳聚糖是一种具有优良的生物相容性的生物可降解材料。研究几种与壳聚糖相关的材料对神经细胞生长的促进作用。实验方法以在材料上培养神经细胞为主。选用胎鼠大脑皮层神经元和神经胶质瘤细胞9L。另外 ,本实验还使用ELISA法测量细胞外基质粘附分子在材料上的吸附量 ,并测量各材料的接触角以研究细胞在材料上的吸附和铺展。结果发现壳聚糖对神经细胞的生长有良好的促进作用 ,而壳聚糖表面涂敷多聚赖氨酸和壳聚糖与多聚赖氨酸混合材料是比壳聚糖更好的促神经细胞生长的生物材料 ,都是很有应用前景的神经修复材料。     

磷铵两性离子改性血管脱细胞支架可以有效体外抗血栓和内皮化

由于自体血管(由同一受体的血管用于血管移植材料)的有限可用性,以及非自体血管(人工制成的血管移植材料)的生长能力不足,组织工程血管越来越受到重视。文中构建了一种磷铵两性离子改性的血管脱细胞支架附以高度生物相容的骨髓源内皮祖细胞为内层的新型血管移植材料。通过一种简便的方法——共沉淀法改性血管脱细胞支架,评价其体外血小板粘附实验、溶血实验、复钙实验和细胞毒性等相关指标。磷铵两性离子改性后抗凝血活性提高,可以有效地促使类似于天然血管腔表面凹凸结构的脱细胞支架表面内皮祖细胞的附着。改性后的脱细胞支架具有与天然血管相似的力学性能,在体外可以有效地构建内皮化。研究结果为血管脱细胞支架通过改性实现体外抗血栓和内皮化方面进行了初步探索。     

小鼠主动脉血管平滑肌原代细胞的分离培养及鉴定

目的:血管平滑肌细胞在人类心血管疾病中具有重要的作用,而作为重要的遗传学研究模式生物的小鼠血管平滑肌材料有限,因此建立一种简单高效的小鼠血管平滑肌原代细胞分离培养方法很重要。方法:分离小鼠主动脉中膜层,胶原酶消化法获得原代平滑肌细胞,免疫荧光方法检测细胞的纯度和分化状态;分离平滑肌细胞特异的报告小鼠的平滑肌细胞,LacZ染色鉴定。结果:用该方法分离的原代平滑肌细胞生长迅速,3d后即可达5×106个。免疫荧光显示,细胞传至第3代后纯度在98%以上,细胞传至8代分化状态没有改变。LacZ染色鉴定报告小鼠分离的3代平滑肌细胞98%以上显示特异的蓝染。两种实验证明,应用此方法分离原代平滑肌细胞可以满足平滑肌体外功能实验的需求。结论:与传统的组织块培养法相比,该方法操作简便、经济,可以获得更多高纯度的血管平滑肌细胞。     

Investigation of MC3T3-E1 cell behavior on the surface of GRGDS-coupled chitosan

The GRGDS (Gly-Arg-Gly-Asp-Ser) peptide has intermediate affinity to alphaVbeta3 and alphaIIbbeta3, which are the integrins most reported to be involved in bone function. In this study, biomimetic chitosan films modified with GRGDS peptide were prepared and were used as a substrate for the in vitro culture of MC3T3-E1 cells in order to investigate the effect of GRGDS modification on MC3T3-E1 cell behavior. The results of electron spectroscopy for chemical analysis (ESCA), attenuated total reflection-Fourier transform infrared spectra (ATR-FTIR), and amino acid analysis (AAA) demonstrated that the chitosan films were successfully modified with GRGDS peptides and that the surface density of the immobilized GRGDS was on the order of 10(-9) mol/cm2. The immobilization of the GRGDS sequence on chitosan as well as the peptide concentration play a significant role in MC3T3-E1 cell behavior. MC3T3-E1 cell attachment, proliferation, migration, differentiation, and mineralization were remarkably greater on GRGDS-coupled chitosan than on unmodified chitosan. Besides, the degree of acceleration of these biological processes was found to be dependent on peptide density. Competitive inhibition of MC3T3-E1 cell attachment using soluble GRGDS peptides indicated that the interaction of MC3T3-E1 cells with the surface of the materials was ligand-specific. Cytoskeleton organization in the fully spread MC3T3-E1 cells was highly obvious on GRGDS-coupled chitosan when compared to the lack of actin fibers noted in the round MC3T3-E1 cells on unmodified chitosan. These results suggest that MC3T3-E1 cell function can be modulated, in a peptide density-dependent manner, by the immobilization of GRGDS peptide on chitosan used for scaffold-based bone tissue engineering.     

A chemical surface modification of chitosan by glycoconjugates to enhance the cell-biomaterial interaction

The use of wheat germ agglutinin (WGA), a lectin molecule, to modify chitosan and enhance the cell-biomaterial interaction was examined. The percentage of living fibroblast cells on the surfaces of tissue culture polystyrene (TCPS) control, WGA-modified chitosan, and unmodified chitosan films increased to 99%, 99%, and 85%, respectively, after seeding for 48 h. DNA staining revealed that a portion of fibroblasts cultivated on chitosan films( )were undergoing apoptosis. In contrast, fibroblasts growing on WGA-modified chitosan film surfaces did not show any indication of apoptosis. The number of fibroblast cells was the highest on the WGA-modified chitosan surfaces, followed by the TCPS and unmodified chitosan surfaces. This WGA-mediated enhancement on the fibroblast cell-biomaterial interaction was cell type dependent. Other types of cells may need different lectin molecules for enhanced interaction with biomaterials. Further, the evaluation of the heat shock protein (HSP) mRNA expression indicated that HSP 90 expression was increased in the fibroblast cells cultivated on chitosan films and decreased to basal levels on the WGA-modified chitosan films. Taken together, our data suggest that the use of WGA and other lectin molecules to enhance the cell-biomaterial interaction via oligosaccharide-mediated cell adhesion is a promising way to improve cell adhesion and proliferation, the two key issues in tissue engineering.     

Cytotoxic activities of water-soluble chitosan derivatives with different degree of deacetylation

Chitosans with different degree of deacetylation (DD) (90% and 50% deacetylated chitosan) were prepared by N-deacetylation followed by grafted onto chitosan to form water-soluble aminoethyl-chitosan (AE-chitosan), and dimetylaminoethyl-chitosan (DMAE-chitosan), diethylaminoethyl-chitosan (DEAE-chitosan). In the present study, cytotoxic activities of the chitosan derivatives were evaluated using three tumor cell lines and two normal cell lines, and structure-activity relationship was suggested. The cytotoxic activity was dependent on their DD and substituted group.     

Solid-state and mechanical properties of aqueous chitosan-amylose starch films plasticized with polyols

The film-forming ability of chitosan and binary mixtures of chitosan and native amylose corn starch (Hylon VII) was evaluated with free films prepared by a casting/solvent evaporation method. Unplasticized and plasticized free chitosan films in aqueous acetic acid and respective films containing a mixture of chitosan and native amylose starch in acetic acid were prepared. Glycerol, sorbitol, and i-erythritol were used as plasticizers. Solid-state and mechanical properties of the films were studied by powder x-ray diffractometry (XPRD), differential scanning calorimetry (DSC), and a materials testing machine. The films composed of a mixture of chitosan and native amylose starch in acetic acid were clear and colorless. A plasticizer concentration of 20% wt/wt (of the polymer weight) ws sufficient to obtain flexible films with all samples tested. X-ray diffraction patterns and DSC thermograms indicated an amorphous state of the films independent of the type of plasticizer used. In conclusion, incorporation of native amylose com starch into chitosan films improves the consistency and the mechanical properties of the films.     

Role of integrin-linked kinase in vascular smooth muscle cells: regulation by statins and angiotensin II

Our goal was to characterize the role of integrin-linked kinase (ILK) in vascular smooth muscle cells (VSMC), which play a crucial role in atherogenesis. Transfection of VSMC with wild-type and dominant-negative ILK cDNA constructs revealed that ILK mediates migration and proliferation of VSMC but has no effect on VSMC survival. The pro-atherogenic mediator angiotensin II increases ILK protein expression and kinase activity while statin treatment down-regulates ILK in VSMC. Functionally, ILK is necessary for angiotensin II-mediated VSMC migration and proliferation. In VSMC transduced with dominant-negative ILK, statins mediate an additive inhibition of VSMC migration and proliferation, while transfection with wild-type ILK is sufficient to overcome the inhibitory effects of statin treatment on VSMC migration and proliferation. In vivo, ILK is expressed in VSMC of aortic sections from wild-type mice where it is down-regulated following statin treatment and up-regulated following induction of atherosclerosis in apoE-/- mice. These data identify ILK as a novel target in VSMC for anti-atherosclerotic therapy.     

Growth inhibitory effect on bacteria of chitosan membranes regulated with deacetylation degree

Antibacterial activity of chitosan membranes was investigated by a conductimetric assay using a Bactometer. The purpose of this investigation was to produce a practical, high-performance membrane for separation engineering. The antibacterial activity of powdered chitosan membrane was evaluated by the minimal inhibitory concentration (MIC). The MIC for Escherichia coli was almost 200 (mg-chitosan/ml-bacterial suspension), and for Staphylococcus aureus it was 40 (mg-chitosan/ml-bacterial suspension). Growth of the gram-positive sample (S. aureus) was more strongly inhibited by chitosan than the gram-negative sample (E. coli). This inhibitory effect was recognized as a bactericidal effect. Antibacterial activity was also observed and depended on the shape and the specific surface area of the powdered chitosan membrane. The influence of the deacetylation degree (DD) of the chitosan on inhibiting the growth of S. aureus was investigated by two methods: incubation using a mannitol salt agar medium, and a conductimetric assay. By both methods, chitosan with a higher DD successfully inhibited growth of S. aureus. Our findings regarding the dominant role of the DD of chitosan will be useful for designing long-life, hygienic, membrane-based processes.     

The enzymatic degradation and swelling properties of chitosan matrices with different degrees of N-acetylation

In the design of chitosan-based drug delivery systems and implantable scaffolds, the biodegradation rate of the chitosan matrix represents a promising strategy for drug delivery and the function of carriers. In this study, we have investigated the degradation of chitosan with different degrees of N-acetylation, with respect to weight loss, water absorption, swelling behavior, molecular weight loss of bulk materials, and reducing sugar content in the media. Chitosan matrices were prepared by compression molding. The results revealed that the initial degradation rate, equilibrium water absorption, and swelling degree increased with decreasing degree of deacetylation (DD) and a dramatic rise began as DD of the chitosan matrix decreased to 62.4%. Chitosan matrices with DD of 52.6%, 56.1%, and 62.4% had the weight half-life of 9.8, 27.3, and more than 56 days, respectively, and the weight half-life of average molecular weight 8.4, 8.8, and 20.0 days, respectively. For chitosan matrices with DD of 71.7%, 81.7%, and 93.5%, both types of half-life exceeded 84 days because of the much slower degradation rate. The dimension of chitosan matrices during degradation was determined by the process of swelling and degradation. These findings may help to design chitosan-based biomedical materials with predetermined degradation timed from several days to months and proper swelling behaviors.