聚合物表面生物修饰对肌腱细胞黏附特性的影响

为了探讨增强肌腱细胞与聚合物材料黏附力学特性的措施 ,采用生物可降解聚合物—乳酸与羟基乙酸共聚物 85 / 15 ,制成透光的薄膜 ,在膜表面裱衬聚赖氨酸的基础上 ,表面裱衬细胞外基质 ( I型胶原蛋白、纤维粘连蛋白 ,以及相应的抗体 )和生长因子 (类胰岛素生长因子 1) ,接种转化人胚肌腱细胞后 ,利用微吸管实验技术测定转化人胚肌腱细胞与聚合物薄膜的黏附力。结果显示 :在聚合物薄膜表面裱衬纤维粘连蛋白或 I型胶原蛋白 ,可明显提高转化人胚肌腱细胞与聚合物薄膜的黏附力 ( P<0 .0 5 ) ,但若在此基础上进一步分别复合裱衬纤维粘连蛋白抗体或 I型胶原蛋白抗体 ,则引起转化人胚肌腱细胞与聚合物薄膜的黏附力明显下降 ( P<0 .0 5 ) ;肌腱细胞对聚合物薄膜的黏附力与细胞外基质蛋白 (纤维粘连蛋白或 I型胶原蛋白 )的裱衬浓度有很好的依赖性 ;I型胶原蛋白和纤维粘连蛋白介导转化人胚肌腱细胞与聚合物薄膜的特异性黏附作用 ;二者复合裱衬浓度达到一定比例时 ,可产生协同作用 ,增强黏附效果 ;这种特异性黏附作用可被相应的抗体分子所抑制 ;生长因子对转化人胚肌腱细胞有明显的促黏附作用。提示 ,材料表面生物修饰可促进转化人胚肌腱细胞与聚合物的黏附作用 ,这对构建组织工程化肌腱具有重要的指导意义     

聚合物表面生物修饰对转化人胚肌腱细胞粘附力学特性的影响

目的探讨增强转化人胚肌腺细胞与聚合物材料粘附力学特性的措施。方法采用生物可降解聚合物一乳酸与羟基乙酸共聚物85／15（PLGA85／15）,制成造光的薄膜,表面被衬细胞gbe质（l型胶原蛋白、纤维粘连蛋白,以及相应的抗体）和生长因子（类胰岛素生长因子1）,接种转化人胚肌健细胞后,利用微吸管实验技术测定转化人胚肌腱细胞与聚合物薄膜的粘附力。结果（1）随着I型胶原蛋白裤衬浓度从0μpg／ml增加到10μg／ml,转化人胚肌位细胞与聚合物薄膜的粘附力从1．57×10-8N增至4.17×10-8N,增幅达170％;若在I型胶原蛋白祛衬基础上,复合核…     

纳米PLLA有序膜制备及其对内皮生长晕细胞的促进作用

通过观察内皮生长晕细胞（EOCs）在纳米PLLA有序膜表面黏附、增殖的情况,为优化组织工程材料提供一种新途径。通过静电纺丝技术制备的纳米PLLA纤维支架,进行低温等离子体技术改性及I型胶原表面涂覆,与EOCs复合培养。采用细胞生长曲线和光镜、荧光显微镜及扫描电镜观察支架材料对种子细胞黏附、增殖、形态特征等方面的影响。结果显示：制得的纳米PLLA纤维孔径为300~400 nm,孔隙率〉90%;有序膜和超级有序膜组吸光度A值与无序膜、单纯细胞组有显著性差异（P〈0.05）;细胞在支架膜上生长良好,纳米无序膜细胞生长较散在、杂乱;有良好空间定向效果的有序纤维及超级有序纤维支架有利于细胞沿纤维定向附着、伸展、增殖,分泌胞外基质,而超级有序膜更有利于保持其结构。内皮生长晕细胞是理想的血管组织工程种子细胞来源;纳米PLLA有序及超级有序膜支架能促进种子细胞在材料表面的黏附、增殖,并能较好地保持细胞的形态,是一种理想的血管组织工程支架材料。     

成纤维细胞在聚羟基丁酸酯表面黏附与种植研究

可降解高分子聚合物广泛应用于组织工程,其表面的黏附特性影响细胞的种植与生长。我们研究了成纤维细胞系NIH3T3在可降解材料聚羟基丁酸酯（PHB）表面的黏附和生长。应用线性变剪切流槽研究NIH3T3细胞在材料表面的黏附力学特性,结果表明,由于在PHB表面的临界脱离剪应力较低,高密度接种时间细胞趋于聚集;在经多聚赖氨酸衣被的表面临界脱离剪应力提高,细胞铺展生长。对三维PHB多孔支架种植细胞实验表明,在PHB材料表面进行多聚赖氨酸衣被,可以有效地提高细胞种植效率。     

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50~60 µm、小孔径10~   20 µm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10~20 µm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~ 60 µm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。     

人牙髓细胞复合不同孔径羟基磷灰石/磷酸三钙支架材料的生物学行为

背景:有文献表明,通过组织工程的方法将人牙髓细胞复合羟基磷灰石/磷酸三钙多孔支架材料修复牙缺损具有可行性。然而究竟多大孔径的支架材料最有利于人牙髓细胞的生长及分化,至今尚无定论。目的:观察人牙髓细胞复合不同孔径羟基磷灰石/磷酸三钙支架材料后黏附、增殖和分化等生物学行为。方法:人牙髓细胞接种至3种不同孔径的羟基磷灰石/磷酸三钙材料上,采用荧光显微镜以及扫描电镜检测细胞在材料表面的黏附生长情况,然后通过细胞的黏附率实验与MTT比色法观察人牙髓细胞在材料表面的黏附与增殖特性。不同孔径的羟基磷灰石/磷酸三钙支架复合人牙髓细胞后分别用生长培养基和矿化诱导液培养,于接种后第4,7,10天检测碱性磷酸酶活性。结果与结论:人牙髓细胞在3种不同孔径支架材料表面和孔隙内均能顺利黏附并增殖。其中100～300μm组支架材料黏附率最高,MTT结果显示接种3d后300～500μm组能较好地促进细胞增殖。培养10d后,复合在100～300μm和300～500μm材料上的人牙髓细胞,其碱性磷酸酶活性显著高于500～700μm组。提示与500～700μm孔径相比,孔径为100～300μm和300～500μm的羟基磷灰石/磷酸三钙材料能更好地促进人牙髓细胞黏附、增殖和分化。     

多孔壳聚糖膜的制备及皮下植入组织学观察

目的 分析交联剂戊二醛对多孔壳聚糖膜孔隙率与孔径的影响,并探讨该多孔膜作为皮下植入式葡萄糖传感器保护膜的可行性.方法 通过致孔剂法利用硅胶和不同量的戊二醛制成多孔壳聚糖膜,并采用密度法和切片法分析其孔隙率与孔径等结构参数,扫描电子显微镜观察其表面形态.将无交联的多孔膜植入到9只SD大鼠皮下,第7、17、45天取材切片染色,观察组织形态学变化,定量分析多孔膜内外胶原沉积与血管增生密度.结果 戊二醛能提高多孔壳聚糖膜的孔隙率,5％交联度时孔隙率最大达76.2％/73.0％(密度法/切片法),但同时降低了多孔膜孔径.使用孔径最大的不交联多孔膜(38.5 μm)皮下植入,膜内胶原沉积含量由第7天的6.74％增加到第45天的22.5％,而Masson染色测量的膜内增生血管密度由0.37％增加到2.56％,与HE染色测量结果一致(由0.11％增加到1.65％).第45天时膜外胶原沉积含量38.3％,是膜内的1.7倍.结论 多孔壳聚糖膜能降低材料-组织界面纤维成分的致密性,增加血管增生,具有良好的组织相容性,有望应用于植入式葡萄糖传感器.     

多肽修饰聚合物PLGA-[ASP-PEG]对骨髓基质细胞黏附特性的影响

探讨在PLGA-[ASP-PEG]表面进行多肽改性后,对骨髓基质细胞在其表面黏附力的影响。在骨支架材料PLGA-[ASP-PEG]表面固定多肽GRGDSPC,用微吸管吸吮法测定骨髓基质细胞不同的时间段在骨支架材料表面的黏附力,并进行扫描电镜观察。结果表明:骨髓基质细胞接种在二种支架材料上4 h时,PLGA-[ASP-PEG]表面黏附力为172.78±15.23 N,多肽改性的PLGA-[SP-PEG]细胞黏附力209.47±92.59 N,二者无明显差异;在12h,多肽改性的PLGA-[ASP-PEG]黏附力576.23±165.74 N,PLGA-[ASP-PEG]黏附力为261.84±100.09 N,前者表面细胞黏附力明显强于后者(P<0.01);在24 h时,二种材料表面的细胞黏附力无明显差异(P>0.05)。扫描电镜观察结果为多肽改性支架材料上表面黏附的细胞数明显多于未改性材料表面黏附的细胞数。在生物材料表面结合多肽可以增强细胞在材料表面的黏附力,从而改善生物材料生物相容性。     

静电纺纳米纤维PLCL/纤维蛋白原人工韧带的生物相容性评价

本研究旨在评价聚乳酸-己内酯（PLCL）/纤维蛋白原纳米纤维膜（P/F-Ns）的安全性和功能性,为临床应用提供理论支撑。通过扫描电镜、材料测试机、接触角测量仪和酶标仪分别研究P/F-Ns的表面形态、力学性能、亲水性和纤维蛋白原含量。通过CCK-8法和实时荧光定量PCR评价Hig-82细胞在P/F-Ns上的黏附、增殖及韧带修复相关基因表达。结果显示,随着纤维蛋白原含量增加,P/F-Ns表面纤维孔隙增大,力学性能下降,亲水性增强,其中P/F-N-2最有利于细胞黏附和增殖。以不含纤维蛋白原的P/F-N-0为对照,含纤维蛋白原的P/F-N-1、P/F-N-2和P/F-N-3上细胞韧带修复相关基因在第3天和第7天表达均上调。与P/F-N-0相比,含纤维蛋白原的P/F-Ns具有更好的生物相容性,能够有效应用于前交叉韧带重建。     

磁性纳米多孔复合支架材料的细胞相容性研究

目的研究磁性纳米多孔复合(n-HA/PLLA/Fe2O3)材料的细胞相容性,探讨细胞在材料表面黏附、增殖、表达等生物学行为,为其医学应用提供实验依据。方法将大鼠成骨细胞与磁性纳米多孔复合材料共培养,采用CCK-8法检测细胞增殖、扫描电镜观察细胞在材料上的黏附、RT-PCR检测I型胶原和骨钙素基因的表达。结果 CCK-8检测显示实验组磁性纳米多孔复合材料上细胞的增殖与空白对照组没有差异性(0.05);扫描电镜观察到细胞在磁性纳米多孔复合材料的表面和孔隙内大量黏附、增殖和生长,随着共培养时间的增加,材料表面的细胞数量明显增多;RT-PCR显示随着共培养时间的增加,I型胶原基因的表达增强(0.05),骨钙素的表达无明显差异(0.05)。结论磁性纳米多孔复合支架材料适于成骨细胞的黏附、生长和分化,具有良好的细胞相容性。     

转化人胚腱细胞在PLA和PLGA表面上的粘附特性研究

为了定量研究转化人胚腱细胞在聚乳酸和聚乳酸与羟基乙酸共聚物８５／１５表面上的粘附特性,我们采用微管吸吮技术测定了单个ＴＨＥＴＣ与ＰＬＡ膜和ＰＬＧＡ８５／１５膜表面的粘附力学特性。结果显示,ＴＨＥＴＣ与ＰＬＡ膜和ＰＬＧＡ８５／１５膜表面的粘附率和粘附力明显大于膜表面裱衬牛血清白蛋白实验组,同时小于膜表面裱衬聚赖氨酸实验组,而且ＰＬＧＡ８５／１５膜更易于ＴＨＥＴＣ粘附。表明ＢＳＡ能抑制ＴＨＥＴＣ在聚全     

Adhesion strength of human tenocytes to extracellular matrix component-modified poly(DL-lactide-co-glycolide) substrates

We report a direct measurement of the adhesion strength of human embryonic tenocytes (HETCs) and transformed human embryonic tenocytes (THETCs) to fibronectin (FN)- and type I collagen (CNI)- modified poly(DL-lactide-co-glycolide) (PLGA) substrates with a micropipette aspiration technique. PLGA substrates were first coated with poly-D-lysine (PDL), and then with various concentrations (1 microg/ml, 2 microg/ml, 5 microg/ml, and 10 microg/ml) of FN and CNI in serum-free F12 media. Anti-FN and Anti-CNI antibodies were used to inhibit attachment of tenocytes to FN- and CNI- modified substrates in a dilution range of 1:5000-1:500 and 1:1500-1:250, respectively. The substrates were employed for incubation of HETCs and THETCs for 30 min at 37 degrees C before the adhesion strength measurements. We found that the adhesion strengths showed a strong dependence on the seeding time and FN or CNI concentrations. Anti-FN and Anti-CNI antibodies significantly compromised adhesion of HETCs and THETCs to the corresponding modified substrates (P < 0.05). These findings show that FN- or CNI-modified polymer substrates offer significant advantages for tissue engineering tendon scaffolds concerning tenocyte adhesion. In addition, HETCs and THETCs bear similar biological behaviors in terms of adhesion, indicating the possibility of using THETCs in place of HETCs in tissue engineering construction of human tendons.     

Persistent adhesion of epithelial tissue is sensitive to polymer topography.

The persistent adhesion of corneal epithelial tissue to the surface of a porous polymer is of interest in the development of a corneal onlay. Using an in vitro model system, this study examined the effect of polymer surface topography on the assembly of basement membrane and hemidesmosomes. Corneal epithelial tissue was grown on polycarbonate surfaces with a range of pore sizes (0.1-3.0 micron, pore diameter) and an equivalent nonporous surface. The ultrastructure of the tissue-polymer interface was evaluated using electron microscopy. On the porous surfaces, the tissue responded to a balance between the size of the discontinuity (pores) and the amount of polymer surface between the pores. Continuous basement membrane and a regular pattern of hemidesmosomal plaque occurred only on the 0.1 micron surface, where both the pores and the total surface area covered by pores were relatively small. The assembly of adhesive structures on surfaces with pore diameters between 0.4-2.0 microns was restricted to regions of polymer between pores. No adhesive structures assembled on the nonporous or on the 3.0-micron surface. These results demonstrate that, in addition to porosity, surface topography is a significant factor in the formation of structures involved in the persistent adhesion of stratified epithelial tissue on a polymer.     

Effect of porosity and surface hydrophilicity on migration of epithelial tissue over synthetic polymer

The relative effects of porosity and surface chemistry on the migration of epithelial tissue over the surface of a polymer were determined in vitro. These studies compared nonporous polymers with those having 0.1-microm diameter track-etched pores and were conducted on polycarbonate and polyester. Epithelial tissue migration over the polymer surface was stimulated by the presence of these pores. The surface chemistries of the polymers were modified by deposition of various polymer films using radio frequency gas deposition, giving a range of surfaces that varied in air:water sessile contact angle (SCA) of between 26 and 100 degrees. Tissue migration on the nonporous surfaces was affected by the surface chemistry, being generally linear as a function of the SCA and higher on hydrophilic than on hydrophobic surfaces but reduced if the hydrophilic surface had a mobile chemistry. The effects of the 0.1-microm diameter pores and the surface hydrophilicity were additive with the maximal level of epithelial tissue migration occurring on a porous, hydrophilic polymer surface.     

Human monocyte adhesion and activation on crystalline polymers with different morphology and wettability in vitro

This study evaluated the effects of crystalline polyamide (Nylon-66), poly(ethylene-co-vinyl alcohol) (PEVA), and poly(vinylidene fluoride) (PVDF) polymers with nonporous and porous morphologies on the ability of monocytes to adhere and subsequently activate to produce IL-1beta, IL-6, and tumor necrosis factor alpha. The results indicated monocyte adhesion and activation on a material might differ to a great extent, depending on the surface morphology and wettability. As the polymer wettability increases, the ability of monocytes to adhere increases but the ability to produce cytokines decreases. Similarly, these polymers, when prepared with porous surfaces, enhance monocyte adhesion but suppress monocyte release of cytokines. Therefore, the hydrophobic PVDF with a nonporous surface stimulates the most activity in adherent monocytes but shows the greatest inhibition of monocyte adhesion when compared with all of the other membranes. In contrast, the hydrophilic Nylon-66, which has a porous surface, is a relatively better substrate for this work. Therefore, monocyte behavior on a biomaterial may be influenced by a specific surface property. Based on this result, we propose that monocyte adhesion is regulated by a different mechanism than monocyte activation. Consequently, the generation of cytokines by monocytes is not proportional to the number of cells adherent to the surface.     

Modulation of peritendinous adhesion formation by alginate solution in a rabbit flexor tendon model

To examine the antiadhesive effect of an alginate solution following tendon surgery, unilateral subtotal laceration of the flexor digitorum communis tendon was created in one hind limb while the other side was left intact in 32 Japanese white rabbits. The lesion was coated with alginate solution in 16 animals and not coated in the other 16. Degree of adhesion formation was assessed histologically and biomechanically by measuring the flexion angle of the first toe when the flexor digitorum tendon was pulled with a specified force at 4 weeks postoperatively. When compared with the control group, the alginate-treated group demonstrated significantly greater toe flexion, with less scar tissue formation at the repair site. Histologically, complete tendon healing with longitudinal remodeling of collagen fibers was observed in the alginate-treated group, while a random pattern of fibers was observed in the control group. Reduction in adhesion formation using alginate solution represents a novel strategy for the management of tendon injury and repair in the clinical setting.     

Electrospun polyphosphazene nanofibers for in vitro rat endothelial cells proliferation

A large variety of natural and synthetic polymers have been explored as scaffolds for the seeding and growth of different types of cells. To fabricate a scaffold that can be used as a synthetic extracellular matrix (ECM), it is important to replicate the nanoscale dimensions of natural ECM. The electrospinning process allows to produce ultrathin fibers so that this method represents a suitable approach to scaffold fabrication for tissue engineering applications. In this work, the feasibility of obtaining flat or tubular matrices from biocompatible poly[(ethyl phenylalanato)(1.4) (ethyl glycinato)(0.6) phosphazene] by electrospinning was evaluated and the effect of process parameters on the diameter of nanofibers was examined. The adhesion and growth of rat neuromicrovascular endothelial cells cultured on sheets and tubes composed by the polymer with an average fiber diameter of 850 +/- 150 nm were also reported. Microscopic examination of the seeded tubes demonstrated that, after 16 days of incubation, endothelial cells formed a monolayer on the whole surface. These results are the first step to demonstrate that tubes of biodegradable polyphosphazenes might be a feasible model to construct human tissues such as vessels or cardiac valves.     

Analysis of the biological response of endothelial and fibroblast cells cultured on synthetic scaffolds with various hydrophilic/hydrophobic ratios: influence of fibronectin adsorption and conformation

In this study we developed polymer scaffolds intended as anchorage rings for cornea prostheses among other applications, and examined their cell compatibility. In particular, a series of interconnected porous polymer scaffolds with pore sizes from 80 to 110 microns were manufactured varying the ratio of hydrophobic to hydrophilic monomeric units along the polymer chains. Further, the effects of fibronectin precoating, a physiological adhesion molecule, were tested. The interactions between the normal human fibroblast cell line MRC-5 and primary human umbilical vein endothelial cells (HUVECs) with the scaffold surfaces were evaluated. Adhesion and growth of the cells was examined by confocal laser scanning microscopy. Whereas MRC-5 fibroblasts showed adhesion and spreading to the scaffolds without any precoating, HUVECs required a fibronectin precoating for adhesion and spreading. Although both cell types attached and spread on scaffold surfaces with a content of up to a 20% hydrophilic monomers, cell adhesion, spreading, and proliferation increased with increasing hydrophobicity of the substrate. This effect is likely due to better adsorption of serum proteins to hydrophobic substrates, which then facilitate cell adhesion. In fact, atomic force microscopy measurements of fibronectin on surfaces representative of our scaffolds revealed that the amount of fibronectin adsorption correlated directly with the hydrophobicity of the surface. Besides cell adhesion we also examined the inflammatory state of HUVECs in contact with the scaffolds. Typical patterns of platelet/endothelial cell adhesion molecule-1 expression were observed at intercellular boarders. HUVECs adhering on the scaffolds retained their proinflammatory response potential as shown by E-selectin mRNA expression after stimulation with lipopolyssacharide (LPS). The proinflammatory activation occurred in most of the cells, thus confirming the presence of a functionally intact endothelium. Little or no expression of the proinflammatory activation markers in the absence of LPS stimulation was observed for HUVECs growing on scaffolds with up to a 20% of hydrophilic component, whereas activation of these markers was observed after stimulation. In conclusion, scaffolds containing up to 20% hydrophilic monomers exhibited excellent cell compatibility toward human fibroblast cell line MRC-5 and human endothelial cells. Atomic force microscopy confirmed that adsorbed serum proteins such as fibronectin probably accounted for the positive correlation of HUVEC adhesion and surface hydrophobicity.     

表面图案化生物可降解聚膦腈制备及对成骨细胞的黏附

背景：纳/微米尺度的拓扑结构可影响细胞在基体上的黏附和增殖。 目的：改变制备条件,考察表面图案化聚(甘氨酸乙酯-丙氨酸乙酯)膦腈(PGAP)膜的表面性质对成骨细胞黏附和增殖的影响。 方法：将PGAP溶于氯仿配成不同浓度溶液,在25 ℃、20%或80%湿度下浇铸成膜。采用SEM、AFM、XRD和水接触角进行表征,通过体外培养,采用黏附、增殖和细胞形态等评价膜表面特性对成骨细胞生物学行为的影响。 结果与结论：高相对湿度环境有利于形成图案化表面,溶液浓度升高使图案化结构变得更加规整。溶液质量浓度120 g/L时,可得到一种蜂巢结构表面,但当其升至160 g/L后,反而不利于图案化表面的形成。高湿度环境下得到的膜表现出极性磷和氮原子在表面富集的现象。这些特性使表面图案化的PGAP膜更易于吸附蛋白质,从而使成骨细胞更易在其上黏附,增殖速度更快,细胞铺展状态也更好。说明溶液浓度和环境湿度是一种简便、可用于控制PGAP膜表面性质的方法,可用于对成骨细胞生物学行为的调控。