Micropatterned surfaces modified with select peptides promote exclusive interactions with osteoblasts

Microcontact printing techniques were used to pattern circles (diameters 10. 50, 100, and 200 microm) of N1[3-(trimethoxysilyl)-propyl]diethylenetriamine (DETA) surrounded by octadecyltrichlorosilane (OTS) borders on borosilicate glass, a model substrate. The DETA regions were further modified by immobilization of either the cell-adhesive peptides Arginine-Glycine-Aspartic Acid-Serine (RGDS) and Lysine-Arginine-Serine-Arginine (KRSR) or the non-adhesive peptides Arginine-Aspartic Acid-Glycine-Serine (RDGS) and Lysine-Serine-Serine-Arginine (KSSR). After four hours under standard cell culture conditions but in the absence of serum, adhesion of either osteoblasts or fibroblasts on surfaces patterned with the non-adhesive peptides RDGS and KSSR was random and low. In contrast, both osteoblasts and fibroblasts adhered and formed clusters onto circles modified with the adhesive peptide RGDS, whereas only osteoblasts adhered and formed clusters onto the circles modified with KRSR, a peptide that selectively promotes adhesion of osteoblasts. These results provide evidence that patterning of select peptides can direct adhesion of specific cell lines exclusively to predetermined regions on material surfaces.     

Micropatterned hot-embossed polymeric surfaces influence cell proliferation and alignment

Micropatterning is a powerful technique to custom-make and precisely control the surface topography of materials, which is determinant for a better interaction with cells. A modification of conventional micropatterning is proposed here to fabricate textured film from stiff and sticky polymers such as poly(lactide(s)-co-glycolide(s)) (PLGA) without the use of supports or solvents. Micropatterned PLGA films with square pits varying in height and channels varying in width were made to study the influence of these topographical parameters on human fibroblasts proliferation, morphology, and alignment. With increasing the square pit height, the cell attachment efficiency increased. After 10 days of culture the micropatterned films supported a significantly higher cell proliferation than smooth films. In particular, cell growth was highly stimulated in 150-mum-wide channels. Fibroblasts were spread with a typical spindle shape in all the films. Cell spreading increased with increasing the textured dimensions. A random cell organization was found for smooth and for square pit samples, and a high alignment was observed along the 150-mum-wide channels. Smaller and bigger channels did not support substantial cell growth, suggesting a possible "recognition" mechanism of the cells for optimal organization. These findings could be useful in tissue engineering applications where higher proliferation rates and eventual random or unidimensional alignments of cells are desirable.     

Micropatterned surfaces for controlling cell adhesion and rolling under flow

Cell adhesion and rolling on the vascular wall is critical to both inflammation and thrombosis. In this study we demonstrate the feasibility of using microfluidic patterning for controlling cell adhesion and rolling under physiological flow conditions. By controlling the width of the lines (50–1000 μm) and the spacing between them (50–100 μm) we were able to fabricate surfaces with well-defined patterns of adhesion molecules. We demonstrate the versatility of this technique by patterning surfaces with 3 different adhesion molecules (P-selectin, E-selectin, and von Willebrand Factor) and controlling the adhesion and rolling of three different cell types (neutrophils, Chinese Hamster Ovary cells, and platelets). By varying the concentration of the incubating solution we could control the surface ligand density and hence the cell rolling velocity. Finally by patterning surfaces with both P-selectin and von Willebrand Factor we could control the rolling of both leukocytes and platelets simultaneously. The technique described in this paper provides and effective and inexpensive way to fabricate patterned surfaces for use in cell rolling assays under physiologic flow conditions.     

Micropatterned surfaces of PDMS as growth templates for HEK 293 cells

In this paper the easy and reliable preparation of precise micropatterns on PDMS surfaces is described and the growth of HEK 293 cells on those patterns during culture over several days is examined. The first patterning approach described is based on soft-lithography and polyelectrolyte multilayer deposition. Two different soft-lithographic techniques are employed for creating surface patterns of PAH, PSS, untreated and oxidized PDMS. The growth behavior of HEK 293 cells is investigated on all the dual combinations of the four surfaces, and decreasing preference of the cells for the surfaces in the order PAH (–NH₂) > ox-PDMS (–OH) >> PSS (–SO₃ ⁻) > PDMS (–CH₃) is revealed. As the second patterning approach a method is introduced, which allows the deposition of gel droplets in a microarray format utilizing differences in the surface wettability. This concept is new and expected to be very useful for various applications. Finally, a speculative explanation for the different cell spreading behavior is provided considering the interplay between individual cell–surface interactions and a permanent cell tractional force.     

Micropatterned surfaces prepared using a liquid crystal projector-modified photopolymerization device and microfluidics

A commercial liquid crystal device projector was modified for photopolymerization using its on-board intense light source and a precision optical control circuit. This device projects reduced images generated by a typical personal computer onto the stage where photopolymerization on a surface occurs. This all-in-one device does not require expensive photomasks and external light sources. However, light scattering and diffraction through glass substrates resulted in undesired reactions in areas corresponding to masked (black) domains in mask patterns, limiting pattern resolution. To overcome this shortcoming, two-step surface patterning was developed. First, three-dimensional microstructures of crosslinked silicone elastomer were fabricated with this device and adhered onto silanized glass substrate surfaces, forming microchannels in patterns on the glass support. Then, acrylamide monomer solution containing photoreactive initiator was flowed into these micromold channels and reacted in situ. The resultant polyacrylamide layer was highly hydrophilic and repelled protein adsorption. Cell seeding on these patterns in serum-supplemented culture medium produced cells selectively adhered to different patterns: cells attached and spread only on unpolymerized silanized glass surfaces, not on the photopolymerized acrylamide surfaces. This technique should prove useful for inexpensive, rapid prototyping of surface micropatterns from polymer materials.     

Evaluation of a peptide motif designed for protein tethering to polymer surfaces

Abstract

In search for peptide motifs that allow us to efficiently tether fusion proteins onto polymer surfaces, we designed a KLKLKLKLKL (KL5) decapeptide in which basic and hydrophobic amino acids were alternately linked. By means of genetic engineering technology together with a bacterial expression system, the KL5 fusions of epidermal growth factor (EGF), basic fibroblast growth factor, and stromal cell-derived factor-1α were prepared together with their control counterparts without KL5. The adsorption experiments were performed for these fusion proteins on the surface of polystyrene, hydrophilized polystyrene, and polycaprolactone by surface plasmon resonance analysis. To understand the results of the binding assays, the structure of the fusion proteins was predicted by ab initio computer simulation and analyzed empirically by circular dichroism spectroscopy. The result of structural analyses suggested that the KL5 peptide is exposed to the outside and has a negligible effect on the structure of the protein partners. However, it was found that the efficiency of KL5 as a peptide motif greatly depends on protein partners. Our results showed that KL5 exerts most effectively its function as a peptide motif when fused to acidic proteins such as EGF. Indeed, the number of living human mesenchymal stem cells determined after 7-day culture was larger on the polystyrene and polycaprolactone surfaces with EGF tethered through the KL5 peptide than control surfaces. According to the results obtained in this study, we conclude that KL5 is useful as a peptide motif for tethering a specific class of protein partners.     

Spatially Guided Angiogenesis by Three-Dimensional Collagen Scaffolds Micropatterned with Vascular Endothelial Growth Factor

Abstract

Successful regeneration of large and highly functionalized tissue and organs depends on the ability to guide blood vessel formation with three-dimensional scaffolds. Angiogenic growth factors have the potential to stimulate blood vessels in scaffolds. However, simply incorporating angiogenic growth factors in a random fashion may lead to uncontrolled blood vessel generation, which ultimately results in poor blood vessel network function and uneven growth of engineered tissue. To control and guide the formation of a blood vessel network in porous scaffolds, we prepared collagen sponges with micropatterned vascular endothelial growth factor (VEGF). VEGF was micropatterned in three-dimensional collagen sponges using micropatterned collagen/VEGF ice lines, which were prepared by a dispersing machine. The VEGF-micropatterned collagen sponges were implanted subcutaneously in nude mice. Following 6 weeks of implantation, the VEGF-micropatterned collagen sponges induced the formation of micropatterned blood vessel networks. More blood vessels were observed in the regions in which VEGF was immobilized than those without VEGF. The micropattern of VEGF determined the micropattern of the regenerated blood vessel network. The spatial immobilization of VEGF in three-dimensional porous scaffolds may be useful to stimulate guided blood vessel formation in a variety of tissue-engineering applications.     

Fasciculation and Defasciculation of Neurite Bundles on Micropatterned Substrates

We describe experiments of fasciculation, i.e., bundling, of chick sensory neurites on 2D striped substrates. By Fourier decomposition, we separate left-going and right-going neurite components from in vitro images, and we find first that neurite bundles orient toward preferred angles with respect to the stripe direction, and second that in vitro bundles travel in leftward and rightward directions nearly uninterrupted by crossings of bundles traveling in the opposing direction. We explore mechanisms that lead to these behaviors, and summarize implications for future models for neurite outgrowth and guidance.     

Endothelial cell interactions with granulocytes: tethering and signaling molecules.

The adhesion of granulocytes to endothelial cells requires regulated expression of molecules on both the endothelial cell and the granulocyte. These pro-adhesive molecules have diverse structures and mechanisms of expression, and act either to tether the two cells together or as signals that induce activation-dependent adhesion events. Combinations of tethering and signaling molecules regulate endothelial-cell-granulocyte interactions at the endothelial surface.     

Micropatterned assembly of silica nanoparticles for a protein microarray with enhanced detection sensitivity

We used an assembly of silica nanoparticles (SNPs) as a three-dimensional template for protein immobilization to prepare a protein microarray with enhanced protein loading capacity and detection sensitivity. SNPs were first modified with 3-aminopropyltriethoxysilane (APTES) for covalent immobilization of protein and micropatterned on poly(ethylene glycol)(PEG)-coated glass slides using elastomeric membranes with an array of holes. Proteins were selectively immobilized only on the SNP region, while the PEG regions served as an effective barrier to protein adsorption. Because of multi-layered SNPs that had curved surface, protein loading in the SNP micropattern was about six times greater than on a planar surface, as observed by fluorescence microscopy, which consequently improved the protein activity and reaction rate. GOX-catalyzed glucose oxidation and the molecular recognition mediated, specific binding between biotin and streptavidin were both successfully assayed using SNP microarrays, with better fluorescence signal and sensitivity than corresponding planar microarrays.     

Micropatterned Structures for Studying the Mechanics of Biological Polymers

Studying the mechanics of nanometer-scale biomolecules presents many challenges; these include maintaining light microscopy image quality and avoiding interference with the laser used for mechanical manipulation, that is, optical tweezers. Studying the pushing forces of a polymerizing filament requires barriers that meet these requirements and that can impede and restrain nanoscale structures subject to rapid thermal movements. We present a flexible technique that meets these criteria, allowing complex barrier geometries with undercut sidewall profiles to be produced on #1 cover glass for the purpose of obstructing and constraining polymerizing filaments, particularly microtubules. Using a two-layer lithographic process we are able to separate the construction of the primary features from the construction of a depth and shape-controlled undercut. The process can also be extended to create a large uniform gap between an SU-8 photoresist layer and the glass substrate. This technique can be easily scaled to produce large quantities of shelf-stable, reusable microstructures that are generally applicable to microscale studies of the interaction of cellular structures with defined microscale features.     

Patterned surfaces as tools to study ligand recognition and synapse formation by T cells

Activation of immune cells is often achieved via ligand-receptor interactions occurring at a cell-cell junction known as an immunological synapse (IS). Synapse structures, probably best studied in the context of T cell-antigen presenting cell (APC) interactions, are characterized by clustering of cell surface receptors and intracellular signaling components, and in some settings, the formation of microscale or submicron patterns of receptors in the cell-cell interface. To help expand our understanding of how synapses form and function, substrates bearing patterned protein ligands are being developed as simplified models of the APC surface. These new tools allow well-defined signal inputs to be delivered to the T cell in order to ask how the physical organization and composition of APC-derived signals control T cell activation.     

Micropatterned cell co-cultures using layer-by-layer deposition of extracellular matrix components

Micropatterned cellular co-cultures were fabricated using three major extracellular matrix components: hyaluronic acid (HA), fibronectin (FN) and collagen. To fabricate co-cultures with these components, HA was micropatterned on a glass substrate by capillary force lithography, and the regions of exposed glass were coated with FN to generate cell adhesive islands. Once the first cell type was immobilized on the adhesive islands, the subsequent electrostatic adsorption of collagen to HA patterns switched the non-adherent HA surfaces to adherent, thereby facilitating the adhesion of a second cell type. This technique utilized native extracellular matrix components and therefore affords high biological affinity and no cytotoxicity. This biocompatible co-culture system could potentially provide a new tool to study cell behavior such as cell-cell communication and cell-matrix interactions, as well as tissue-engineering applications.     

The CHEVI tethering complex: facilitating special deliveries

VPS33B and VIPAR comprise the two known components of the recently christened class C Homologues in Endosome–Vesicle Interaction (CHEVI) complex, thought to act as a tethering complex in endosomal trafficking distinct from the HOPS and CORVET complexes in mammalian cells. A recent paper in The Journal of Pathology further explores the role of the CHEVI complex in the biogenesis of α‐granules in megakaryocytes, identifying two novel interactors of this complex: α‐tubulin and SEC22B, and demonstrating that VPS33B expression is required for the localization of SEC22B and the α‐granule cargo VWF to proplatelets in megakaryocytes. These findings advance the current knowledge of the function of the CHEVI complex in α‐granule biogenesis and together with studies in other systems, corroborate its role in the specialized delivery of cargo in different cell types. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

The heterogenization of tumour cells with tuberculin. I. The coupling of tuberculin to cell surfaces using con-A as ligand

The introduction of an antigenic determinant strongly recognized by T cells on to a cell enhances the immune response to weak cellular antigens. Tuberculin (PPD) is a particularly suitable antigenic determinant for this purpose since it behaves in many ways as a 'T-cell hapten'. It has been found that direct chemical coupling of PPD to cell surfaces damages their antigenicity, but that this can be circumvented by coupling PPD to the lectin Concanavalin A and using this as the ligand for binding PPD to the cell. Techniques for preparing Con-A/PPD using little glutaraldehyde or SPDP as cross-linking agents are described.     

Optically controlled ligand delivery, 1. Synthesis of water-soluble copolymers containing photocleavable bonds

To verify the possibility of developing a ligand delivery system which is controlled by light pulses, we synthesized copolymers of N-(2-hydroxypropyl)methacrylamide ( 1 ) containing sidechains terminated in ligands (Boc-Gly, fluorescein, tetramethylrhodamine) bound via photocleavable 2-nitrobenzyl groups. Copolymers in solution were exposed to light of wavelength ≈? 360 nm which resulted in release of the bound ligands. Depending on the experimental conditions (type of solvent, presence of oxygen) changes in the structure of released fluorochromes were observed (photofading effect). These effects were quantified by determining the binding constants of released modified fluorochromes with monoclonal antifluorescyl anti-bodies.     

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.     

Soluble CD40 ligand levels during controlled ovarian hyperstimulation--a possible culprit of systemic inflammation

AIM: To investigate the behavior and association of serum sex-steroids and serum CD40 ligand in patients undergoing controlled ovarian hyperstimulation (COH) for in vitro fertilization (IVF). DESIGN: Prospective, observational study. SETTING: The IVF unit of an academic medical center. PATIENTS AND METHODS: Blood was drawn three times during the COH cycle from 17 patients undergoing the long gonadotropin-releasing hormone-analog protocol: (i) day on which adequate suppression was obtained (Day-S); (ii) day of or prior to administration of human chorionic gonadotropin (Day-hCG); and (iii) day of ovum pick-up (Day-OPU). Levels of sex steroids and serum CD40 ligand were compared among the three time points. RESULTS: During gonadotropin treatment, serum ovarian sex steroids (estradiol, progesterone, free testosterone and androstenedione) significantly increased while CD40 ligand levels nonsignificantly decreased. After hCG administration, there was a significant increase in the levels of serum CD40 ligand, ovarian androgens, and progesterone, with a significant decrease in estradiol levels. No correlations were observed between CD40 ligand and ovarian sex-steroid levels or other treatment variables. CONCLUSION: The administration of hCG leads to activation of systemic inflammation, as reflected by CD40 ligand levels. This, in turn, may lead to the development of ovarian hyperstimulation syndrome via several mechanisms, including an increase in several angiogenic factors.     

MC3T3-E1成骨细胞在微格式化表面的黏附

细胞在材料表面的黏附对细胞的增殖和分化志重要作用。格式化表面提供了对细胞在基底的空间分布和黏附进行控制的方法。本文利用微制作利用微制作形成的格式模板，分别以微接触转印法和微流道法形成格式化表面，使MC3T3－E1成骨细胞以一定的格式黏附于表面上，在微接触转印法形成的含二氯二甲基硅烷（DMS）的疏水区域和不含DMS的亲水区域相间隔的表面，细胞优先在亲水区域黏附，在微流道法形成的胶原和白蛋白格式化表面，细胞优先黏附于含胶原区域，结果还表明微格式化表面可以用于研究表面的物理化学性质对细胞的黏附等功能的影响。