表面改性及微沟槽技术对肌腱细胞生长与取向的影响

利用光刻技术制作微格式模板,微接触转印法制作微沟槽PDMS表面,微流道技术裱衬不同浓度的Ⅰ型胶原于微沟槽表面上,比较其对细胞生长形态及生长取向的影响.对照组为未裱衬胶原的微沟槽、平板PDMS及裱衬胶原的平板PDMS.种植SD大鼠肌腱细胞在材料上,于37℃孵箱中培养48 h.采用MTT比色法测定不同浓度的胶原对肌腱细胞的生长增殖的影响.通过倒置相差显微镜、扫描电镜、荧光显微镜观察细胞生长的形态,取向情况.结果显示:Ⅰ型胶原修饰的PDMS微沟槽材料比未裱衬胶原的对照组具有明显的促细胞生长的作用(P＜0.05),并且随着胶原浓度的增加作用越明显.有微沟槽表面的材料对细胞的生长形态和生长取向有明显影响.提示,Ⅰ型胶原修饰的微沟槽材料不仅能规范肌腱细胞的生长取向,而且能促进肌腱细胞的生长,从而得到理想的细胞生长形态,该结果对工程化肌腱的构建有重要的指导意义.     

利用自组装单分子层（SAM）研究细胞和表面的相互作用

研究细胞和表面相互作用具有重要意义,利用长链的烷硫醇在金表面或烷基硅烷在羟基化表面形成的自组装单分子层及其微格式化的表面允许以分子水平研究并控制细胞和表面的相互作用,我们概述了自组装单分子层及其格式化表面研究细胞和表面相互作用的最新进展。     

神经芯片研究新进展：微接触印刷将神经元和硅片融为一体

神经芯片是一种利用微电极阵列实现神经细胞与电子器件信息传导的生物芯片,是人工神经代偿器件的接口.它通过把活的神经元和硅电路有机地连接在一起.实现对细胞无损伤的长时程记录,并且可以施加人为刺激.目前这一领域的关键问题在于如何使神经细胞在芯片上按照电极点的位置黏附生长,建立通讯联系并提高芯片材料的信噪比.利用微加工工艺有望达到这一目的 .微接触印刷技术在其中应用最为广泛.它应用聚二甲基硅氧烷为印章,以生物大分子或有机聚合物为"墨水",把主模板上的精细图案转印到硅片材料上,从而实现对材料表面黏附底物的改性和结构的微加工.本综述简要介绍了神经芯片的研究进展,详细阐述了微接触印刷技术的研究现状,并且初步探讨了这一工艺在神经芯片领域中的最新应用.     

聚醚醚酮表面多孔羟基化改性对MC3T3-E1细胞黏附、增殖的影响

目的对聚醚醚酮(polyetheretherketone,PEEK)薄片表面进行多孔化和羟基化改性,观察PEEK表面形貌和生物活性的变化,并探讨该改性方法对前成骨MC3T3-E1细胞黏附、增殖的影响。方法超声波环境下浓硫酸处理PEEK表面,在其表面形成大量微孔结构;经湿化学法将PEEK表面的酮类基团还原成羟基基团,改善其表面化学活性,提升PEEK薄片的生物相容性。利用扫描电子显微镜(SEM)、傅里叶变换红外光谱仪(FTIR)及静态水接触角检测改性前后材料表面形貌、化学基团及亲水性的变化。未处理PEEK、多孔化PEEK、羟基化PEEK、多孔羟基化PEEK与MC3T3-E1细胞共培养,评价表面改性后PEEK薄片对细胞黏附、增殖的影响。结果 SEM结果显示浓硫酸处理后的PEEK薄片表面形成密集的空隙大小均匀的微孔结构,FT-IR结果证实羟基化改性成功地在PEEK表面还原出了大量羟基基团。同时,表面多孔化和羟基化改性均可有效提升PEEK材料表面的亲水性能。在体外细胞实验中,不同改性的PEEK材料与MC3T3-E1细胞共培养后结果显示,多孔化、羟基化和多孔羟基化改性均可显著促进细胞黏附和伸展,同时随着时间的延长,其促进细胞增殖的功能也逐步增强。结论表面多孔羟基化改性能有效提高PEEK材料表面的生物学活性和亲水性能,进而显著促进细胞的黏附和增殖。     

三维动静态培养软骨源性微组织的细胞行为及软骨形成能力

背景：与传统二维培养相比,三维培养软骨微组织具有更大的优势,但仍需进一步探索更有利的三维培养方式。目的：评价2种三维培养方式下微组织的细胞行为及促软骨形成能力。方法：通过化学脱细胞方法和组织粉碎方法制备软骨源性微载体,采用DNA定量和核染色验证脱细胞是否成功,通过组织学染色观察脱细胞前后基质保留情况,采用扫描电子显微镜和CCK-8方法对微载体进行表征;通过三维静态培养法和三维动态培养法将软骨源性微载体与人脂肪间充质干细胞结合构建软骨源性微组织,利用扫描电子显微镜、活死染色、RT-q PCR等手段检测两组微组织的细胞活力及成软骨能力。结果与结论：(1)成功制备软骨源性微载体,与脱细胞前相比,脱细胞后DNA含量显著降低(P <0.001);扫描电子显微镜观察微载体表面有胶原包绕,保持天然软骨细胞外基质特征;CCK-8法检测表明微载体无细胞毒性且能够促进细胞增殖;(2)扫描电子显微镜及活死染色结果显示,相比三维静态组,三维动态组微组织细胞具有更舒展的形态,细胞与细胞间、细胞与基质间、基质与基质间形成广泛的连接;(3)RT-qPCR结果表明两组微组织SOX9、蛋白聚糖、Ⅱ型胶原表达在培养...     

RGD肽表面修饰聚苯乙烯及其细胞相容性研究

目的以聚苯乙烯二维平面为模板研究了蛋白表面修饰技术,构建具有生物活性的生物材料表面。方法采用物理包被法依靠疏水作用在PS表面架构明胶、胶原和RGD（精氨酸-甘氨酸-天冬氨酸）多肽的生物活性层。通过光电子能谱（XPS）分析修饰表面的元素含量变化,N元素含量显著提高,说明蛋白分子在表面存在。Bradford方法定量分析明胶、胶原和RGD多肽的表面吸附量。结果XPS证实了表面N原子的引入,存在酰胺键,确定蛋白分子存在于PS表面。结论动态接触角下降显著,证明修饰表面的亲水性得到提高。并在明胶、胶原和RGD多肽修饰表面接种人表皮细胞,对比考察其对细胞行为的影响,提高了细胞的黏附和增殖能力。     

整合素beta1亚单位对肝癌细胞与IV型胶原黏附与趋化行为的影响

采用微吸管实验技术 ,测定肝细胞癌 (Hepatocellular carcinom a,HCC)细胞与 IV型胶原裱衬表面的黏附力 ;进一步加入针对整合素 beta1亚单位 (CD2 9)的单克隆抗体 (Anti- CD2 9)处理 HCC细胞 ,观察 Anti- CD2 9对细胞与 IV型胶原裱衬表面的黏附力的影响。采用双微吸管实验法进行 HCC细胞趋化实验 ,在两侧微管内加入相同浓度 (6 0 0μg/ ml )的 IV型胶原 ,并引导微管尖端与同一细胞紧密接触 ,动态观察细胞两侧伪足形成过程 ;在一侧微吸管内加入 2 0μg/ ml Anti- CD2 9,考察 beta1整合素亚单位阻断对 HCC细胞伪足形成的影响。利用流式细胞仪对 HCC细胞表面整合素 beta1亚单位的表达进行分析。结果表明 ,HCC细胞与 5μg/ ml IV型胶原裱衬表面之间的黏附力为 932± 134(× 10 - 1 0 N ,n=6 0 ) ,加入 5μg/ ml Anti- CD2 9黏附力减小到 4 4 9± 119(× 10 - 1 0 N,n=6 0 ) ;加入 10 μg/ ml Anti- CD2 9时黏附力减小到 2 2 0± 78(× 10 - 1 0 N,n=5 5 )。双微吸管趋化实验表明 :两侧微吸管加入相同浓度 IV型胶原 ,细胞向两侧微吸管均有伪足形成 ;在此基础上 ,微吸管加入 Anti- CD2 9的一侧 ,HCC细胞伪足生长曲线呈现明显的抑制 ,而未加入 Anti- CD2 9的微吸管一侧与加入的对侧相比 ,该侧细胞的伪足明显增     

壳聚糖球形多孔微载体支持下人肝细胞L-02的高浓度细胞培养

背景：微载体培养技术作为一项体外高浓度细胞培养技术,近年来已在肝细胞的体外培养中得到应用。 目的：对壳聚糖球形多孔微载体培养的人肝细胞L-02进行定时的形态学观察。 方法：以自制的壳聚糖球形多孔微载体样本为支架来培养人肝细胞L-02设为实验组;无壳聚糖球形多孔微载体支持下人肝细胞的培养设为对照组。对两组细胞进行定时的细胞计数,并对实验组进行形态学观察,包括倒置相差生物显微镜观察和扫描电子显微镜观察。 结果：两组培养的细胞数量均呈现前3 d增长,在第3天细胞数量达到最高值;而且实验组3个样本培养的细胞数明显高于对照组无微载体培养的细胞数量(P < 0.05),实验组各样本之间差异无显著性意义(P > 0.05);倒置相差生物显微镜下动态观察,可见前3 d微载体表面黏附生长的肝细胞则逐渐增多,第3天可见大部分微载体表面有许多肝细胞黏附成团,总的存活率均在90%以上,且肝细胞保持着良好的形态学结构;扫描电子显微镜观察,微载体表面、切面和内部均可看到有许多球状肝细胞紧密黏附。提示,以自制的壳聚糖球形多孔微载体作为一种支架,在体外三维环境下可以进行高浓度细胞培养。     

胶原改良聚乳酸电纺丝支架用于组织工程化输尿管的体外构建***

背景：聚乳酸是一种应用广泛的细胞支架材料,但其疏水性和缺乏细胞识别信号影响了在组织工程器官构建中的应用。 目的：探讨Ⅰ型胶原蛋白改良聚乳酸电纺丝支架体外构建组织工程化输尿管的可行性。 方法：用Ⅰ型胶原蛋白醋酸溶液冻干法处理聚乳酸电纺丝,使Ⅰ型胶原蛋白吸附于电纺丝纤维表面,制成胶原改良电纺丝支架。将分离培养的输尿管上皮细胞分别接种于改良聚乳酸电纺丝支架和未处理的聚乳酸电纺丝支架上。 结果与结论：MTT检测显示输尿管上皮细胞在改良支架中生长良好,细胞整体活性在各时间点均明显优于未处理的聚乳酸电纺丝支架上的细胞。扫描电镜观察发现细胞在改良支架表面黏附良好,接种后5 d,支架表面大部分已被增殖的输尿管上皮细胞覆盖。说明胶原改良聚乳酸电纺丝支架能明显提高种子细胞的黏附和增殖活性,可用于体外构建组织工程化输尿管。 关键词：输尿管;电纺丝;聚乳酸;胶原;黏附;增殖;组织工程 doi:10.3969/j.issn.1673-8225.2012.12.002     

重组人vWF A3-GPI融合蛋白在CHO细胞膜上的表达及其生物学活性

目的 在CHO中表达人vWF A3-GPI融合蛋白并测定其生物学活性,以期获得能够锚定至细胞表面,并引导其黏附于受损血管壁的分子.方法 构建真核表达质粒pcDNA3.1(-)-vWF A3-GPI,转染CHO细胞.采用流式细胞术和免疫荧光激光共聚焦显微镜观察其对细胞膜的锚定作用.应用CCK8法测定其结合胶原的能力及经不同浓度PI-PLC处理后的变化情况.结果 vWF A3-GPI融合蛋白能够锚定在CHO细胞膜上,并具有很好的胶原结合活性;PI-PIC处理后,其胶原结合活性降低,降低程度与PI-PLC浓度成正比.结论 CHO中高效表达的人vWF A3-GPI融合蛋白具有良好的生物学功能,能有效地锚定至细胞表面,并保持良好的胶原结合效应,可作为引导细胞黏附于受损血管壁的分子.     

Patterning proteins and cells using soft lithography

This review describes the pattering of proteins and cells using a non-photolithographic microfabrication technology, which we call 'soft lithography' because it consists of a set of related techniques, each of which uses stamps or channels fabricated in an elastomeric ('soft') material for pattern transfer. The review covers three soft lithographic techniques: microcontact printing, patterning using microfluidic channels, and laminar flow patterning. These soft lithographic techniques are inexpensive, are procedurally simple, and can be used to pattern a variety of planar and non-planar substrates. Their successful application does not require stringent regulation of the laboratory environment, and they can be used to pattern surfaces with delicate ligands. They provide control over both the surface chemistry and the cellular environment. We discuss both the procedures for patterning based on these soft lithographic techniques, and their applications in biosensor technology, in tissue engineering, and for fundamental studies in cell biology.     

Patterned networks of mouse hippocampal neurons on peptide-coated gold surfaces

Patterned networks of hippocampal neurons were generated on peptide-coated gold substrates prepared by microscope projection photolithography and microcontact printing. A 19 amino acid peptide fragment of laminin A (PA22-2) that includes the IKVAV cell adhesion domain was used to direct patterns of cell adhesion in primary culture. Microscale grid patterns of peptide were deposited on gold-coated glass cover slips by soft lithography using "stamps" fashioned from polydimethylsiloxane. Strong coordination bonding between gold atoms on the surface and the sulfur atoms of the N-terminal cysteine residues supported stable adhesion of the peptide, which was confirmed by immunofluorescence using anti-IKVAV antiserum. Dispersed hippocampal cells isolated from neonatal mouse pups were grown on peptide-patterned gold substrates for 7 days. Neurons preferentially adhered to peptide-coated regions of the gold surface and restricted their processes to the peptide patterns. Whole cell recordings of neurons grown in patterned arrays revealed an average membrane potential of -50 mV, as well as the presence of voltage-gated ion conductances. Peptide-modified gold surfaces serve as convenient and effective substrates for growing ordered neural networks that are compatible with existing multi-electrode array recording technology.     

Biological surface engineering: a simple system for cell pattern formation.

Biological surface engineering using synthetic biological materials has a great potential for advances in our understanding of complex biological phenomena. We developed a simple system to engineer biologically relevant surfaces using a combination of self-assembling oligopeptide monolayers and microcontact printing (muCP). We designed and synthesized two oligopeptides containing a cell adhesion motif (RADS)n (n = 2 and 3) at the N-terminus, followed by an oligo(alanine) linker and a cysteine residue at the C-terminus. The thiol group of cysteine allows the oligopeptides to attach covalently onto a gold-coated surface to form monolayers. We then microfabricated a variety of surface patterns using the cell adhesion peptides in combination with hexa-ethylene glycol thiolate which resist non-specific adsorption of proteins and cells. The resulting patterns consist of areas either supporting or inhibiting cell adhesion, thus they are capable of aligning cells in a well-defined manner, leading to specific cell array and pattern formations.     

Directing cell migration using micropatterned and dynamically adhesive polymer brushes

Micropatterning techniques, such as photolithography and microcontact printing, provide robust tools for controlling the adhesive interactions between cells and their extracellular environment. However, the ability to modify these interactions in real time and examine dynamic cellular responses remains a significant challenge. Here we describe a novel strategy to create dynamically adhesive, micropatterned substrates, which afford precise control of cell adhesion and migration over both space and time. Specific functionalization of micropatterned poly(ethylene glycol methacrylate) (POEGMA) brushes with synthetic peptides, containing the integrin-binding arginine–glycine–aspartic acid (RGD) motif, was achieved using thiol–yne coupling reactions. RGD activation of POEGMA brushes promoted fibroblast adhesion, spreading and migration into previously non-adhesive areas, and migration speed could be tuned by adjusting the surface ligand density. We propose that this technique is a robust strategy for creating dynamically adhesive biomaterial surfaces and a useful assay for studying cell migration.     

Fabrication of patterned cell co-cultures on albumin-based substrate: Applications for microfluidic devices

A surface coated with cross-linked albumin film resists the adhesion of cells, and subsequent exposure to UV irradiation or electrostatic adsorption of a cationic polymer switches the surface from non-adherent to adherent. Taking advantage of this unique property of cross-linked albumin, the authors fabricated patterned cell co-cultures with desired patterns and cell types. In this scheme, the cell-adherent region was initially created in the cell-non-adhesive albumin substrate, on which a first cell type was attached. Subsequently, the remaining region was also changed to adherent for the attachment of secondary cells in the same manner, thereby allowing distinctly localized co-cultures. As a proof of concept demonstration of the feasibility of this approach, a patterned co-culture of Neuro-2a cells with L929 cells was successfully prepared on the substrate. Furthermore, combining this technique with a microfluidic technique, a micropatterned co-culture of PA6 cells with 3T3 fibroblasts was created inside microfluidic devices. This approach could potentially be a useful tool for fundamental investigations of cell–cell interactions and for tissue engineering applications.     

Poly(dimethylsiloxane) thin films as biocompatible coatings for microfluidic devices: cell culture and flow studies with glial cells

Oxygen plasma treatment of poly(dimethylsiloxane) (PDMS) thin films produced a hydrophilic surface that was biocompatible and resistant to biofouling in microfluidic studies. Thin film coatings of PDMS were previously developed to provide protection for semiconductor-based microoptical devices from rapid degradation by biofluids. However, the hydrophobic surface of native PDMS induced rapid clogging of microfluidic channels with glial cells. To evaluate the various issues of surface hydrophobicity and chemistry on material biocompatibility, we tested both native and oxidized PDMS (ox-PDMS) coatings as well as bare silicon and hydrophobic alkane and hydrophilic oligoethylene glycol silane monolayer coated under both cell culture and microfluidic studies. For the culture studies, the observed trend was that the hydrophilic surfaces supported cell adhesion and growth, whereas the hydrophobic ones were inhibitive. However, for the fluidic studies, a glass-silicon microfluidic device coated with the hydrophilic ox-PDMS had an unperturbed flow rate over 14 min of operation, whereas the uncoated device suffered a loss in rate of 12%, and the native PDMS coating showed a loss of nearly 40%. Possible protein modification of the surfaces from the culture medium also were examined with adsorbed films of albumin, collagen, and fibrinogen to evaluate their effect on cell adhesion.     

Selective Surface Modification in Silicon Microfluidic Channels for Micromanipulation of Biological Macromolecules

Interactions between biological macromolecules and micrometer- and sub-micrometer-scale surface structures are directly influenced by the surface wettability, chemical reactivity and surface charge. Understanding these interactions is crucial for developing integrated microsystems for biological and biomedical processing and analysis. We report development of selective surface modification techniques based on microcontact printing and polyelectrolyte adsorption. These techniques were applied to lithographically patterned silicon microfluidic channels and flat silicon substrates to create surface microstructures with contrasting wetting properties and surface charges. These controls enabled us to devise various techniques for controlled loading and processing of biomaterials in the channels. Solutions containing long chain biological macromolecules DNA and microtubules were directly loaded into the microchannels by using a micromanipulator/microinjector system. Structural arrangements of these linear macromolecules, which were probed by using fluorescence and laser scanning confocal microscopy, were found to be quite different from bulk solutions. As expected, the filamentous molecules were observed to align linearly along the channels, with the degree of alignment dependent on channel width as well as the length of the molecule. This molecular alignment, which is induced by both the surface confinement effect and capillary flow during sample loading, may be used to enhance processing of biological materials in silicon biomedical microdevices. It also opens up the possibility of carrying out direct combinatorial structural characterization of proteins in the microchannels utilizing X-ray diffraction, which so far has not been possible.     

Microcontact printing of proteins on oxygen plasma-activated poly(methyl methacrylate)

This paper describes a method for microcontact printing protein solutions onto polymer substrates temporarily activated by oxygen plasma. Following plasma treatment, poly(dimethyl siloxane) (PDMS) stamps were coated with an aqueous laminin solution then placed in direct contact with plasma-treated poly(methyl methacrylate) (PMMA) substrates. This process resulted in well defined laminin stripes on the PMMA surface when printing was performed within 45min of the plasma treatment. Axonal outgrowth from embryonic chick dorsal root ganglia (DRG) was largely confined to the stamped pattern, while over 90% of primary rat Schwann cells adhered to the protein stamped areas on the PMMA substrates. Oxygen-plasma treatment of the PMMA surface was necessary to deposit proteins that direct axonal outgrowth from chick DRG and Schwann cell adherence.     

Protein adsorption and cell attachment to patterned surfaces

To better understand the events involved in the generation of defined tissue architectures on biomaterials, we have examined the mechanism of attachment of human bone-derived cells (HBDC) to surfaces with patterned surface chemistry in vitro. Photolithography was used to generate alternating domains of N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS) and dimethyldichlorosilane (DMS). At 90 min after seeding, HBDC were localized preferentially to the EDS regions of the pattern. Using sera specifically depleted of adhesive glycoproteins, this spatial organization was found to be mediated by adsorption of vitronectin (Vn) from serum onto the EDS domains. In contrast, fibronectin (Fn) was unable to adsorb in the face of competition from other serum components. These results were confirmed by immunostaining, which also revealed that both Vn and Fn were able to adsorb to EDS and DMS regions when coated from pure solution, i.e., in the absence of competition. In this situation, each protein was able to mediate cell adhesion across a range of surface densities. Cell spreading was constrained on the EDS domains, as indicated by cell morphology and the lack of integrin receptor clustering and focal adhesion formation. This spatial constraint may have implications for the subsequent expression of differentiated function.     

A probabilistic approach to measure the strength of bone cell adhesion to chemically modified surfaces

Patterned surfaces with alternating regions of amino silanes [N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS)] and alkyl silanes [dimethyldichlorosilane (DMS)] have been used to alter the kinetics of spatial distribution of cellsin vitro. In particular, we have previously observed the preferential spatial distribution of bone cells on the EDS regions of EDS/DMS patterned surfaces (10). In this study, we examined whether the mechanism of spatial distribution of cells on the EDS regions was adhesion mediated. Homogeneous layers of EDS and DMS were immobilized on quartz substrates and characterized by contact angle, X-ray photoelectron spectroscopy, and spectroscopic ellipsometry. The strength of bone cell attachment to the modified substrates was examined using a radial flow apparatus, within either 20 min or 2 hr of cell incubation in the presence of serum. A Weibull distribution was chosen to characterize the strength of cell-substratum adhesion. Within 20 min of cell exposure, the strength of adhesion was significantly larger on EDS and clean surfaces, compared with DMS surfaces (p<0.0001). Within 2 hr of cell incubation, there was no statistical difference between the strength of cell adhesion to EDS, DMS, and clean surfaces. The results of this study suggest that the surface chemistry mediates adhesion-based spatial cell arrangement through a layer of adsorbed serum proteins.