Enhancing the Activity of Surface Immobilized Antimicrobial Peptides Using Thiol-Mediated Tethering to Poly(ethylene glycol).

Considering the need for versatile surface coatings that can display multiple bioactive signals and chemistries, the use of more novel surface modification methods is starting to emerge. Thiol-mediated conjugation of biomolecules is shown to be quite advantageous for such purposes due to the reactivity and chemoselectivity of thiol functional groups. Herein, the immobilization of poly(ethylene glycol) (PEG) and antimicrobial peptides (AMPs) to silica colloidal particles based on thiol-mediated conjugation techniques, along with an assessment of the antimicrobial potential of the functionalized particles against Pseudomonas aeruginosa and Staphylococcus aureus is investigated. Immobilization of PEG to thiolated Si particles is performed by either a two-step thiol-ene "photo-click" reaction or a "one-pot" thiol-maleimide type conjugation using terminal acrylate or maleimide functional groups, respectively. It is demonstrated that both immobilization methods result in a significant reduction in the number of viable bacterial cells compared to unmodified samples after the designated incubation periods with the PEG-AMP-modified colloidal suspensions. These findings provide a promising outlook for the fabrication of multifunctional surfaces based upon the tethering of PEG and AMPs to colloidal particles through thiol-mediated biocompatible chemistry, which has potential for use as implant coatings or as antibacterial formulations that can be incorporated into wound dressings to prevent or control bacterial infections.

Biomimicking nanofibrous gelatin microspheres recreating the stem cell niche for their ex-vivo expansion and in-vivo like differentiation for injectable stem cell transplantation.

Stem cells based novel treatment modality for degenerative and immune dysfunction diseases created a huge demand of suitable carriers to support ex-vivo production of quality stem cells, and effective in-vivo transplantation of stem cells and their fate. In spite of promising candidature of nanofibrous microspheres (NFM) to recreate native stem cell niches to be used for possible scaling-up for ex-vivo stem cells expansion, it remains fairly unexplored. A systematic study on the stem cell-NFM interaction comparative with commercial microspheres (CM) has been performed for the first time. Gelatin NFM with variable physicochemical properties such as size, surface properties, surface chemistry, and variable degradability were prepared using microemulsion coupled with thermally induced phase separation (TIPS) method. Effect of physicochemical properties of NFM and their cellular interaction such as binding, morphology, metabolic activity and proliferation studies were performed using human bone marrow-derived mesenchymal stem cells (hBMSCs), human dental follicle stem cells (hDFSCs) and human gingival fibroblast (HGF) cells and compared with the commercial and solid microspheres. Gelatin NFM supports excellent cell binding, proliferation, metabolic activities and chemical cues specific differentiation. All out-turns indicate that NFM stand to be an outstanding candidate for ex-vivo cells' expansion and injectable carriers for stem cell transplantation.

Colloidal Self-Assembled Patterns Maintain the Pluripotency and Promote the Hemopoietic Potential of Human Embryonic Stem Cells.

The generation of blood cells in a significant amount for clinical uses is still challenging. Human pluripotent stem cells-derived hemopoietic cells (hPSC-HCs) are a promising cell source to generate blood cells. Previously, it has been shown that the attached substrates are crucial in the maintenance or differentiation of hPSCs. In this study, a new family of artificial extracellular matrix (ECM) called colloidal self-assembled patterns (cSAPs: #1-#5) was used for the expansion of mouse and human PSCs. The optimized cSAP (i.e., #4 and #5) was selected for subsequent hemopoietic differentiation of human embryonic stem cells (hESCs). Results showed that the hematopoietic potential of hESCs was enhanced approx 3-4 folds on cSAP #5 compared to the flat control. The cell population of hematopoietic progenitors (i.e., CD34+CD43+ cells) and erythroid progenitors (i.e., CD71+GPA+ cells) were enhanced 4 folds at day 8 and 3 folds at day 14. RNA sequencing analysis of cSAP-derived hESCs showed that there were 300 genes up-regulated and 627 genes down-regulated compared to the flat control. The enriched signaling pathways, including up-regulation (i.e., Toll-like receptor, HIF-1a, and Notch) or down-regulation (i.e., FAs, MAPK, JAK/STAT, and TGF-ß) were classic in the maintenance of hESC phenotype Real time PCR confirmed that the expression of focal adhesion (PTK2, VCL, and CXCL14) and MAPK signaling (CAV1) related genes was down-regulated 2-3 folds compared to the flat control. Altogether, cSAP enhances the pluripotency and the hematopoietic potential of hESCs that subsequently generates more blood-like cells. This study reveals the potential of cSAPs on the expansion and early-stage blood cell lineage differentiation of hPSCs.

Decontamination-Induced Modification of Bioactivity in Essential Oil-Based Plasma Polymer Coatings.

Plasma polymer coatings fabricated from Melaleuca alternifolia essential oil and its derivatives have been previously shown to reduce the extent of microbial adhesion on titanium, polymers, and other implantable materials used in dentistry. Previous studies have shown these coatings to maintain their performance under standard operating conditions; however, when used in e.g., a dental implant, these coatings may inadvertently become subject to in situ cleaning treatments, such as those using an atmospheric pressure plasma jet, a promising tool for the effective in situ removal of biofilms from tissues and implant surfaces. Here, we investigated the effect of such an exposure on the antimicrobial performance of the Melaleuca alternifolia polymer coating. It was found that direct exposure of the polymer coating surface to the jet for periods less than 60 s was sufficient to induce changes in its surface chemistry and topography, affecting its ability to retard subsequent microbial attachment. The exact effect of the jet exposure depended on the chemistry of the polymer coating, the length of plasma treatment, cell type, and incubation conditions. The change in the antimicrobial activity for polymer coatings fabricated at powers of 20-30 W was not statistically significant due to their limited baseline bioactivity. Interestingly, the bioactivity of polymer coatings fabricated at 10 and 15 W against Staphylococcus aureus cells was temporarily improved after the treatment, which could be attributed to the generation of loosely attached bioactive fragments on the treated surface, resulting in an increase in the dose of the bioactive agents being eluted by the surface. Attachment and proliferation of Pseudomonas aeruginosa cells and mixed cultures were less affected by changes in the bioactivity profile of the surface. The sensitivity of the cells to the change imparted by the jet treatment was also found to be dependent on their origin culture, with mature biofilm-derived P. aeruginosa bacterial cells showing a greater ability to colonize the surface when compared to its planktonic broth-grown counterpart. The presence of plasma-generated reactive oxygen and nitrogen species in the culture media was also found to enhance the bioactivity of polymer coatings fabricated at power levels of 10 and 15 W, due to a synergistic effect arising from simultaneous exposure of cells to reactive oxygen and nitrogen species (RONS) and eluted bioactive fragments. These results suggest that it is important to consider the possible implications of inadvertent changes in the properties and performance of plasma polymer coatings as a result of exposure to in situ decontamination, to both prevent suboptimal performance and to exploit possible synergies that may arise for some polymer coating-surface treatment combinations.

Conductive, Acid-Doped Polyaniline Electrospun Nanofiber Gas Sensing Substrates Made Using a Facile Dissolution Method.

A novel dissolution method that allows for the total solvation of high-concentration, high-molecular-weight polyaniline (PANi) doped with (+)-camphor-10-sulfonic acid (CSA) is reported. Preparation of 12-16 wt % 65,000 Da PANi solutions in N,N-dimethylformamide is achievable using a simple one-pot method. Doped polyaniline solutions in common organic solvents were processed into nanofibers using a convenient single-nozzle electrospinning technique. The electrospinning of PANi-CSA into nanofibrous membranes generated substrates that were subsequently employed in colorimetric gas sensing. These substrates demonstrated linearity of response upon exposure to 50-5500 ppm ammonia at ambient (50 ± 10% RH) and high (80% RH) humidity.

Harnessing Colloidal Self-Assembled Patterns (cSAPs) to Regulate Bacterial and Human Stem Cell Response at Biointerfaces In Vitro and In Vivo.

The generation of complex physicochemical signals on the surface of biomedical materials is still challenging despite the fact that a broad range of surface modification methods have been developed over the last few decades. Colloidal self-assembled patterns (cSAPs) are combinations of unique colloids differing in size and surface chemistry acting as building blocks that can be programmed to generate surface patterns with exquisite control of complexity. This study reports on producing a variety of pre-modified colloids for the fabrication of cSAPs as well as post-assembly modifications to yield complex surfaces. The surface of cSAPs presents hierarchical micro- and nanostructures, localized hydrophilic/hydrophobic characteristics, and tunable surface functionality imparted by the individual colloids. The selected cSAPs can control bacterial adhesion (S. aureus, P. aeruginosa, and E. coli) and affect the cell cycle of human bone marrow stem cells (hBMSCs). Moreover, in a mouse subcutaneous model, cSAPs with selective [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium (SBMA) modification can reduce the inflammatory response after being challenged with bacteria. This study reveals that functionalized cSAPs are versatile tools for controlling cellular responses at biointerfaces, which is instructive for biomaterials or biodevices.

Indirect co-culture of lung carcinoma cells with hyperthermia-treated mesenchymal stem cells influences tumor spheroid growth in a collagen-based 3-dimensional microfluidic model.

BACKGROUND: Mesenchymal stem cells (MSCs) have paradoxically been reported to exert either pro- or anti-tumor effects in vitro. Hyperthermia, in combination with chemotherapy, has tumor-inhibiting effects; however, its role, together with MSCs, so far is not well understood. Furthermore, a lot of research is conducted using conventional 2-dimensional in vitro models that do not mimic the actual tumor microenvironment. AIM: In light of this fact, an indirect method of co-culturing human amniotic membrane-derived MSCs (AMMSCs) with collagen-encapsulated human lung carcinoma cells (A549) was performed using a 3-dimensional (3D) tumor-on-chip device. METHODS: The conditioned medium of AMMSCs (AMMSC-CM) or heat-treated AMMSCs (heat-AMMSC-CM) was utilized to create indirect co-culture conditions. Tumor spheroid growth characterization, immunocytochemistry and cytotoxicity assays, and anti-cancer peptide (P1) screening were performed to determine the effects of the conditioned medium. RESULTS: The A549 cells cultured inside the 3D microfluidic chip developed into multicellular tumor spheroids over five days of culture. The AMMSC-CM, contrary to previous reports claiming its tumor-inhibiting potential, led to significant proliferation of tumor spheroids. Heat-AMMSC-CM led to reductions in both spheroid diameter and cell proliferation. The medium containing the P1 peptide was found to be the least cytotoxic to tumor spheroids in co-culture compared with the monoculture and heat-co-culture groups. CONCLUSIONS: Hyperthermia, in combination with the anticancer peptide, exhibited highest cytotoxic effects. This study highlights the growing importance of 3D microfluidic tumor models for testing stem-cell-based and other anti-cancer therapies.

Binary Colloidal Crystal (BCC) Substrates for Controlling the Fate of Mouse Embryonic Stem Cells.

Understanding the interactions of stem cells with surface topography can give us an invaluable tool in controlling stemness and fate of stem cells for further use in biomedical applications. In this study, we have fabricated topographical features using a class of cell culture substrates called binary colloidal crystals (BCCs), that are made by self-assembly of mixtures of spherical micron sized silica (Si) and nanometer sized polystyrene (PS) or poly (methyl methacrylate) (PMMA) particles. The substrates formed are arrays of ordered, hexagonally packed large Si particles inter-dispersed with the PS particles that are stabilized by gentle heating, which melts the PS or PMMA forming substrates suitable for cell culture. BCC substrates were used for culture of mouse embryonic stem cells (mESCs). Compared to tissue culture plates, COM1 (Si5-PMMA0.4), COM2 (Si5-PS0.4) and COM4 (Si2-PSC0.22) have shown to provide a better support for mESC proliferation in the presence of the cytokine leukemia inhibitory factor (LIF). The behavior of mESCs with the BCCs in presence and absence of LIF, was further explored and it was found that interaction of mESCs with the culture substrate can be controlled by tuning surface topography and roughness, which is determined by the size and type of particles used in making BCCs. Furthermore, it was shown that limiting cell-surface interactions and controlling colony shape can promote stemness maintenance on COM1 and COM2 substrates as indicated by better proliferation and higher expression of pluripotency genes including Nanog both in presence and in absence of LIF. Together with higher expression of GATA6 gene, it can be stated that these surfaces can be used for endodermic priming of mESCs. Therefore, we believe that these surfaces, especially COM1 and COM2 surfaces can be beneficial as stem cell culture systems for further use in biomedical research.

3D hepatic mimics - the need for a multicentric approach.

The liver is a center of metabolic activity, including the metabolism of drugs, and consequently is prone to drug-induced liver injury. Failure to detect hepatotoxicity of drugs during their development will lead to the withdrawal of the drugs during clinical trials. To avoid such clinical and economic consequences, in vitro liver models that can precisely predict the toxicity of a drug during the pre-clinical phase is necessary. This review describes the different technologies that are used to develop in vitro liver models and the different approaches aimed at mimicking different functional aspects of the liver at the fundamental level. This involves mimicking of the functional and structural units like the sinusoid, the bile canalicular system, and the acinus.

Harnessing the perinuclear actin cap (pnAC) to influence nanocarrier trafficking and gene transfection efficiency in skeletal myoblasts using nanopillars.

Gene transfection is important in biotechnology and is used to modify cells intrinsically. It can be conducted in cell suspension or after cell adhesion, where the efficiency is dependent on many factors such as the type of nanocarrier used and cell division processes. Anchor-dependent cells are sensitive to the substrate they are attached to and adapt their behavior accordingly, including plasmid trafficking during gene transfection. Previously, it was shown in our group that the cytoskeleton is an essential factor in influencing gene transfection in skeletal myoblasts using nanogrooves as a substrate. In this study, the effect of the cytoskeleton on gene transfection efficiency of skeletal myoblasts was studied using various nanopillars and nanocarriers. Nanopillars with different diameters (200-1000 nm) and depths (200 or 400 nm) were fabricated using colloidal self-assembly and reactive ion etching. All surfaces were treated with oxygen plasma or polydopamine (PD) to further control cell morphology. Plasmid DNA was delivered into cells using jetPRIME or Lipofectamine 3000 nanocarriers. After screening hundreds of images, two distinguishable F-actin distributions were found, i.e., cells with or without a perinuclear actin cap (pnAC). Cells attached to nanopillars, especially the deep pillars, had a smaller spreading area, shorter F-actin, more 3D-like cell nuclei, and a lower percentage of pnAC, which lead to a higher gene transfection efficiency using jetPRIME. On the other hand, cells attached to the shallow nanopillars or flat surfaces had a larger spreading area, longer F-actin, more 2D-like cell nuclei, and a higher percentage of pnAC that facilitates gene transfection using Lipofectamine. The effects of cell density, cytoskeleton (cytoD), and focal adhesions (RGD) on gene transfection were also studied, and the results were consistent with our hypothesis that F-actin distribution is one of the critical factors in gene transfection. In conclusion, pnAC plays a vital role in the intracellular trafficking of nanocarrier/plasmid complexes and this study provides new insights into gene transfection in anchor-dependent cells. STATEMENT OF SIGNIFICANCE: This study provides a new perspective in gene transfection using attached cells where perinuclear actin cap (pnAC) is an essential factor involved in transfection efficiency. A series of nanopillars were used to harness cell and cytoskeleton morphology. Two distinguishable cytoskeletal structures were found including cells with or without pnAC. 2D-like cells with pnAC facilitate gene delivery using liposome-based nanocarriers, while 3D-like cells without pnAC benefit gene delivery using cationic polymer-based nanocarriers. This study reveals the importance of the cytoskeleton during gene transfection that is beneficial in tissue transfection.

Selective Cytotoxicity of a Novel Trp-Rich Peptide against Lung Tumor Spheroids Encapsulated inside a 3D Microfluidic Device.

There is a globally rising healthcare need to develop new anticancer therapies as well as to test them on biologically relevant in vitro cancer models instead of overly simplistic 2D models. To address both these needs, a 3D lung cancer spheroid model is developed using human A549 cells trapped inside a collagen gel in a compartmentalized microfluidic device and homogenously sized (35-45 µm) multicellular tumor spheroids are obtained in 5 days. The novel tryptophan-rich peptide P1, identified earlier as a potential anticancer peptide (ACP), shows enhanced cytotoxic efficacy against A549 tumor spheroids (>75%) in clinically relevant low concentrations, while it does not affect human amniotic membrane mesenchymal stem cells at the same concentrations (<15%). The peptide also inhibits the formation of tumor spheroids by reducing cell viability as well as lowering the proliferative capacity, which is confirmed by the expression of cell proliferation marker Ki-67. The ACP offers a novel therapeutic strategy against lung cancer cells without affecting healthy cells. The microfluidic device used is likely to be useful in helping develop models for several other cancer types to test new anticancer agents.

On-chip anticancer drug screening - Recent progress in microfluidic platforms to address challenges in chemotherapy.

There is an increasing need for advanced and inexpensive preclinical models to accelerate the development of anticancer drugs. While costly animal models fail to predict human clinical outcomes, in vitro models such as microfluidic chips ('tumor-on-chip') are showing tremendous promise at predicting and providing meaningful preclinical drug screening outcomes. Research on 'tumor-on-chips' has grown enormously worldwide and is being widely accepted by pharmaceutical companies as a drug development tool. In light of this shift in philosophy, it is important to review the recent literature on microfluidic devices to determine how rapidly the technology has progressed as a promising model for drug screening and aiding cancer therapy. We review the past five years of successful developments and capabilities in microdevice technology (cancer models) for use in anticancer drug screening. Microfluidic devices that are being designed to address current challenges in chemotherapy, such as drug resistance, combinatorial drug therapy, personalized medicine, and cancer metastasis are also reviewed in detail. We provide a perspective on how personalized 'tumor-on-chip', as well as high-throughput microfluidic platforms based on patient-specific tumor cells, can potentially replace the more expensive and 'non-human' animal models in preclinical anticancer drug development.

Binary Colloidal Crystal Layers as Platforms for Surface Patterning of Puroindoline-Based Antimicrobial Peptides.

The ability of bacteria to form biofilms and the emergence of antibiotic-resistant strains have prompted the need to develop the next generation of antibacterial coatings. Antimicrobial peptides (AMPs) are showing promise as molecules that can address these issues, especially if used when immobilized as a surface coating. We present a method that explores how surface patterns together with the selective immobilization of an AMP called PuroA (FPVTWRWWKWWKG-NH2) can be used to both kill bacteria and also as a tool to study bacterial attachment mechanisms. Surface patterning is achieved using stabilized self-assembled binary colloidal crystal (BCC) layers, allowing selective PuroA immobilization to carboxylated particles using N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide (EDC) hydrochloride/N-hydroxysuccinimide (NHS) coupling chemistry. Covalent immobilization of PuroA was compared with physical adsorption (i.e., without the addition of EDC/NHS). The AMP-functionalized colloids and BCC layers were characterized by X-ray photoelectron spectroscopy, ζ potentials, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Surface antimicrobial activity was assessed by viability assays using Escherichia coli. MALDI-TOF MS analysis revealed that although not all of PuroA was successfully covalently immobilized, a relatively low density of PuroA (1.93 × 1013 molecules/cm2 and 7.14 × 1012 molecules/cm2 for covalent and physical immobilization, respectively) was found to be sufficient at significantly decreasing the viability of E. coli by 70% when compared to that of control samples. The findings provide a proof of concept that BCC layers are a suitable platform for the patterned immobilization of AMPs and the importance of ascertaining the success of small-molecule grafting reactions using surface-MALDI, something that is often assumed to be successful in the field.

Tuning the Density of Poly(ethylene glycol) Chains to Control Mammalian Cell and Bacterial Attachment.

Surface modification of biomaterials with polymer chains has attracted great attention because of their ability to control biointerfacial interactions such as protein adsorption, cell attachment and bacterial biofilm formation. The aim of this study was to control the immobilisation of biomolecules on silicon wafers using poly(ethylene glycol)(PEG) chains by a "grafting to" technique. In particular, to control the polymer chain graft density in order to capture proteins and preserve their activity in cell culture as well as find the optimal density that would totally prevent bacterial attachment. The PEG graft density was varied by changing the polymer solubility using an increasing salt concentration. The silicon substrates were initially modified with aminopropyl-triethoxysilane (APTES), where the surface density of amine groups was optimised using different concentrations. The results showed under specific conditions, the PEG density was highest with grafting under "cloud point" conditions. The modified surfaces were characterised with X-ray photoelectron spectroscopy (XPS), ellipsometry, atomic force microscopy (AFM) and water contact angle measurements. In addition, all modified surfaces were tested with protein solutions and in cell (mesenchymal stem cells and MG63 osteoblast-like cells) and bacterial (Pseudomonas aeruginosa) attachment assays. Overall, the lowest protein adsorption was observed on the highest polymer graft density, bacterial adhesion was very low on all modified surfaces, and it can be seen that the attachment of mammalian cells gradually increased as the PEG grafting density decreased, reaching the maximum attachment at medium PEG densities. The results demonstrate that, at certain PEG surface coverages, mammalian cell attachment can be tuned with the potential to optimise their behaviour with controlled serum protein adsorption.

Modulation of human mesenchymal and pluripotent stem cell behavior using biophysical and biochemical cues: A review.

In vitro manipulation of human stem cells is a critical process in regenerative medicine and cellular therapies. Strategies and methods to maintain stem cells and direct them into specific lineages are ongoing challenges in these fields. To date, a number of studies have reported that besides biochemical stimulation, biophysical cues in the form of surface patterning and external stimulation also influence stem cell attachment, proliferation, and differentiation, and can be used in cell reprogramming and the maintenance of pluripotency. While biochemical cues are generally effective and easy to deliver, biophysical cues have many other advantages for scalability as they are cost efficient, have a longer lifetime, and can be easily defined. However, different protocols and cell sources utilized in a variety of studies have led to difficulties in obtaining clear conclusions about the effects of the biophysical environment on stem cells. In addition, the examination of different types of external stimulation is time consuming and limited by available fabrication techniques, resulting in a delay in commercialization and clinical applications. In this review, we aim to summarize the most important biophysical cues and methods for the culture of human stem cells, including mesenchymal and pluripotent stem cells, to facilitate their adoption in stem cell biology. The standard classical protocols of using biochemical cues will also be discussed for comparison. We believe that combining biochemical and biophysical stimulation has the greatest potential to generate functionally mature cells at a scalable and inexpensive rate for diverse applications in regenerative medicine and cell therapy. Biotechnol. Bioeng. 2017;114: 260-280. © 2016 Wiley Periodicals, Inc.

Stimulation of Early Osteochondral Differentiation of Human Mesenchymal Stem Cells Using Binary Colloidal Crystals (BCCs).

A new surface based on self-assembly of two colloids into well-defined nanostructures, so-called binary colloidal crystals (BCCs), was fabricated for stem cell culture. The facile fabrication process are able to cover large surface areas (>3 cm-diameter, i.e. > 7 cm(2)) with ordered surface nanotopographies that is often a challenge particularly in biomaterials science. From our library, four different combinations of BCCs were selected using mixtures of silica, polystyrene and poly(methyl methacrylate) particles with sizes in the range from 100 nm to 5 µm. Cell spreading, proliferation, and surface-induced lineage commitment of human adipose-derived stem cells (hADSCs) was studied using quantitative real time polymerase chain reaction (qRT-PCR) and immunostaining. The results showed that BCCs induced osteo- and chondro- but not adipo-gene expression in the absence of induction medium suggesting that the osteochondral lineage can be stimulated by the BCCs. When applying induction media, higher osteo- and chondro-gene expression on BCCs was found compared with tissue culture polystyrene (TCPS) and flat silica (Si) controls, respectively. Colony forming of chondrogenic hADSCs was found on BCCs and TCPS but not Si controls, suggesting that the differentiation of stem cells is surface-dependent. BCCs provide access to complex nanotopographies and chemistries, which can find applications in cell culture and regenerative medicine.

Response of MG63 osteoblast-like cells to ordered nanotopographies fabricated using colloidal self-assembly and glancing angle deposition.

Ordered surface nanostructures have attracted much attention in different fields including biomedical engineering because of their potential to study the size effect on cellular response and modulation of cell fate. However, the ability to fabricate large-area ordered nanostructures is typically limited due to high costs and low speed of fabrication. Herein, highly ordered nanostructures with large surface areas (>1.5 × 1.5 cm(2)) were fabricated using a combination of facile techniques including colloidal self-assembly, colloidal lithography, and glancing angle deposition (GLAD). An ordered tantalum (Ta) pattern with 60-nm-height was generated using colloidal lithography. A monolayer of colloidal crystal, i.e., hexagonal close packed 720 nm polystyrene particles, was self-assembled and used as a mask. Ta patterns were subsequently generated by evaporation of Ta through the mask. The feature size was further increased by 100 or 200 nm using GLAD, resulting in the fabrication of four different surfaces (FLAT, Ta60, GLAD100, and GLAD200). Cell adhesion, proliferation, and mineralization of MG63 osteoblast-like cells were investigated on these ordered nanostructures over a 1 week period. Our results showed that cell adhesion, spreading, focal adhesion formation, and filopodia formation of the MG63 osteoblast-like cells were inhibited on the GLAD surfaces, especially the initial (24 h) attachment, resulting in a lower cell density on the GLAD surfaces. After 1 week culture, alkaline phosphatase activity and the amount of Ca was higher on the GLAD surfaces compared with Ta60 and FLAT controls, suggesting that the GLAD surfaces facilitate differentiation of osteoblasts. This study demonstrates that ordered Ta nanotopographies synthesized by combining colloidal lithography with GLAD can improve the mineralization of osteoblast-like cells providing a new platform for biomaterials and bone tissue engineering.

Modulation of human mesenchymal stem cell behavior on ordered tantalum nanotopographies fabricated using colloidal lithography and glancing angle deposition.

Ordered surface nanostructures have attracted much attention in biotechnology and biomedical engineering because of their potential to modulate cell-surface interactions in a controllable manner. However, the ability to fabricate large area ordered nanostructures is limited because of high costs and low speed of fabrication. Here, we have fabricated ordered nanostructures with large surface areas (1.5 × 1.5 cm(2)) using a combination of facile techniques including colloidal self-assembly, colloidal lithography and glancing angle deposition (GLAD). Polystyrene (722 nm) colloids were self-assembled into a hexagonally close-packed (hcp) crystal array at the water-air interface, transferred on a biocompatible tantalum (Ta) surface and used as a mask to generate an ordered Ta pattern. The Ta was deposited by sputter coating through the crystal mask creating approximately 60-nm-high feature sizes. The feature size was further increased by approximately 200-nm-height respectively using GLAD, resulting in the fabrication of four different surfaces (FLAT, Ta60, GLAD100, and GLAD200). Cell adhesion, proliferation, and osteogenic differentiation of primary human adipose-derived stem cells (hADSCs) were studied on these ordered nanostructures for up to 2 weeks. Our results suggested that cell spreading, focal adhesion formation, and filopodia extension of hADSCs were inhibited on the GLAD surfaces, while the growth rate was similar between each surface. Immunostaining for type I collagen (COL1) and osteocalcin (OC) showed that there was higher osteogenic components deposited on the GLAD surfaces compared to the Ta60 and FLAT surfaces after 1 week of osteogenic culture. After 2 weeks of osteogenic culture, alkaline phosphatase (ALP) activity and the amount of calcium was higher on the GLAD surfaces. In addition, osteoblast-like cells were confluent on Ta60 and FLAT surfaces, whereas the GLAD surfaces were not fully covered suggesting that the cell-cell interactions are stronger than cell-substrate interactions on GLAD surfaces. Visible extracellular matrix deposits decorated the porous surface can be found on the GLAD surfaces. Depth profiling of surface components using a new Ar cluster source and X-ray photoelectron spectroscopy (XPS) showed that deposited extracellular matrix on GLAD surfaces is rich in nitrogen. The fabricated ordered surface nanotopographies have potential to be applied in diverse fields, and demonstrate that the behavior of human stem cells can be directed on these ordered nanotopographies, providing new knowledge for applications in biomaterials and tissue engineering.

Polymerizable peptide copolymer coatings for the control of biointerfacial interactions.

The effective control over biointerfacial interactions is essential for a broad range of biomedical applications in vitro and in vivo such as biosensors, cell culture tools and implantable devices. Here, our aim was to develop a coating strategy that is transferable between different substrate materials and can effectively suppress nonspecific protein adsorption and hence reduce cell attachment while also presenting bioactive signals to enable specific cell-material interactions. In a first step an allylamine plasma polymer coating was applied, followed by the covalent immobilization of a macroinitiator carrying iniferter functionalities in the side chains. Subsequently, copolymers with different molar ratios of acrylamide and a polymerizable peptide containing the sequence Arg-Gly-Asp (RGD) were grafted via surface initiated free radical polymerization. X-ray photoelectron spectroscopy (XPS) was used to confirm the success of each coating step. The cellular response to these coatings was evaluated using L929 mouse fibroblast cell culture assays for up to 24 h. Cell attachment was significantly reduced on acrylamide homopolymer coatings and negative control surfaces representing a polymerizable peptide containing the nonbioactive Arg-Ala-Asp (RAD) sequence. In contrast, cell attachment was increased with increasing polymerizable RGD peptide ratios in the copolymer. The combination of acrylamide-terminated peptide sequences in combination with acrylamide provides a simple and versatile route to surfaces that combine low nonspecific protein adsorption and the display of controlled densities of bioactive signals and is expected to be translated into a number of biomedical applications in vitro and in vivo.

Surface chemistry of grafted expanded poly(tetrafluoroethylene) membranes modifies the in vitro proinflammatory response in macrophages.

A series of surface-modified expanded poly(tetrafluoroethylene) membranes showed varied levels of in vitro macrophage proinflammatory response. Membranes containing a mixture of phosphate and hydroxyl groups (as determined by X-ray photoelectron spectroscopy analysis) stimulate greater macrophage activation than samples containing a mixture of phosphate and carboxylic acid segments. The types of proteins that adsorbed irreversibly from serum onto the two samples with the highest and lowest cellular response were investigated using surface-matrix-assisted laser desorption ionisation time-of-flight mass spectrometry. Distinct differences in the number and type of proteins that adsorbed were observed between these samples. A correlation was found between the main protein components adsorbed onto the surfaces and the resulting in vitro proinflammatory response. This study strongly supports the hypothesis that the cellular response is not controlled directly by surface properties but is mediated by specific protein adsorption events. This in turn highlights the importance of better understanding and controlling the properties of intelligent surface-modified biomaterials.