型胶原在聚-L-乳酸(PLLA)表面的化学接枝和涂层及其对软骨细胞生长的促进作用

采用接枝-涂层技术在聚-L-乳酸(PLLA)膜表面接枝聚甲基丙烯酸(PMAA)得到稳定的PLLA-g-PMAA胶原涂层.利用水溶性碳化二亚胺作为缩合剂,使PLLA-g-PMAA表面的羧基和胶原分子中的氨基发生缩合反应,从而将型胶原接枝在PLLA表面;同时保留材料表面物理涂层的胶原溶液,获得了稳定的胶原涂层.XPS谱图证实了接枝反应的发生.分别用比色法和茚三酮法测定了PLLA-g-PMAA表面的羧基密度和PLLA膜表面胶原的接枝量和涂层量.细胞培养结果表明,改性后的PLLA膜表面软骨细胞的铺展性能和增长速度明显改善.     

聚乳酸的层层自组装修饰及其内皮细胞相容性研究

通过胺解反应在生物降解聚(L-乳酸)表面引入带有正电荷的自由氨基,并通过静电吸引层层(Layer-by-layer,LBL)自组装技术将具有良好生物相容性的硫酸软骨素(CS)和细胞外基质成分型胶原组装到该PLLA材料表面.通过反应性荧光探针标记、紫外-可见吸收光谱以及荧光能量转移等测试技术跟踪并表征了自组装过程的进行.组装层的厚度开始随组装层数的增加而线性增加,而后增加变缓.内皮细胞的体外培养证明,表面组装CS和胶原(以胶原为最外层)以后,细胞的增殖率和细胞活性显著提高,材料的细胞相容性得到明显改善.细胞体现了充分铺展的多角形内皮细胞形貌,而且局部已融合形成了一单层内皮细胞层.     

New cartilage formation in vivo using chondrocytes seeded on poly(L-lactide)

A chondrocyte-poly(L-lactide) (PLLA) composite was employed in an attempt to regenerate cartilage. Rat chondrocytes showed good proliferation on adherent polymer substrates like PLLA and type II collagen production under the in vitro condition. The collagen production became maximal on the 10th day after cultivation. From the in vivo study, seeded chondrocytes spread throughout the scaffold dispersively and a lot of small blood vessels were observed around the matrix when bFGF was coated on the PLLA scaffold. When the chondrocyte-PLLA composite was transplanted into nude mouse, injection of an immunosuppressive agent was not necessary even in an xenograft implantation. A thin layer fibrous capsule was observed surrounding all of the implanted composites, and the inflammatory response of the host to the implants was mild. From the clear histological staining with thionin, it was obvious that the implanted cells exhibited their phenotypes and formed new matured cartilage.     

Preparation and biodegradation of electrospun PLLA/keratin nonwoven fibrous membrane

As a kind of natural protein, wool keratin was used to improve the cell affinity of poly(l-lactic acid) (PLLA). After small keratin particles were prepared from keratin solution by spray-drying process, they were blended with PLLA solution. PLLA/keratin nonwoven fibrous membrane was produced by electrospinning the blend solutions. The release rate of keratin from the composite membrane was detected by Fourier transform infrared (FTIR) after PLLA/keratin membranes were degraded in PBS up to 4 weeks. The chemical compositions of the PLLA/keratin surface were examined by X-ray photoelectron spectroscope. Although more than half of the keratin was removed from PLLA/keratin membrane during the first few hours of degradation, some keratin particles were still embedded in the PLLA fibers. Osteoblast cells were used to evaluate the cellular behaviors of the composite membrane. After 7 days culturing, more cells were observed on PLLA/keratin membranes than on pure PLLA membranes. MTT assay and alkaline phosphatase (ALP) activity results suggested that keratin could improve the interactions between osteoblast cells and the polymeric membranes.     

Length scale heterogeneity in lateral gradients of poly(N-isopropylacrylamide) polymer brushes prepared by surface-initiated atom transfer radical polymerization coupled with in-plane electrochemical potential gradients

We report the preparation and characterization of poly(N-isopropylacrylamide) (PNIPAAm) polymer brushes exhibiting controlled lateral variations in the patchiness of polymer chains. These gradients were achieved through an atom transfer radical polymerization (ATRP) grafting-from approach utilizing surfaces on which the spatial profile of the initiator density was carefully controlled. Initiator density gradients were formed on Au by first preparing a hexadecanethiol (HDT) density gradient, by reductive desorption using a laterally anisotropic electrochemical gradient. The bare areas in the original HDT gradient were then back-filled with a disulfide initiator, (BrC(CH3)2COO(CH2)11S)2. The initiator coverage was characterized by X-ray photoelectron spectroscopy (XPS). Then, surface-initiated ATRP was utilized to transfer the initiator density gradient into gradients of PNIPAAm chain density. Ellipsometry, surface plasmon resonance (SPR), and atomic force microscopy (AFM) were used to characterize these PNIPAAm density gradients. The defining characteristic of the PNIPAAm gradients is the evolution of the morphology from discontinuous mushroom structures at extremely low grafting densities to heterogeneous patchy structures at intermediate grafting densities. The size of the patchy domains gradually increases, until at a high grafting density region, the morphology evolves to a smoother, presumably more extended, structure.     

Morphological Characterization of Electrospun Nano-Fibrous Membranes of Biodegradable Poly(L-lactide) and Poly(lactide-co-glycolide)

Hydrophobic biodegradable polyesters, poly(L-lactide) (PLLA) and poly(lactide-co-glycolide) (PLGA), were electrospun on different types of collectors to induce morphological changes in the nanofibrous membrane. On the metal collector smooth nonwoven membranes were obtained for both PLLA and PLGA, while on the water reservoir the surface of the membranes became rough due to shrinkage and slow charge dissipation. When NaCl was added to water to enhance the conductivity, the roughness of the membrane surface was changed, yet the shrinkage remained relatively unchanged. The crystallization of PLLA electospun material on the metal plate was suppressed because of the rapid solvent evaporation, however, upon annealing above the glass transition temperature for 24 hr the PLLA membrane became crystallized. When electrospun on the water reservoir, the PLLA membrane remained amorphous. Crystalline PLLA was obtained by electrospinning on the methanol reservoir due to the swelling of nanofibers by methanol.     

Chitosan modified poly(L-lactide) microspheres as cell microcarriers for cartilage tissue engineering

The surfaces of poly(l-lactide) (PLLA) microspheres were modified by chitosan via a method of hydrolysis and grafting-coating to improve their compatibility to chondrocytes. The PLLA microspheres with a diameter of 74-150mum were fabricated by an oil/water emulsion solvent evaporation method, followed by hydrolysis in alkaline solution to produce a larger number of carboxyl groups. Using water-soluble carbodiimide as a coupling reagent, chitosan was covalently grafted onto the microspheres. Due to the physical entanglement and insolubility at neutral pH, unbonded chitosan molecules were stably remained to yield a large amount of coated chitosan. Biological performance of the control PLLA and the chitosan-coated PLLA microspheres were assessed by in vitro culture of rabbit auricular chondrocytes. After 24h and 7d culture, the chitosan-coated PLLA microspheres, especially the ones with larger chitosan amount, exhibited stronger ability to promote cell attachment and proliferation, and maintain the secretion function of the chondrocytes. Therefore, the chitosan-coated PLLA microspheres can be potentially used as the injectable cell microcarriers for chondrogenesis in cartilage tissue engineering.     

Cell adhesive and growth behavior on electrospun nanofibrous scaffolds by designed multifunctional composites

Nanostructured biocomposite scaffolds of poly(l-lactide) (PLLA) blended with collagen (coll) or hydroxyapatite (HA), or both for tissue engineering application, were fabricated by electrospinning. The electrospun scaffolds were characterized for the morphology, chemical and tensile properties by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), water contact angle (WCA), Fourier transform infrared (FTIR) measurement, and tensile testing. Electrospun biocomposite scaffolds of PLLA and collagen or (and) HA in the diameter range of 200-700 nm mimic the nanoscale structure of the extracellular matrix (ECM) with a well-interconnection pore network structure. The presence of collagen in the scaffolds increased their hydrophility, and enhanced cell attachment and proliferation, while HA improved the tensile properties of the scaffolds. The biocompatibility of the electrospun scaffolds and the viability of contacting cells were evaluated by 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) nuclear staining and by fluorescein diacetate (FDA) and propidium iodide (PI) double staining methods. The results support the conclusion that 293T cells grew well on composite scaffolds. Compared with pure PLLA scaffolds a greater density of viable cells was seen on the composites, especially the PLLA/HA/collagen scaffolds.     

Immobilized streptavidin gradients as bioconjugation platforms

Surface density gradients of streptavidin (SAV) were created on solid surfaces and demonstrated functionality as a bioconjugation platform. The surface density of immobilized streptavidin steadily increased in one dimension from 0 to 235 ng cm(-2) over a distance of 10 mm. The density of coupled protein was controlled by its immobilization onto a polymer surface bearing a gradient of aldehyde group density, onto which SAV was covalently linked using spontaneous imine bond formation between surface aldehyde functional groups and primary amine groups on the protein. As a control, human serum albumin was immobilized in the same manner. The gradient density of aldehyde groups was created using a method of simultaneous plasma copolymerization of ethanol and propionaldehyde. Control over the surface density of aldehyde groups was achieved by manipulating the flow rates of these vapors while moving a mask across substrates during plasma discharge. Immobilized SAV was able to bind biotinylated probes, indicating that the protein retained its functionality after being immobilized. This plasma polymerization technique conveniently allows virtually any substrate to be equipped with tunable protein gradients and provides a widely applicable method for bioconjugation to study effects arising from controllable surface densities of proteins.     

Vapor permeation of ethanol-water mixtures using sodium alginate membranes with crosslinking gradient structure

For the vapor permeation of ethanol-water mixtures, two types of dense sodium alginate (SA) membranes have been prepared: a nascent SA membrane and crosslinked SA membranes with glutaraldehyde (GA). In the vapor permeation of the concentrated ethanol-water mixtures through the SA membranes, the effects of feed temperature, cell temperature and crosslinking density in the membrane were investigated on the membrane performance, and a comparison of vapor permeation process was made with pervaporation. SA membranes having different crosslinking gradients have been fabricated by exposing the nascent membrane to different GA content of reaction solutions. The extent of the gradient was controlled by the exposing time. The permeation performance of the membranes will be discussed with the extent of the gradient. An optimal crosslinking gradient was determined in terms of flux and membrane stability. The separation of ethanol-water mixtures through the membrane with the optimal crosslinking gradient was carried out by vapor permeation and the permeation performance will be discussed, and compared with pervaporation.     

Growth cone response to ephrin gradients produced by microfluidic networks

A microfluidic network (μFN) etched into a silicon wafer was used to deliver protein solutions containing different concentrations of the axonal guidance molecule ephrinA5 onto a silicone stamp. In a subsequent microcontact printing (μCP) step, the protein was transferred onto a polystyrene culture dish. In this way, stepwise substrate-bound concentration gradients of ephrinA5 were fabricated spanning a total distance of 320 μm. We tested the response of chick retinal ganglion cell (RGC) axons, which are guided in vivo by ephrin gradients, to these in vitro gradients. Temporal, but not nasal axons stop at a distinct zone in the gradient, which is covered with a certain surface density of substrate-bound ephrinA5. Within the temporal RGC population, all axons respond uniformly to the gradients tested. The position of the stop zone depends on the slope of the gradient with axons growing further into the gradient in shallow gradients than in steep gradients. However, axons stop at lower ephrinA5 concentrations in shallow gradients than in steep gradients, indicating that the growth cone can adjust its sensitivity during the detection of a concentration gradient of ephrinA5. Susanne Lang and Anne C. von Philipsborn contributed equally to this work: S.L. performed the experiments; A.P. evaluated the data and wrote the paper.     

明胶/聚乳酸复合纤维膜的制备、表征及作为角膜细胞载体的评价

利用静电纺丝技术制备了明胶与聚乳酸的复合纤维膜, 研究了组分配比对复合膜的表面性能、孔隙结构和力学性能的影响, 并以复合膜为组织工程支架进行兔角膜上皮细胞的体外培养. 采用扫描电子显微镜、免疫荧光染色和噻唑蓝四氮唑溴化物(MTT)比色法综合评价了细胞在支架表面的黏附与增殖能力. 结果表明, 纺丝溶液的组分对纤维的直径分布和表面亲水性有显著影响, 不同组分配比的复合纤维膜均具有高孔隙率的通孔结构; 以明胶为基材可维持复合膜的细胞黏附性; 与聚乳酸复合可以明显提高复合膜的力学性能.     

High‐Throughput Method for Determining Modulus of Polymer Blends

Summary: A method for rapidly determining the modulus of polymer blends was developed. A polymer blend gradient library of poly(L ‐lactic acid) (PLLA) and poly(D ,L ‐lactic acid) (PDLLA) was created in the form of a strip‐shaped film and characterized with FTIR microspectroscopy. Nanoindentation measurements were made along the gradients to obtain modulus data over a wide range of PLLA‐PDLLA blend compositions. This novel, high‐throughput approach to material characterization provides engineers with a technique to accelerate the development of materials.

Deposition of the polymer composition gradient.     

Anisotropic hydrogel thickness gradient films derivatized to yield three-dimensional composite materials

We report the preparation of in-plane density gradients of amino-terminated molecules and gold particles through derivatization of laterally varying thickness gradients of poly(acrylic acid) (PAA) or poly(acrylamide) (PAAm) films. PAA and PAAm gradients were formed by Zn(II)-catalyzed electropolymerization of acrylic acid (AA) or acrylamide (AAm) in the presence of an in-plane electrochemical potential gradient applied to Au or indium-tin-oxide (ITO) working electrodes. PAA thickness gradients were converted into density gradients of fluorocarbons or biocompatible groups by derivatizing with NH(2)CH(2)(CF(2))(6)CF(3) or an Arg-Gly-Asp (RGD)-containing peptide, respectively. X-ray photoelectron spectroscopy (XPS) and XPS imaging were used to characterize the modified PAA gradients. Transition regions as narrow as 104 mum were achieved for fluorocarbon gradients. PAAm gradients were treated with gold particles to form a density gradient of gold particles. Surface plasmon resonance imaging and scanning electron microscopy (SEM) as well as UV-visible absorption measurements were used to characterize the gold particle density gradients. It is likely that the gold particles were attached both on the surface and inside the PAAm film.     

Endothelial cell migration on surface-density gradients of fibronectin, VEGF, or both proteins

Cell migration is essential to many physiological processes, including angiogenesis, which is critical to the success of implanted biomaterials and tissue-engineered constructs. Gradients play an important role in cell migration. Previous work on cell migration has been mostly executed either in the concentration gradients of stimuli (e.g., VEGF) in bulk or hydrogels or on the surface-density gradients of ECM proteins (e.g., fibronectin) or small ligands (e.g., RGD). Little work has been done to investigate how cell migration responds to the surface-density gradients of growth factors. No work has been done to study how the surface gradients of both adhesive proteins and growth factors influence cell migration. In this work, we studied the effect of the surface-density gradients of fibronectin (FN), VEGF, or both proteins on endothelial cell migration. Gradients with different slopes were prepared to study how the gradient slope affects cell migration. The gradients were generated by first forming a counter-propagating C15COOH/C11OH self-assembled monolayer (SAM) gradient using a surface electrochemistry approach, followed by activating the -COOH moieties and covalently immobilizing proteins onto the surface. Fourier transform infrared spectra and X-ray photoelectron spectroscopy were used to characterize the SAM and protein gradients, respectively. A free cell migration assay using bovine aortic endothelial cells was performed on various gradient surfaces or on surfaces with uniform protein density. Results showed that cells on the surface-density gradients of FN, VEGF, or both proteins moved faster along the gradient direction than on the respective uniform control surface after 24-h cell culture. It is also shown that for each protein or protein combination, the directional cell displacement was not statistically different between two gradients with different slopes. Results show that the directional cell migration was increased by about 2-fold on the VEGF gradient as compared to the FN gradient and was further increased by another 2-fold on the combined gradients of both proteins as compared to the VEGF gradient alone. This is the first work to create surface-density gradients of VEGF and the first study to generate a combined surface gradient of growth factor and ECM protein to investigate their effect on cell migration on surfaces. This work broadens our understanding of the directional movement of endothelial cells. Our findings provide useful information for directing cell migration into tissue-engineered constructs and can be potentially used for those applications where cell migration is critical, such as angiogenesis.     

Ultrathin gradient films using thiol‐ene polymerizations

The application of surface‐attached, thiol‐ene polymer films for controlling material properties in a gradient fashion across a surface was investigated. Thiol‐ene films were attached to the surface by first depositing a thiol‐terminated self‐assembled monolayer and performing a thiol‐ene photopolymerization reaction on the surface. Property gradients were created either by creating and modifying a gradient in the surface thiol density in the SAM or by changing the polymerization conditions or both. Film thickness was modified across the substrate by changing either the density of the anchoring thiol functional groups or by changing the reaction conditions such as exposure time. Thicker films (1–11 nm) were obtained by polymerizing acrylate polymer brushes from the surface with varying exposure time (0–60 s). The two factors, that is, the surface thiol density and the exposure time, were combined in orthogonal directions to obtain thiol‐ene films with a two‐dimensional thickness gradient with the maximum thickness being 4 nm. Finally, a thiol‐acrylate Michael type addition reaction was used to modify the surface thiol density gradient with the cell‐adhesive ligand, Arg‐Gly‐Asp‐Ser (RGDS), which subsequently yielded a gradient in osteoblast density on the surface. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 7027–7039, 2006     

Controlling adhesion force by means of nanoscale surface roughness

Control of adhesion is a crucial aspect in the design of microelectromechanical and nanoelectromechanical devices. To understand the dependence of adhesion on nanometer-scale surface roughness, a roughness gradient has been employed. Monomodal roughness gradients were fabricated by means of silica nanoparticles (diameter ～12 nm) to produce substrates with varying nanoparticle density. Pull-off force measurements on the gradients were performed using (polyethylene) colloidal-probe microscopy under perfluorodecalin, in order to restrict interactions to van der Waals forces. The influence of normal load on pull-off forces was studied and the measured forces compared with existing Hamaker-approximation-based models. We observe that adhesion force reaches a minimum value at an optimum particle density on the gradient sample, where the mean particle spacing becomes comparable with the diameter of the contact area with the polyethylene sphere. We also observe that the effect on adhesion of increasing the normal load depends on the roughness of the surface.     

Construction of a tethered poly(ethylene glycol) surface gradient for studies of cell adhesion kinetics

Surface gradients can be used to perform a wide range of functions and represent a novel experimental platform for combinatorial discovery and analysis. In this work, a gradient in the coverage of a surface-immobilized poly(ethylene glycol) (PEG) layer is constructed to interrogate cell adhesion on a solid surface. Variation of surface coverage is achieved by controlled transport of a reactive PEG precursor from a point source through a hydrated gel. Immobilization of PEG is achieved by covalent attachment of the PEG molecule via direct coupling chemistry to a cystamine self-assembled monolayer on gold. This represents a simple method for creating spatial gradients in surface chemistry that does not require special instrumentation or microfabrication procedures. The structure and spatial distribution of the PEG gradient are evaluated via ellipsometry and atomic force microscopy. A cell adhesion assay using bovine arteriole endothelium cells is used to study the influence of PEG thickness and chain density on biocompatibility. The kinetics of cell adhesion are quantified as a function of the thickness of the PEG layer. Results depict a surface in which the variation in layer thickness along the PEG gradient strongly modifies the biological response.     

紫外辐射接枝法修饰聚乳酸表面及其细胞相容性

通过紫外辐射接枝在聚乳酸膜表面引入聚丙烯酸的方法使聚乳酸材料表面的亲水性和细胞相容性得到改善,研究了各种处理条件对材料表面的羧基密度、表面形态和表面接触角的影响,同时还考察了紫外辐射接枝聚丙烯酸的聚乳酸表面的成骨细胞相容性.红外光谱分析和羧基密度测定结果表明:通过紫外光引发接枝,聚丙烯酸被成功接枝到聚乳酸表面,而且接枝密度受接枝时间和聚丙烯酸质量分数的影响很大.接触角和原子力显微镜研究结果表明:接枝聚丙烯酸后的聚乳酸表面的亲水性和粗糙度明显增加,能够促进成骨细胞的生长.     

Cell migration and polarity on microfabricated gradients of extracellular matrix proteins

This paper explores the effects of the surface density and concentration profiles of extra cellular matrix proteins on the migration of rat intestinal IEC-6 cells. Microfluidic devices were used to create linear, immobilized gradients of laminin. This study investigated both the impact of the steepness and local concentrations on the directedness of cell migration. The bulk concentrations of proteins in the feed streams in the mixing device determined the gradient profile and the local concentration of laminin in the device. Two sets of gradients were used to explore cell migration directedness: (i) gradients with similar change in local concentration, i.e., the same gradient steepness, and (ii) different gradients with similar local concentrations. Cells migrated up the gradients, independent of the steepness of the gradients used in this study. At the same local laminin concentration, the migration rate was independent of the gradient steepness. However, cell directedness decreased significantly at high laminin densities.