Rapid Generation of Cell Gradients by Utilizing Solely Nanotopographic Interactions on a Bio‐Inert Glass Surface

The control of cell gradients is critical for understanding many biological systems and realizing the unique functionality of biomimetic implants. Herein, we report a nanotopographic gradient strategy that can rapidly generate cell gradients on a nanodendritic silica substrate without any chemical modification. We can achieve controllable cell gradients within only half an hour of cell incubation solely induced by the topographic effect of the gradient nanodendrites. We also demonstrate that cell gradients can be modulated by the combination of nanotopographic and chemical gradients. The results reveal that the enhanced topographic interactions between the nanodentritic structure and nanoscaled filopodia of the cells mainly contribute to the generation of cell gradients.     

Engineering Substrate Topography at the Micro‐ and Nanoscale to Control Cell Function

The interaction of mammalian cells with nanoscale topography has proven to be an important signaling modality in controlling cell function. Naturally occurring nanotopographic structures within the extracellular matrix present surrounding cells with mechanotransductive cues that influence local migration, cell polarization, and other functions. Synthetically nanofabricated topography can also influence cell morphology, alignment, adhesion, migration, proliferation, and cytoskeleton organization. We review the use of in vitro synthetic cell–nanotopography interactions to control cell behavior and influence complex cellular processes, including stem‐cell differentiation and tissue organization. Future challenges and opportunities in cell–nanotopography engineering are also discussed, including the elucidation of mechanisms and applications in tissue engineering.     

Cell Electrophoresis in Percoll Density Gradients

The existence of surface charges on living cells was recognized very early by Jurgensen in 1860 when he reported a migration of erythrocytes toward the anode(1). Since then, numerous attempts at qualitatively or quantitatively describing these charges have been made, which are now generally accepted as arising from the carboxyls of sialic acid residues(2). But because of serious theoretical and practical limitations of previous designs, these surface charges are not often measured.     

Electrospun Alginate Nanofibers with Controlled Cell Adhesion for Tissue Engineeringa

Alginate, a natural polysaccharide that has shown great potential as a cell scaffold for the regeneration of many tissues, has only been nominally explored as an electrospun biomaterial due to cytotoxic chemicals that have typically been used during nanofiber formation and crosslinking. Alginate cannot be electrospun by itself and is often co‐spun with poly(ethylene oxide) (PEO). In this work, a cell adhesive peptide (GRGDSP) modified alginate (RA) and unmodified alginate (UA) were blended with PEO at different concentrations and blending ratios, and then electrospun to prepare uniform nanofibers. The ability of electrospun RA scaffolds to support human dermal fibroblast cell attachment, spreading, and subsequent proliferation was greatly enhanced on the adhesion ligand‐modified nanofibers, demonstrating the promise of this electrospun polysaccharide material with defined nanoscale architecture and cell adhesive properties for tissue regeneration applications.

     

调控细胞迁移和组织再生的生物材料研究

组织再生材料为细胞、组织的生长提供必要的物质基础,维持再生组织的形状和力学性能,并实现与周围组织的有机整合。其中,材料-细胞的相互作用是组织再生材料的核心问题。组织再生材料表界面的物理结构和化学性能可以直接影响细胞的黏附、铺展、增殖、迁移和分化等行为,进而影响组织修复和再生的效果。多数组织和器官具有立体结构,并具有更为精细的微结构。因此,三维组织再生材料体系的构建及其微结构调控是另外一个重要问题。本文结合本课题组近年的工作,综合国内外最新研究成果,重点介绍了生物材料表界面物理结构和理化性质对微粒吞噬、细胞黏附的影响、梯度材料对细胞黏附和定向迁移的作用、3D水凝胶中的细胞迁移行为及特点,以及用于皮肤和软骨组织修复与再生的植入材料,最后对生物材料在组织再生中的研究与应用进行了展望。     

Development of a Piezoelectric PVDF‐TrFE Fibrous Scaffold to Guide Cell Adhesion,Proliferation, and Alignment

Severe peripheral nervous system injuries currently hold limited therapeutic solutions. Existing clinical techniques such as autografts, allografts, and newer nerve guidance conduits have shown variable outcomes in functional recovery, adverse immune responses, and in some cases low or minimal availability. This can be attributed in part to the lack of chemical, physical, and electrical cues directing both nerve guidance and regeneration. To address this pressing clinical issue, electrospun nanofibers and microfibers composed of piezoelectric polyvinylidene flouride‐triflouroethylene (PVDF‐TrFE) have been introduced as an alternative template for tissue engineered biomaterials, specifically as it pertains to their relevance in soft tissue and nerve repair. Here, biocompatible scaffolds of PVDF‐TrFE are fabricated and their ability to generate an electrical response to mechanical deformations and produce a suitable regenerative microenvironment is examined. It is determined that 20% (w/v) PVDF‐TrFE in (6:4) dimethyl formamide (DMF):acetone solvent maintains a desirable piezoelectric coefficient and the proper physical and electrical characteristics for tissue regeneration. Further, it is concluded that scaffolds of varying thickness promoted the adhesion and alignment of Schwann cells and fibroblasts. This work offers a prelude to further advancements in nanofibrous technology and a promising outlook for alternative, autologous remedies to peripheral nerve damage.     

Polymethacrylate Networks as Substrates for Cell Culture

Summary: methacrylate networks have a long history of applications in medical technology and much is known of their non-fouling properties. However, in recent times it has become clear that the swollen nature of these materials may provide some advantages if they are used as scaffolds in tissue engineering. In general however these hydrogels are resistant to protein adsorption and human cells do not easily adhere. In this work we provide an overview of several strategies that are designed to improve the cell-adhesive properties of hydrogels while maintaining their useful properties, mainly ease of diffusion of nutrients and growth factors. We describe our early attempts at modifying hydrogels based on 2,3-propandiol -1-methacrylate, with either hydrophobic units or acid groups. Modification with lauryl methacrylate produced an improvement but acid modification failed to provide surfaces that were conducive to cell culture. Much better scaffolds were prepared by amination of epoxy functional 2,3-propandiol-1-methacrylate networks. Optimized materials in this class were shown to be good substrates for the co-culture of bovine keratocytes with human corneal epithelial cells. We also describe the synthesis and biological properties of methacrylate conetworks, which phase separate during synthesis to give porous amphiphilic materials. Optimization of these materials produces materials that perform as well as tissue culture plastic so that confluent sheets of human dermal fibroblasts can be produced using standard culture techniques.     

Polyelectrolytes Multilayers to Modulate Cell Adhesion: A Study of the Influence of Film Composition and Polyelectrolyte Interdigitation on the Adhesion of the A549 Cell Line

Polyelectrolyte multilayers (PEMs) with different polycation/polyanion pairs are fabricated by the layer‐by‐layer technique employing synthetic, natural, and both types of polyelectrolytes. The impact of the chemical composition of PEMs on cell adhesion is assessed by studying cell shape, spreading area, focal contacts, and cell proliferation for the A549 cell line. Cells exhibit good adhesion on PEMs containing natural polycations and poly(sodium 4‐styrenesulfonate) (PSS) as polyanion, but limited adhesion is observed on PEMs fabricated from both natural polyelectrolytes. PEMs are then assembled, depositing a block of natural polyelectrolytes on top of a stiffer block with PSS as polyanion. Cell adhesion is enhanced on top of the diblock PEMs compared to purely natural PEMs. This fact could be explained by the interdigitation between polyelectrolytes from the two blocks. Diblock PEM assembly provides a simple means to tune cell adhesion on biocompatible PEMs.

     

Vapor‐Based Polymer Gradients

Chemical vapor deposition (CVD) co‐polymerization was used to fabricate polymer coatings, which comprise of reactive surface composition gradients. Two functionalized derivatives of [2.2]paracyclophane were fed into a two‐source CVD system at a 180 ° angle, then copolymerized and deposited as a polymer gradient. Infrared and X‐ray photoelectron spectroscopy (XPS) confirmed the compositional changes within the bulk polymer and at the surface. By manipulating process parameters, gradients of tailored compositional slope can be deposited on a wide range of substrates. We also were able to selectively immobilize fluorescence‐labeled ligands onto the reactive polymer gradients, making CVD‐based gradient surfaces a flexible platform for fabricating biomolecular substrates.

     

基于纳米金密度的物理-化学双重梯度构建及其用于神经元轴突响应研究

神经元轴突分化和生长等行为受到物理和化学梯度调控。本文通过调节纳米金和基底之间的静电作用及化学因子在基底上的吸附,在玻璃基底上构建了高分辨率的纳米金密度梯度及物理-化学双重梯度,用来研究海马神经元轴突导向和生长规律。统计结果显示,轴突更倾向沿着纳米金密度增加的方向生长,表明纳米金密度梯度对轴突生长具有导向作用。引入化学因子梯度后,神经元轴突分化及生长等行为受到物理-化学梯度协同调控。本文建立的方法及研究结果可望为神经元发育和修复研究提供重要信息以及有力的工具。     

Interaction of hepatocytes with polyelectrolyte complex(I): the effect of nonspecific interaction on adhesion of the cell

The adhesion of hepatocytes to polyelectrolyte complex (PEC), prepared by mixing of aqueous solutions of polycation and polyanion, is discussed. Four PECs, poly((dimethyliminio)ethylene(dimethyliminio)methylene‐1,4‐phenylenemethylene dichloride)–poly(acrylic acid)2X–(PAA), 2X‐poly(acrylic acid–co‐2‐ethylhexylacrylate(COA), 2X‐poly(acrylicacid–co‐butylacrylate) (CBA) and 2X‐poly(acrylic acid–co‐laurylacrylate)(CLA) were prepared. Hydrophobic properties of these PECs increased in the order of 2X–PAA < 2X–COA < 2X–CLA ≤ 2X–CBA. About all the hepatocytes adhered rapidly to various PECs even in the absence of serum, while fewer cells adhered to polystrene (tissue culture grade) dishes. At 37 °C (biological condition) about 70–80% of cells adhered to a collagen‐coated dish, but at 4 °C (nonbiological condition), no cell adhered to it. Nonactive cells (prepared by a single cycle of freezing and thawing) did not adhere to collagen, either. On the contrary about 40% of cells adhered to PEC‐coated dishes even at 4 °C, and nonactive cells also adhered to them. Cytocharosine B and colchicine, which are known as inhibitors of the polymerization of intracellular matrix, did not prevent cell adhesion to PECs. From these results it was suggested that hepatocytes adhered to PEC‐coated dishes mainly through a nonbiological interaction. Copyright © 1999 John Wiley & Sons, Ltd.     

Generation of Large‐Scale DNA Hydrogels with Excellent Blood and Cell Compatibility

Hemocompatibility and cytocompatibility of biomaterials codetermine the success of tissue engineering applications. DNA, the natural component of our cells, is an auspicious biomaterial for the generation of designable scaffolds with tailorable characteristics. In this study, a combination of rolling circle amplification and multiprimed chain amplification is used to generate hydrogels at centimeter scale consisting solely of DNA. Using an in vitro rotation model and fresh human blood, the reaction of the hemostatic system on DNA hydrogels is analyzed. The measurements of hemolysis, platelets activation, and the activation of the complement, coagulation, and neutrophils using enzyme‐linked immunosorbent assays demonstrate excellent hemocompatibility. In addition, the cytocompatibility of the DNA hydrogels is tested by indirect contact (agar diffusion tests) and material extract experiments with L929 murine fibroblasts according to the ISO 10993‐5 specifications and no negative impact on the cell viability is detected. These results indicate the promising potential of DNA hydrogels as biomaterials for versatile applications in the field of regenerative medicine.

     

Regulation of Stem Cell Differentiation by Control of Retinoic Acid Gradients in Hydrospun 3D Scaffold

Morphogen gradients have been associated with differential gene expression and are implicated in the triggering and regulation of developmental biological processes. This study focused on creating morphogenic gradients through the thickness of hydrospun scaffolds. Specifically, electrospun poly(ε‐caprolactone) fibers were loaded with all‐trans‐retinoic acid (ATRA), and designed to release ATRA at a predetermined rate. Multilayered scaffolds designed to present varied initial ATRA concentrations were then exposed to flow conditions in a bioreactor. Gradient formation was verified by a simple convection‐diffusion mathematical model approving establishment of a continuous solute gradient across the scaffold. The biological value of the designed gradients in scaffolds was evaluated by monitoring the fate of murine embryonal carcinoma cells embedded within the scaffolds. Cell differentiation within the different layers matched the predictions set forth by the theoretical model, in accordance with the ATRA gradient formed across the scaffold. This tool bears powerful potential in establishing in vitro simulation models for better understanding the inner workings of the embryo.

     

Switchable Substrates for Analyzing and Engineering Cellular Functions

Cellular activity is highly dependent on the extracellular environment, which is composed of surrounding cells and extracellular matrices. This focus review summarizes recent advances in chemically and physically engineered switchable substrates designed to control such cellular microenvironments by application of an external stimulus. Special attention is given to their molecular design, switching strategies, and representative examples for bioanalytical and biomedical applications.     

生物阴极微生物燃料电池不同阴极材料产电特性

以葡萄糖(COD初始浓度为2000 mg/L, COD为化学需氧量)为阳极燃料底物, 考察了碳纤维刷和柱状活性碳颗粒作为生物阴极微生物燃料电池(MFC)阴极材料的产电性能. 研究结果表明, 碳纤维刷MFC的启动时间比碳颗粒MFC的长, 达到稳定状态后的恒负载(300 Ω)电压(0.324 V)比碳颗粒阴极MFC的(0.581 V)低. 极化分析结果表明, 碳纤维刷MFC和碳颗粒MFC的最大功率密度分别为24.7 W/m3(117.2 A/m3)和50.3 W/m3(167.2 A/m3). 电化学交流阻抗谱(EIS)测定结果表明, 由于电极材料对微生物生长和分布状态存在不同的影响, 使得碳纤维刷阴极MFC的极化内阻大于碳颗粒阴极MFC的极化内阻.     

Analysis of Static Molecular Gradients in a High-Throughput Drug Screening Microfluidic Assay

In this study, we thoroughly analyzed molecular gradient generation, its stability over time, and linearity in our high-throughput drug screening microfluidic assay (HTS). These parameters greatly affect the precision and accuracy of the device’s analytical protocol. As part of the research, we developed a mathematical model of dependence of the concentration profile on the initial concentrations of active substances in reservoirs and the number of tilts, as well as the dependence of the active substance concentration profiles in the culture chambers on the concentration profile of the reference dye in the indicator chamber. The mean concentration prediction error of the proposed equations ranged from 1.4% to 2.4% for the optimized parameters of the procedure and did not increase with the incubation time. The concentration profile linearity index, Pearson’s correlation coefficient reached −0.997 for 25 device tilts. The observed time stability of the profiles was very good. The mean difference between the concentration profile after 5 days of incubation and the baseline profile was only 7.0%. The newly created mathematical relationships became part of the new HTS biochip operating protocols, which are detailed in the article.     

Hydrogels with Dynamically Controllable Mechanics and Biochemistry for 3D Cell Culture Platforms

Many cell-matrix interaction studies have proved that dynamic changes in the extracellular matrix(ECM)are crucial to maintain cellular properties and behaviors.Thus,developing materials that can recapitulate the dynamic attributes of the ECM is highly desired for threedimensional(3 D)cell culture platforms.To this end,we sought to develop a hydrogel system that would enable dynamic and reversible turning of its mechanical and biochemical properties,thus facilitating the control of cell culture to imitate the natural ECM.Herein,a hydrogel with dynamic mechanics and a biochemistry based on an addition-fragmentation chain transfer(AFCT)reaction was constructed.Thiol-modified hyaluronic acid(HA)and allyl sulfide-modifiedε-poly-L-lysine(EPL)were synthesized to form hydrogels,which were non-swellable and biocompatible.The reversible modulus of the hydrogel was first achieved through the AFCT reaction;the modulus can also be regulated stepwise by changing the dose of UVA irradiation.Dynamic patterning of fluorescent markers in the hydrogel was also realized.Therefore,this dynamically controllable hydrogel has great potential as a 3 D cell culture platform for tissue engineering applications.     

Evaluation of a Gelatin-Modified Poly(ε-Caprolactone) Film as a Scaffold for Lung Disease

Lung transplantation is a necessary step for the patients with the end stage of chronic obstructive pulmonary disease. The use of artificial lungs is a promising alternative to natural lung transplantation which is complicated and is restricted by low organ donations. For successful lung engineering, it is important to choose the correct combination of specific biological cells and a synthetic carrier polymer. The focus of this study was to investigate the interactions of human lung epithelial cell line NCl-H292 that is involved in lung tissue development with the biodegradable poly(ε-caprolactone) before and after its chemical modification to evaluate potential for use in artificial lung formation. Also, the effect of polymer chemical modification on its mechanical and surface properties has been investigated. The poly(ε-caprolactone) surface was modified using aminolysis followed by immobilization of gelatin. The unmodified and modified polymer surfaces were characterized for roughness, tensile strength, and NCl-H292 metabolic cell activity. The results showed for the first time the possibility for NCI-H292 cells to adhere on this polymeric material. The resazurin assay showed that the metabolic activity at 24?h postseeding of 80% in the presence of the unmodified and greater than 100% in the presence of the modified polymer was observed. The roughness of the poly(ε-caprolactone) increased from 4 to 26?nm and the film strength increased from 0.01 to 0.045 kN when the material was chemically modified. The results obtained to date show potential for using modified poly(ε-caprolactone) as a scaffold for lung tissue engineering.     

Preparation of Gradient Surfaces by Using a Simple Chemical Reaction and Investigation of Cell Adhesion on a Two‐Component Gradient

This article describes a simple method for the generation of multicomponent gradient surfaces on self‐assembled monolayers (SAMs) on gold in a precise and predictable manner, by harnessing a chemical reaction on the monolayer, and their applications. A quinone derivative on a monolayer was converted to an amine through spontaneous intramolecular cyclization following first‐order reaction kinetics. An amine gradient on the surface on a scale of centimeters was realized by modulating the exposure time of the quinone‐presenting monolayer to the chemical reagent. The resulting amine was used as a chemical handle to attach various molecules to the monolayer with formation of multicomponent gradient surfaces. The effectiveness of this strategy was verified by cyclic voltammetry (CV), matrix assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry (MS), MS imaging, and contact‐angle measurements. As a practical application, cell adhesion was investigated on RGD/PHSRN peptide/peptide gradient surfaces. Peptide PHSRN was found to synergistically enhance cell adhesion at the position where these two ligands are presented in equal amounts, while these peptide ligands were competitively involved in cell adhesion at other positions. This strategy of generating a gradient may be further expandable to the development of functional gradient surfaces of various molecules and materials, such as DNA, proteins, growth factors, and nanoparticles, and could therefore be useful in many fields of research and practical applications.