蒸汽灭菌对聚醚酯材料及其血管细胞相容性的影响

探讨了蒸汽灭菌对聚乙二醇对苯二甲酸酯/聚对苯二甲酸丁二醇酯多嵌段共聚物(PEGT/PBT)性能的改变而导致对血管细胞相容性的影响.血管细胞能在紫外辐照灭菌的PEGT/PBT膜片上较好地黏附生长,而在蒸汽灭菌的膜片表面几乎无法黏附生长.使用差示扫描量热分析、静态接触角、光电子能谱、表面羧基测定、核磁共振和扫描电镜等分析测试方法对灭菌前后膜片的本体性能和表面性能进行表征.结果表明,蒸汽灭菌没有改变材料表面形貌和材料的组成.但是在蒸汽灭菌过程中PEGT/PBT链段发生重新取向,亲水性软段PEGT和材料的端羧基在表面富集,使得材料表面亲水性增加、硬段的结晶度有所增加.可能由于端羧基和表面聚乙二醇增多导致蛋白在材料表面的吸附减少,而致使血管细胞无法在蒸汽灭菌的膜片上黏附生长.     

聚己二酸丁二醇酯-对苯二甲酸丁二醇酯/Ⅰ型胶原取向纤维促进前交叉韧带断裂后腱-骨愈合

背景:材料表面的微/纳米结构对细胞的行为具有调控作用,肌腱组织主要由平行的胶原纤维组成,因此取向纤维结构对腱-骨愈合具有一定的促进作用.目的:探索聚己二酸丁二醇酯-对苯二甲酸丁二醇酯[poly(butylene adipate-co-terephthalate),PBAT]/Ⅰ型胶原取向纤维支架的生物相容性和成骨诱导活...     

新型骨材料填充器的研制及其生物相容性研究*

目的探讨采用聚对苯二甲酸乙二醇酯（PET）为基质的新型骨材料填充器的研制及其生物相容性。方法将聚对苯二甲酸乙二醇酯制成网孔状的填充料袋,结合输送器械,获得可膨胀性的骨材料扩张及填充系统。按照GB／T16886标准通过急性全身毒性试验、溶血试验和致敏试验系统评价聚对苯二甲酸乙二醇酯注射聚甲基丙烯酸甲脂（PMMA）复合材料（PET／PMMA）的生物相容性。结果PET／PMMA复合材料急性毒性试验实验组及阴性对照组小鼠活动、体重及呼吸均无明显差异;致敏豚鼠试验组无红斑及水肿;溶血率为1．24％,无溶血性。结论本课题组研制的新型骨材料填充器,经检验其具有良好的生物相容性。     

纤维连接蛋白表面修饰对促进新型PET材料人工韧带（LARS）细胞黏附的实验研究

目的通过体外实验探讨采用表面水解法处理纤维连接蛋白表面修饰增进新型PET材料人工韧带生物相容性的有效性。方法采用碱性水解法表面处理后再与纤维连接蛋白反应修饰该新型人工韧带,使用红外透射光谱分析和X线衍射分析技术测定材料表面蛋白结合含量,采用大鼠成纤维细胞与聚脂纤维人工韧带材料体外共培养的方法,观察细胞粘附、增殖以及分化情况,对比采用纤维连接蛋白表面修饰的材料和未处理的材料的生物相容性的差别。结果红外透射光谱分析和X线衍射分析技术证实通过纤维连接蛋白表面修饰后该新型聚脂纤维人工韧带表面蛋白含量明显提高,采用纤维连接蛋白预处理的聚脂材料上细胞能够早期粘附,附着生长情况更好,数量更多。结论采用碱性水解表面处理纤维连接蛋白修饰的方法简便、有效,能够增进新型PET材料人工韧带的生物相容性。     

硅烷化玻璃酸酯在皮肤抗皱剂中细胞水平的功能研究

硅烷化玻璃酸酯复合物(silanol hyaluronate compound,SHAC)是一种透明质酸衍生物。类人胶原和肌肽是当前市场上主流的抗皱成分。本研究将SHAC用于皮肤抗皱剂中,研究其对成纤维细胞的影响。SHAC分别与类人胶原和肌肽复配,作用于体外培养的L929细胞,检测正常细胞的增殖活性和UVB损伤细胞的修复能力。结果表明:(1)肌肽能促进L929细胞的增殖,具有抑制UVB照射细胞引起的氧化损伤的能力。类人胶原蛋白没有明显效果。(2)SHAC分别与类人胶原、肌肽制备复配制剂,均能够显著促进细胞的增殖,抑制UVB照射引起的细胞损伤。SHAC能增强类人胶原和肌肽的功效,在开发皮肤抗皱功能的产品领域前景良好。     

辛二酰苯胺氧肟酸对血管生成的抑制作用及其机制

目的研究辛二酰苯胺氧肟酸(SAHA)对人脐静脉内皮细胞(HUVEC)生物学特性的影响及对血管生成的作用。方法 CCK-8法检测SAHA对HUVEC增殖能力的影响。TranswellTM小室法、基质胶体外成管实验、Western blot法、基质胶体内血管生成实验分别观察15μmol/L SAHA处理HUVEC 48 h后HUVEC迁移能力的变化;HUVEC在基质胶上形成管腔样结构的能力;HUVEC细胞中血管内皮生长因子(VEGF)信号通路相关蛋白表达的变化以及小鼠体内基质胶栓中新生血管的数量。结果 SAHA能够抑制HUVEC的增殖,抑制作用呈剂量、时间依赖性;与对照组相比较,SAHA处理组迁移过膜的HUVEC细胞数明显降低;HUVEC形成的管腔样结构SAHA处理组明显少于对照组;SAHA抑制HUVEC中VEGF相关信号通路;SAHA处理后小鼠体内的基质胶栓中新生的血管数明显少于对照组。结论 SAHA能够抑制HUVEC增殖及体内外血管形成能力,并且其可能机制是SAHA阻断了VEGF相关的信号通路。     

含紫杉醇可降解乙交酯/丙交酯共聚物材料能抑制动脉平滑肌细胞生长

目的研究含药物紫杉醇可降解支架材料--乙交酯/丙交酯共聚物(PLLGA)对人脐动脉平滑肌细胞(SMC)作用,检测此药物降解材料是否是血管内支架的理想材料.方法选取人脐动脉原代平滑肌细胞植块培养,得到传代细胞.然后与含不同紫杉醇药物浓度的降解材料一起培养,显微镜下观察降解材料远近处细胞形态发展的状态,观察金属支架组、Ⅰ组不含紫杉醇只含PLLGA对照组,Ⅱ～Ⅳ组分别含1、2、3μg紫杉醇PLLGA可降解材料对细胞生长的影响,并进行计数统计.结果降解材料对照组及金属支架组对平滑肌细胞生长无影响,含1、2、3μg紫杉醇的降解材料组细胞生长状态不佳.统计分析可见,72 h,对照组、金属支架组与各用药组有显著统计学意义(P＜0.01),但24 h无统计学意义(P＞0.05).结论此紫杉醇降解材料(PLLGA)可作为抑制平滑肌细胞生长的血管内支架材料.     

两种软骨组织工程新型复合支架材料初步研究

目的 利用聚谷氨酸苄酯-co-聚谷氨酸(PBLG-co-PGA)改性2种常规的生物可降解材料(胶原与壳聚糖),比较其性能及生物相容性,探索其用于软骨组织工程的可行性.方法 用PBLG-co-PGA改性胶原和壳聚糖,对改性前后的材料进行接触角、体外降解性能、拉伸强度、细胞黏附率及细胞相容性等性能的检测和比较.结果 随PBLG-co-PGA含量的增加,PBLG-co-PGA/胶原、PBLG-co-PGA/壳聚糖复合材料的降解速度减慢,接触角逐渐减小,PBLG-co-PGA/胶原复合材料的拉伸强度增加.选用PBLG-co-PGA质量分数为30％的复合材料,以兔软骨细胞进行细胞黏附率和细胞相容性实验,与无PBLG-co-PGA的单纯胶原或单纯壳聚糖材料比较,细胞黏附率差异无统计学意义(P＞0.05);但兔软骨细胞在PBLG-co-PGA/壳聚糖复合材料上生长增殖优于其他3种材料(P＜0.05).结论 经PBLG-co-PGA改性后的壳聚糖和胶原材料,可以调节材料的降解速度,改善壳聚糖亲水性,增加胶原拉伸强度,并且有良好的细胞相容性.其中PBLG-co-PGA/壳聚糖复合材料具有更好的促细胞增殖特性,可以作为一种新的组织工程软骨支架材料.     

西罗莫司-聚三亚甲基碳酸酯改性镁合金的降解和药物释放行为

背景：冠状动脉镁合金支架腐蚀速率及药物释放的双重调控是目前镁合金支架需要解决的重要课题。可降解载药聚合物涂层作为一种有前途的改性策略引发了广泛关注。目的：研究可降解载药聚合物涂层修饰对镁合金降解的影响,以及镁合金降解对可降解载药聚合物涂层药物释放的影响。方法：制备不同的药物聚合物溶液,溶液S1：西罗莫司0.01 g、聚三亚甲基碳酸酯0.005 g、二氯甲烷1 mL;溶液S2：西罗莫司0.01 g、聚三亚甲基碳酸酯0.01 g、二氯甲烷1 mL;S3：西罗莫司0.01 g、聚三亚甲基碳酸酯0.02 g、二氯甲烷1 mL;S2-1：西罗莫司0.01 g、聚三亚甲基碳酸酯0.01 g、0.001 g聚乙二醇400、二氯甲烷1 mL;S2-2：西罗莫司0.01 g、聚三亚甲基碳酸酯0.01 g、0.002 g聚乙二醇400、二氯甲烷1 mL。将5种药物聚合物溶液涂覆在AZ31镁合金表面制备涂层,对比涂层在镁合金基底上的药物释放行为差异,以及各涂层对镁合金腐蚀能力的影响;探索基底对西罗莫司-聚三亚甲基碳酸酯涂层的影响;考察各修饰镁合金的体外细胞相容性与血液相容性。结果与结论：(1)西罗莫司-聚三...     

霉酚酸酯对血管内皮细胞与淋巴细胞粘附及其表达CD40L的影响

目的：观察霉酚酸酯（MMF）对细胞因子刺激下人脐静脉内皮细胞（mWEC）CD40L的影响。方法：正常分娩人脐带经胶原酶消化后，分离出内皮细胞，培养至3～5代，用于细胞粘附和CIMOL表达试验。以TNF-α和／或霉酚酸（船A）处理HUVEC 20小时后，用虎红法研究对淋巴细胞与内皮细胞粘附作用。以TNF-α、rIFN-γ、LPS分别和MPA同时作用HUVEC 24小时。加不同浓度的MPA与LPS诱导HUVEC 24小时，用Cell-ELISA检测CIMOL的表达。结果：（1）MMF能抑制静息及TNF-α激活的内皮细胞与淋巴细胞间的粘附作用。（2）三种细胞因子均可明显诱导内皮细胞表达CD40L分子。（3）MMF不能抑制静息状态下内皮细胞CD40L的表达，但抑制TNF-α、rIFN-γ和LPS诱导CIMOL的表达作用，且MMF抑制LPS诱导的内皮细胞CD40L表达呈剂量依赖效应。结论：MMF通过抑制内皮细胞表达CIMOL而影响淋巴细胞与内皮细胞的相互作用，这可能是MMF抗排斥反应机制之一。     

The regulation of expanded human nasal chondrocyte re-differentiation capacity by substrate composition and gas plasma surface modification

Optimizing re-differentiation of clinically relevant cell sources on biomaterial substrates in serum containing (S+) and serum-free (SF) media is a key consideration in scaffold-based articular cartilage repair strategies. We investigated whether the adhesion and post-expansion re-differentiation of human chondrocytes could be regulated by controlled changes in substrate surface chemistry and composition in S+ and SF media following gas plasma (GP) treatment. Expanded human nasal chondrocytes were plated on gas plasma treated (GP+) or untreated (GP-) poly(ethylene glycol)-terephthalate-poly(butylene terephthalate) (PEGT/PBT) block co-polymer films with two compositions (low or high PEG content). Total cellularity, cell morphology and immunofluorescent staining of vitronectin (VN) and fibronectin (FN) integrin receptors were evaluated, while post-expansion chondrogenic phenotype was assessed by collagen types I and II mRNA expression. We observed a direct relationship between cellularity, cell morphology and re-differentiation potential. Substrates supporting high cell adhesion and a spread morphology (i.e. GP+ and low PEG content films), resulted in a significantly greater number of cells expressing alpha5beta1 FN to alpha(V)beta3 VN integrin receptors, concomitant with reduced collagen type II/ImRNA gene expression. Substrates supporting low cell adhesion and a spherical morphology (GP- and high PEG content films) promoted chondrocyte re-differentiation indicated by high collagen type II/I gene expression and a low percentage of alpha5beta1 FN integrin expressing cells. This study demonstrates that cell-substrate interactions via alpha5beta1 FN integrin mediated receptors negatively impacts expanded human nasal chondrocyte re-differentiation capacity. GP treatment promotes cell adhesion in S+ media but reverses the ability of low PEG content PEGT/PBT substrates to maintain chondrocyte phenotype. We suggest alternative cell immobilization techniques to GP are necessary for clinical application in articular cartilage repair.     

Effect of biologically active coating on biocompatibility of Nitinol devices designed for the closure of intra-atrial communications

Anti-thrombogenicity and rapid endothelialisation are prerequisites for the use of closure devices of intra-atrial communications in order to reduce the risk of cerebral embolism. The purpose of this study was therefore to assess the effect of bioactive coatings on biocompatibility of Nitinol coils designed for the closure of intra-atrial communications. Nitinol coils (n = 10, each) and flat Nitinol bands (n = 3, each) were treated by basic coating with poly(amino-p-xylylene-co-p-xylylene) and then coated with either heparin, r-hirudin or fibronectin. Anti-thrombogenicity was studied in vitro in a dynamic model with whole blood by partial thromboplastin time (PTT), platelet binding and thrombin generation, respectively, and cytotoxicity by hemolysis. Endothelialisation was studied on Nitinol bands with human umbilical venous endothelial cells (HUVEC) by 3-(4,5-dimethylthiazole-2yl)-2,5-triphenyl tetrazolium (MTT) assay and immnuofluorescence analysis of Ki67, vinculin, fibronectin and von Willebrand Factor. Uncoated or coated devices did not influence hemolysis and PTT. r-Hirudin (but not heparin) and fibronectin coating showed lower platelet binding than uncoated Nitinol (p < 0.005, respectively). Heparin and r-hirudin coating reduced thrombin formation (p < 0.05 versus Nitinol, respectively). HUVEC adhesion, proliferation, and matrix formation decreased in the order: fibronectin coating > uncoated Nitinol > r-hirudin coating > heparin coating > basic coating. MTT assay corroborated these findings. In conclusion, r-hirudin and fibronectin coating, by causing no acute cytotoxicity, decreasing thrombogenicity and increasing endothelialisation improve in vitro biocompatibility of Nitinol devices designed for the closure of intra-atrial communications.     

RGD肽对内皮细胞在聚酯材料表面粘附、增殖的影响

RGD是许多粘附蛋白结构中的高度保守序列，与细胞在生物材料表面的粘附、增殖密切相关。本研究在聚酯薄膜表面分别预衬纤维粘连蛋白和共价接枝RGD三肽，然后在不同聚酯材料上种植体外培养的人脐静脉内皮细胞，结果显示RGD可明显促进细胞在材料表面的粘附和增殖，与纤维粘连蛋白相比，RGD促进细胞粘附的作用更为明显，而在细胞增殖方面，二者的作用无显著性差异。本研究为改进生物材料的表面设计，促进心血管移植物的内皮化提供了一个切实可行的思路。     

Evaluation of chondrogenesis within PEGT: PBT scaffolds with high PEG content

Porous poly(ethylene glycol) terephthalate:poly (butylene terephthalate) (PEGT:PBT) scaffolds with high PEG molecular weight (1000 g/mole) and PEGT content (60%) were fabricated using two different processes-paraffin templating and compression molding-for cartilage engineering applications. This polymer composition has previously been shown to enable chondrocyte adhesion and maintain differentiated phenotype in 2D monolayer culture. The influence of 3D polymer scaffold processing on the formation of cartilaginous tissue was studied by seeding primary immature bovine chondrocytes within cylindrical scaffolds in mixed flask reactors for 3 days, followed by cultivation in culture plates for a total of 10 or 24 days. Tissue-polymer constructs were evaluated morphologically by SEM and histology, and quantitatively for cellularity, total collagen, and glycosaminoglycan content, all of which remained statistically equivalent for each time point tested, irrespective of fabrication method. These data demonstrate that the polymers engineered for this study were able to support chondrogenesis independent of scaffold fabrication process, with the influence of pore architecture lessened by the highly hydrated scaffold microenvironments induced by high PEG content.     

Tissue engineering of fish skin: behavior of fish cells on poly(ethylene glycol terephthalate)/poly(butylene terephthalate) copolymers in relation to the composition of the polymer substrate as an initial step in constructing a robotic/living tissue hybrid

This study presents the development of a biosynthetic fish skin to be used on aquatic robots that can emulate fish. Smoothness of the external surface is desired in improving high propulsive efficiency and maneuvering agility of autonomous underwater vehicles such as the RoboTuna (Triantafyllou, M., and Triantafyllou, G. Sci. Am. 272, 64, 1995). An initial step was to determine the seeding density and select a polymer for the scaffolds. The attachment and proliferation of chinook salmon embryo (CHSE-214) and brown bullhead (BB) cells were studied on different compositions of a poly(ethylene glycol terephthalate) (PEGT) and poly(butylene terephthalate) (PBT) copolymer (Polyactive). Polymer films were used, cast of three different compositions of PEGT/PBT (weight ratios of 55/45, 60/40, and 70/30) and two different molecular masses of PEGT (300 and 1000 Da). When a 55 wt% and a 300-Da molecular mass form of PEGT was used, maximum attachment and proliferation of CHSE-214 and BB cells were achieved. Histological studies and immunostaining indicate the presence of collagen and cytokeratins in the extracellular matrix formed after 14 days of culture. Porous scaffolds of PEGT/PBT copolymers were also used for three-dimensional tissue engineering of fish skin, using BB cells. Overall, our results indicate that fish cells can attach, proliferate, and express fish skin components on dense and porous Polyactive scaffolds.     

Bilayered biodegradable poly(ethylene glycol)/poly(butylene terephthalate) copolymer (Polyactive) as substrate for human fibroblasts and keratinocytes.

The purpose of this study was to find an optimal polymer matrix and to optimize the culture conditions for human keratinocytes and fibroblasts for the development of a human skin substitute. For this purpose porous, dense bilayers made of a block copolymer of poly(ethylene glycol terephthalate) (PEGT) and poly(butylene terephthalate) (PBT; Polyactivetrade mark) with a PEGT/PBT weight ratio of 55/45 and a PEG molecular weight (MW) of 300, 600, 1000, or 4000 Da were used. The best performance was achieved with PEGT/PBT copolymer with MW of PEG 300 D (300PEG55PBT45). When fibroblasts were seeded into the porous underlayer and cultured for 3 weeks in medium supplemented with 100 microg/mL ascorbic acid, all pores were filled with fibroblasts and with extracellular matrix, which was judged from the presence of collagen types I, III, and IV, and laminin. When seeded onto the dense top layer of the bilayered (cell free or fibroblast populated) copolymer matrix, human keratinocytes grew out into confluent sheets. After subsequent lifting to the air-liquid interface, a multilayered epithelium with a morphology corresponding to that of the native epidermis was formed. Some differences could still be observed: the expression and localization of some differentiation specific proteins was different and close to that seen in hyperproliferative epidermis; a basal lamina and anchoring zone were absent.     

Membranes as substrates for hepatocyte adhesion in liver support bioreactors

Fourteen membranes out of cellulose (CuprophanR), polyamide and polypropylene were compared in a cytocompatibility test using the cytokinetics and cytomorphology of primary hepatatocytes as parameters. Additionally, the impact of coating the membranes with collagen or fibronectin was investigated. Hepatocytes were not able to attach in acceptable amounts on investigated cellulose membranes. On polyamide and polypropylene membranes a sufficient cell seeding was possible. Coating with collagen or fibronectin improves the attachment and spreading on all membranes. Differences between collagen and fibronectin were detected, observing the morphology of the cells: on collagen, most of the cells spread, whilst on fibronectin, most of the cells spread and flattened polygonally. If the adhesion of hepatocytes prolongs their metabolic function, a large adhesion surface in bioreactors is necessary. To reach a high surface area for cell adhesion in bioreactors one possibility is the use of polyamide and polypropylene membranes.     

Tissue engineering of bovine articular cartilage within porous poly(ether ester) copolymer scaffolds with different structures

The potential of porous poly(ether ester) scaffolds made from poly(ethylene glycol) terephthalate: poly(butylene terephthalate) (PEGT:PBT) block copolymers produced by various methods to enable cartilaginous tissue formation in vitro was studied. Scaffolds were fabricated by two different processes: paraffin templating (PT) and compression molding (CM). To determine whether PEGT:PBT scaffolds are able to support chondrogenesis, primary bovine chondrocytes were seeded within cylindrical scaffolds under dynamic seeding conditions. On day 3, constructs were transferred to six-well plates and evaluated for glycosaminoglycan (GAG) distribution (3, 10, and 24 days), type II collagen distribution, cellularity, and total collagen and GAG content (10 and 24 days). It was observed that better cell distribution during infiltration within PT scaffolds allowed greater chondrogenesis, and at later time points, than in CM scaffolds. The amount of GAG remained constant for all groups from 10 to 24 days, whereas collagen content increased significantly. These data suggest that PEGT:PBT scaffolds are suitable for cartilage tissue engineering, with the PT process enabling greater chondrogenesis than CM.     

The use of PEGT/PBT as a dermal scaffold for skin tissue engineering

Human skin equivalents (HSEs) were engineered using biodegradable-segmented copolymer PEGT/PBT as a dermal scaffold. As control groups, fibroblast-populated de-epidermized dermis, collagen, fibrin and hybrid PEGT/PBT-collagen matrices were used. Two different approaches were used to generate full-thickness HSE. In the 1-step approach, keratinocytes were seeded onto the fibroblast-populated scaffolds and cultured at the air-liquid (A/L) interface. In the 2-step approach, fully differentiated epidermal sheets were transferred onto fibroblast-populated scaffolds and cultured at the A/L. In a 1-step procedure, keratinocytes migrated into the porous PEGT/PBT scaffold. This was prevented by incorporating fibroblast-populated collagen into the pores of the PEGT/PBT matrix or using the 2-step procedure. Under all experimental conditions, fully differentiated stratified epidermis and basement membrane was formed. Differences in K6, K16, K17, collagen type VII, laminin 5 and nidogen staining were observed. In HSE generated with PEGT/PBT, the expression of these keratins was higher, and the deposition of collagen type VII, laminin 5 and nidogen at the epidermal/matrix junction was retarded compared to control HSEs. Our results illustrate that the copolymer PEGT/PBT is a suitable scaffold for the 2-step procedure, whereas the incorporation of fibroblast-populated collagen or fibrin into the pores of the scaffold is required for the 1-step procedure.