Fabrication of Functional Polymer Structures through Bottom-Up Selective Vapor Deposition from Bottom-Up Conductive Templates.

An electrically induced bottom-up process was introduced for the fabrication of multifunctional nanostructures of polymers. Without requiring complicated photolithography or printing techniques, the fabrication process first produced a conducting template by colloidal lithography to create an interconnected conduction pathway. By supplying an electrical charge to the conducting network, the conducting areas were enabled with a highly energized surface that generally deactivated the adsorbed reactive species and inhibited the vapor deposition of poly- p-xylylene polymers. However, the template allowed the deposition of ordered poly- p-xylylene nanostructures only on the confined and negative areas of the conducting template, in a relatively large centimeter-scale production. The wide selection of functionality and multifunctional capability of poly- p-xylylenes naturally rendered the synergistic and orthogonal chemical reactivity of the resulting nanostructures. With only a few steps, the construction of a nanometer topology with the functionalization of multiple chemical conducts can be achieved, and the selected deposition process represents a state-of-the-art nanostructure fabrication in a simple and versatile approach from the bottom up.

Topologically Controlled Cell Differentiation Based on Vapor-Deposited Polymer Coatings.

In addition to the widely adopted method of controlling cell attachment for cell patterning, pattern formation via cell proliferation and differentiation is demonstrated using precisely defined interface chemistry and spatial topology. The interface platform is created using a maleimide-functionalized parylene coating (maleimide-PPX) that provides two routes for controlled conjugation accessibility, including the maleimide-thiol coupling reaction and the thiol-ene click reaction, with a high reaction specificity under mild conditions. The coating technology is a prime tool for the immobilization of sensitive molecules, such as growth factor proteins. Conjugation of fibroblast growth factor 2 (FGF-2) and bone morphogenetic protein (BMP-2) was performed on the coating surface by elegantly manipulating the reaction routes, and confining the conjugation reaction to selected areas was accomplished using microcontact printing (µCP) and/or UV irradiation photopatterning. The modified interface provides chemically and topologically defined signals that are recognized by cultured murine preosteoblast cells for proliferation (by FGF-2) and osteogenesis (by BMP-2) activities in specific locations. The reported technique additionally enabled synergistic pattern formation for both osteogenesis and proliferation activities on the same interface, which is difficult to perform using conventional cell attachment patterns. Because of the versatility of the coating, which can be applied to a wide range of materials and on curved and complex devices, the proposed technology is extendable to other prospective biomaterial designs and material interface modifications.

Efficient reduction of graphene oxide catalyzed by copper.

We report that copper thin films deposited on top of graphene oxide (GO) serve as an effective catalyst to reduce GO sheets in a diluted hydrogen environment at high temperature. The reduced GO (rGO) sheets exhibit higher effective field-effect hole mobility, up to 80 cm(2) V(-1) s(-1), and lower sheet resistance (13 kΩ â–¡(-1)) compared with those reduced by reported methods such as hydrazine and thermal annealing. Raman and XPS characterizations are addressed to study the reduction mechanism on graphene oxide underneath copper thin films. The level of reduction in rGO sheets is examined by Raman spectroscopy and it is well correlated with hole mobility values. The conductivity enhancement is attributed to the growth of the graphitic domain size. This method is not only suitable for reduction of single GO sheets but also applicable to lower the sheet resistance of Langmuir-Blodgett assembled GO films.

A new cell line (NTU-SE) from pupal tissues of the beet armyworm, Spodoptera exigua (Lepidoptera: Noctuidae), is highly susceptible to S. exigua multiple nucleopolyhedrovirus (SeMNPV) and Autographa californica MNPV (AcMNPV).

A new continuous cell line, NTU-SE, was established from the pupal tissues of an economically important pest, the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae). This cell line contains four major morphologic types: round, polymorphic, spindle-shaped, and comma-shaped cells. The population doubling time of this new line in TNM-FH medium supplemented with 8% fetal bovine serum (FBS) at 28°C is 35.5h. The chromosomal spread from NTU-SE cells is typical to the chromosomal morphology of lepidopteran cell lines. Confidently, NTU-SE cell line is a new cell line that exhibits distinct isozyme patterns of esterase, lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) from those of the other insect cell lines. In addition, the DNA sequence of the nuclear ribosomal internal transcribed spacer (ITS) region of NTU-SE cells is above 96% identical to that sequence of S. exigua larvae, as compared to only 66% identical to that of S. litura larvae. The NTU-SE cell line is highly susceptible to S. exigua multiple nucleopolyhedrovirus (SeMNPV) and Autographa californica MNPV (AcMNPV). Therefore, a highly virulent SeMNPV strain, SeMNPV-1, had been successfully isolated and propagated in NTU-SE cells. We conclude that the NTU-SE cell line will be a useful tool for the selection and mass production of highly virulent SeMNPV strains for the S. exigua biocontrol and the baculovirus based recombinant protein expression systems.

Direct formation of wafer scale graphene thin layers on insulating substrates by chemical vapor deposition.

Direct formation of high-quality and wafer scale graphene thin layers on insulating gate dielectrics such as SiO(2) is emergent for graphene electronics using Si-wafer compatible fabrication. Here, we report that in a chemical vapor deposition process the carbon species dissociated on Cu surfaces not only result in graphene layers on top of the catalytic Cu thin films but also diffuse through Cu grain boundaries to the interface between Cu and underlying dielectrics. Optimization of the process parameters leads to a continuous and large-area graphene thin layers directly formed on top of the dielectrics. The bottom-gated transistor characteristics for the graphene films have shown quite comparable carrier mobility compared to the top-layer graphene. The proposed method allows us to achieve wafer-sized graphene on versatile insulating substrates without the need of graphene transfer.

Camphorsulfonic Acid-Mediated One-Pot Tandem Consecutive via the Ugi Four-Component Reaction for the Synthesis of Functionalized Indole and 2-Quinolone Derivatives by Switching Solvents.

A camphorsulfonic acid-mediated one-pot tandem consecutive approach was developed to synthesize functionalized indole and 2-quinolone derivatives from the Ugi four-component reaction by switching solvents. A reaction of the Ugi adduct in an aprotic solvent undergoes 5-exo-trig cyclization to form an indole ring. In a protic solvent, however, the Ugi adduct undergoes an alkyne-carbonyl metathesis reaction to form a 2-quinolone ring.

Vapor construction and modification of stem cell-laden multicomponent scaffolds for regenerative therapeutics.

Tissue engineering based on the combined use of isolated cells, scaffolds, and growth factors is widely used; however, the manufacture of cell-preloaded scaffolds faces challenges. Herein, we fabricated a multicomponent scaffold with multiple component accommodations, including bioactive molecules (BMs), such as fibroblast growth factor-2 (FGF-2) and l-ascorbic acid 2-phosphate (A2-P), and living cells of human adipose-derived stem cells (hASCs), within one scaffold construct. We report an innovative fabrication process based on vapor-phased construction using iced templates for vapor sublimation. Simultaneously, the vaporized water molecules were replaced by vapor deposition of poly-p-xylylene (PPX, USP Class VI, highly compatible polymer, FDA-approved records), forming a three-dimensional and porous scaffold matrix. More importantly, a multicomponent modification was achieved based on using nonvolatile solutes, including bioactive molecules of FGF-2 and A2-P, and living cells of hASCs, to prepare iced templates for sublimation. Additionally, the fabrication and construction resulted in a multicomponent scaffold product comprising the devised molecules, cells, and vapor-polymerized poly-p-xylylene as the scaffold matrix. The clean and dry fabrication process did not require catalysts, initiators or plasticizers, and potentially harmful solvents, and the scaffold products were produced in simple steps within hours of the processing time. Cell viability analysis showed a high survival rate (approximately 86.4%) for the accommodated hASCs in the fabricated scaffold product, and a surprising multilineage differentiation potential of hASCs was highly upregulated because of synergistic guidance by the same accommodated FGF-2 and A2-P components. Proliferation and self-renewal activities were also demonstrated with enhancement of the multicomponent scaffold product. Finally, in vivo calvarial defect studies further revealed that the constructed scaffolds provided blood vessels to grow into the bone defect areas with enhancement, and the induced conduction of osteoblast growth also promoted bone healing toward osseointegration. The reported scaffold construction technology represents a prospective tissue engineering scaffold product to enable accommodable and customizable versatility to control the distribution and composition of loading delicate BMs and living hASCs in one scaffold construct and demonstrates unlimited applications in tissue engineering repair and regenerative medicine applications.

Ice-templated synthesis of multicomponent porous coatings via vapour sublimation and deposition polymerization.

A multicomponent vapour-deposited porous (MVP) coating with combined physical and biochemical properties was fabricated based on a chemical vapour sublimation and deposition process. Multiple components are used based on their natural thermodynamic properties, being volatile and/or nonvolatile, resulting in the sublimation of water vapour (from an iced template), and a simultaneous deposition process of poly-p-xylylene occurs upon radical polymerization into a disordered structure, forming porous coatings of MVP on various substrates. In terms of physical properties, the coating technology exhibits adjustable hydrophobicity by tuning the surface morphology by timed control of the sublimation of the iced template layer from a substrate. However, by using a nonvolatile solution during fabrication, an impregnation process of the deposited poly-p-xylylene on such a solution with tuning contact angles produces an MVP coating with a customizable elastic modulus based on deformation-elasticity theory. Moreover, patterning physical structures with adjustable pore size and/or porosity of the coatings, as well as modulation and compartmentalization to introduce necessary boundaries of microstructures within one MVP coating layer, can be achieved during the proposed fabrication process. Finally, with a combination of defined solutions comprised of both volatile and nonvolatile multicomponents, including functional biomolecules, growth factor proteins, and living cells, the fabrication of the resultant MVP coating serves devised purposes exhibiting a variety of biological functions demonstrated with versatility for cell proliferation, osteogenesis, adipogenesis, odontogenesis, spheroid growth of stem cells, and a complex coculture system towards angiogenesis. Multicomponent porous coating technology is produced based on vapour sublimation and deposition upon radical polymerization that overturns conventional vapour-deposited coatings, resulting in only dense thin films, and in addition, the versatility of adjusting coating physical and chemical properties by exploiting the volatility mechanism of iced solution templates and accommodation of solute substances during the fabrication process. The MVP coating and the proposed fabrication technique represent a simple approach to provide a prospective interface coating layer for materials science and are attractive for unlimited applications.

Vapor-phased fabrication and modulation of cell-laden scaffolding materials.

Bottom-up approaches using building blocks of modules to fabricate scaffolds for tissue engineering applications have enabled the fabrication of structurally complex and multifunctional materials allowing for physical and chemical flexibility to better mimic the native extracellular matrix. Here we report a vapor-phased fabrication process for constructing three-dimensional modulated scaffold materials via simple steps based on controlling mass transport of vapor sublimation and deposition. We demonstrate the fabrication of scaffolds comprised of multiple biomolecules and living cells with built-in boundaries separating the distinct compartments containing defined biological configurations and functions. We show that the fabricated scaffolds have mass production potential. We demonstrate overall >80% cell viability of encapsulated cells and that modulated scaffolds exhibit enhanced cell proliferation, osteogenesis, and neurogenesis, which can be assembled into various geometric configurations. We perform cell co-culture experiments to show independent osteogenesis and angiogenesis activities from separate compartments in one scaffold construct.

Genomic sequencing and analyses of Lymantria xylina multiple nucleopolyhedrovirus.

BACKGROUND: Outbreaks of the casuarina moth, Lymantria xylina Swinehoe (Lepidoptera: Lymantriidae), which is a very important forest pest in Taiwan, have occurred every five to 10 years. This moth has expanded its range of host plants to include more than 65 species of broadleaf trees. LyxyMNPV (L. xylina multiple nucleopolyhedrovirus) is highly virulent to the casuarina moth and has been investigated as a possible biopesticide for controlling this moth. LdMNPV-like virus has also been isolated from Lymantria xylina larvae but LyxyMNPV was more virulent than LdMNPV-like virus both in NTU-LY and IPLB-LD-652Y cell lines. To better understand LyxyMNPV, the nucleotide sequence of the LyxyMNPV DNA genome was determined and analysed. RESULTS: The genome of LyxyMNPV consists of 156,344 bases, has a G+C content of 53.4% and contains 157 putative open reading frames (ORFs). The gene content and gene order of LyxyMNPV were similar to those of LdMNPV, with 151 ORFs identified as homologous to those reported in the LdMNPV genome. Two genes (Lyxy49 and Lyxy123) were homologous to other baculoviruses, and four unique LyxyMNPV ORFs (Lyxy11, Lyxy19, Lyxy130 and Lyxy131) were identified in the LyxyMNPV genome, including a gag-like gene that was not reported in baculoviruses. LdMNPV contains 23 ORFs that are absent in LyxyMNPV. Readily identifiable homologues of the gene host range factor-1 (hrf-1), which appears to be involved in the susceptibility of L. dispar to NPV infection, were not present in LyxyMNPV. Additionally, two putative odv-e27 homologues were identified in LyxyMNPV. The LyxyMNPV genome encoded 14 bro genes compared with 16 in LdMNPV, which occupied more than 8% of the LyxyMNPV genome. Thirteen homologous regions (hrs) were identified containing 48 repeated sequences composed of 30-bp imperfect palindromes. However, they differed in the relative positions, number of repeats and orientation in the genome compared to LdMNPV. CONCLUSION: The gene parity plot analysis, percent identity of the gene homologues and a phylogenetic analysis suggested that LyxyMNPV is a Group II NPV that is most closely related to LdMNPV but with a highly distinct genomic organisation.

Parylene-Based Porous Scaffold with Functionalized Encapsulation of Platelet-Rich Plasma and Living Stem Cells for Tissue Engineering Applications.

A scaffold was fabricated to synergistically encapsulate living human adipose-derived stem cells (hASCs) and platelet-rich plasma (PRP) based on a vapor-phase sublimation and deposition process. During the process, ice templates were prepared using sterile water as the solvent and were used to accommodate the sensitive living cells and PRP molecules. Under controlled processing conditions, the ice templates underwent vapor sublimation to evaporate water molecules, while at the same time, vapor-phase deposition of poly-p-xylylene (Parylene, USP Class VI highly biocompatible) occurred to replace the templates, and the final construction yielded a scaffold with Parylene as the matrix, with simultaneously encapsulated living hASCs and PRP molecules. Evaluation of the fabricated synergistic scaffold for the proliferation activities toward the encapsulated hASCs indicated significant augmentation of cell proliferation contributed by the PRP ingredients. In addition, osteogenic activity in the early stage by alkaline phosphatase expression and later stage with calcium mineralization indicated significant enhancement toward osteogenetic differentiation of the encapsulated hASCs, which were guided by the PRP molecules. By contrast, examinations of adipogenic activity by lipid droplet formation revealed an inhibition of adipogenesis with decreased intracellular lipid accumulation, and a statistically significant downregulation of adipogenic differentiation was postulated for the scaffold products when compared to the osteogenetic results and the control experiments. The reported fabrication method featured a clean and simple process to construct scaffolds that combined delicate living hASCs and PRP molecules inside the structure. The resultant synergistic scaffold and the selected commercially available hASCs and PRP are emerging as tissue engineering tools that provide multifunctionality for tissue repair and regeneration.

Clickable and Photo-Erasable Surface Functionalities by Using Vapor-Deposited Polymer Coatings.

A prospective design for interface properties is enabled to perform precise functionalization, erasure capability for existing properties, reactivation of surface functionality to a second divergent property. A vapor-deposited, 2-nitro-5-(prop-2-yn-1-yloxy)methylbenzyl carbamate-functionalized poly-para-xylylene coating is synthesized in this study to realize such tasks by offering the accessibility of the azide/alkyne click reaction, an integrated photochemical decomposition/cleavage moiety, and the reactivation sites of amines behind the cleavage that allow the installation of a second surface function. With the benefits from the mild processing conditions used for the coatings and the rapid response of the photochemical reaction, the creation of sophisticated interface properties and localized chemical compositions was elegantly demonstrated with a hybrid functionality including a confined hydrophlic/hydrophobic wetting property and/or a cell adherent/repellent platform on such a coating surface.

Defined cell adhesion for silicon-based implant materials by using vapor-deposited functional coatings.

The field of implantable electronics relies on using silicon materials due to the merits of a well-established fabrication process and favorable properties; of particular interest is the surface modification of such materials. In the present study, we introduce a surface modification technique based on coatings of functionalized Parylene on silicon substrates, where the modified layers provide a defined cell adhesion capability for the resultant silicon materials/devices. Functionalization of Parylene was achieved during a one-step chemical vapor deposition (CVD) polymerization process, forming NHS ester-functionalized Parylene, and subsequent RGD attachment was enabled via a conjugation reaction between the NHS ester and amine groups. The modification procedures additionally provided a clean and gentle approach to avoid thermal excursions, intense irradiation, chemicals, or solvents that might damage delicate structures or sensitive molecules on the devices. The modification layers exhibited excellent mechanical strength on the substrate, meeting the high standards of the American Society for Testing and Materials (ASTM), and the resultant cell adherence property was verified by a centrifugation assay and the analysis of attached cell morphologies; the results collectively demonstrated robust and sustainable modification layers of the NHS ester-functionalized Parylene and confirmed that the cell-adherence property imparted by using this facile modification technique was effective. The modification technology is expected to benefit the design of prospective interface properties for silicon-based devices and related industrial products.

Characterization of Mechanical Stability and Immunological Compatibility for Functionalized Modification Interfaces.

Surface modification layers are performed on the surfaces of biomaterials and have exhibited promise for decoupling original surface properties from bulk materials and enabling customized and advanced functional properties. The physical stability and the biological compatibility of these modified layers are equally important to ensure minimized delamination, debris, leaching of molecules, and other problems that are related to the failure of the modification layers and thus can provide a long-term success for the uses of these modified layers. A proven surface modification tool of the functionalized poly-para-xylylene (PPX) system was used as an example, and in addition to the demonstration of their chemical conjugation capabilities and the functional properties that have been well-documented, in the present report, we additionally devised the characterization protocols to examine stability properties, including thermostability and adhesive strength, as well as the biocompatibility, including cell viability and the immunological responses, for the modified PPX layers. The results suggested a durable coating stability for PPXs and firmly attached biomolecules under these stability and compatibility tests. The durable and stable modification layers accompanied by the native properties of the PPXs showed high cell viability against fibroblast cells and macrophages (MΦs), and the resulting immunological activities created by the MΦs exhibited excellent compatibility with non-activated immunological responses and no indication of inflammation.

Enhanced Growth Activities of Stem Cell Spheroids Based on a Durable and Chemically Defined Surface Modification Coating.

Surface modification layers are needed for the precise definition of surface chemistries and are equally important for durable and stable adhesive properties to ensure long-term stability and effective performance for biotechnological applications. This study demonstrates a robust modification layer that is synthesized based on chemical vapor deposition copolymerization, and the resultant coating layer is composed of the side-by-side presentation of N-hydroxysuccinimide ester and maleimide functionalities with a controlled ratio to define the immobilization accessibility of chitosan and growth factor protein (FGF-2) molecules on the substrate surface for enhancing cellular activities of stem cells. Characterizations of the copolymer modification layer showed excellent durability, including adhesive strength and thermal stability, and the layer is free of concerns for delamination and/or unacceptable deformation/debris formation that can cause potential toxicity to the surrounding biological environment. Modifications using the copolymer layer on the cell culture surface have demonstrated synergistic activity by chitosan to support the formation of spheroids and by FGF-2 to enhance the proliferation of human adipose-derived stem cells (ADSCs) within the spheroids while increasing the spheroid size and cell numbers. Healthy and flourishing growth activities were discovered for ADSCs on the modified culture surfaces, and the results are useful for potential and related stem cell research and the interfaces of biomaterials.

Surface modification: activation and deactivation of osteogenic differentiation based on detachable growth factor protein.

An advanced material interface is modified by using a substrate-independent coating of detachable poly-para-xylylene, enabling dynamical control of the immobilization and detachment of biomolecules, and a previously installed biological function is deactivated or tuned with reduced activity. The induction of osteogenesis activity, and subsequent deactivation of such osteogenesis activity, is demonstrated.

The 5' untranslated region of Perina nuda virus (PnV) possesses a strong internal translation activity in baculovirus-infected insect cells.

A bicistronic baculovirus expression vector and fluorescent protein-based assays were used to identify the sequences that possess internal translation activity in baculovirus-infected insect cells. We demonstrated that the 5' untranslated region (5'UTR; 473 nucleotides) of Perina nuda virus (PnV) and the 5'UTR (579 nucleotides) of Rhopalosiphum padi virus (RhPV), but not the IRES sequence of Cricket paralysis virus, have internal translation activity in baculovirus-infected Sf21 cells. In addition, we found that including the first 22 codons of the predicted PnV open reading frame (ORF; a total of 539 nucleotides) enhanced internal translation activity by approximately 18 times. This is the first report of internal translation activity for a baculovirus expression system (BEVS) in the iflavirus 5' sequence and may facilitate the development of polycistronic baculovirus transfer vectors that can be used in BEVS for the production of multiple protein complexes.

Stepwise and Programmable Cell Differentiation Pathways of Controlled Functional Biointerfaces.

An advanced control of biomaterial surfaces was created to enable the stepwise and switchable activities of the immobilized growth factor (GF) proteins for a programmed manipulation over cell differentiation pathways. The GF protein was immobilized on an advanced vapor-based coating of poly[(4-2-amide-2'-amine-dithiobisethyl-p-xylylene)-co-(p-xylylene)], and the equipped disulfide exchange mechanism of the coating enables the detachment and/or the displacement of the previously installed GF to reinstall a second GF protein. In this study, the controlled immobilization and displacement of the fibroblast growth factor (FGF-2) and bone morphogenetic protein (BMP-2) were demonstrated on cell culture substrates, and the resulting surfaces provided a programmable induction of cellular responses in proliferation and osteogenesis toward the cultured murine preosteoblasts (MC3T3-E1). A depreciated or stopped activity of the previously induced biological function, i.e., proliferation or osteogenesis, was found for MC3T3-E1 on the modified surface after the cleavage of the corresponding GF. In addition, with the approach to devise the displacement and reinstallation of FGF-2/BMP-2 proteins, a combined induction of proliferation and osteogenesis can be initiated in a timed latency to resolve programmable biological activities.

Multifunctional nanoparticles with controllable dimensions and tripled orthogonal reactivity.

Multifunctional nanoparticles featuring three distinct and orthogonal functionalities for performing catalyst-free click reactions of azide-alkyne and maleimide-thiol and atom transfer radical polymerization (ATRP) are fabricated using a simple chemical vapor deposition copolymerization approach with the flexibility to control the particle size and geometry.

Controlling multi-function of biomaterials interfaces based on multiple and competing adsorption of functional proteins.

Multifunctional biomaterial surfaces can be created by controlling the competing adsorption of multiple proteins. To demonstrate this concept, bone morphogenetic protein 2 (BMP-2) and fibronectin were adsorbed to the hydrophobic surface of polychloro-para-xylylene. The resulting adsorption properties on the surface depended on the dimensional and steric characteristics of the selected protein molecule, the degree of denaturation of the adsorbed proteins, the associated adsorption of interphase water molecules within the protein layers, and the aggregation of proteins in a planar direction with respect to the adsorbent surface. Additionally, a defined surface composition was formed by the competing adsorption of multiple proteins, and this surface composition was directly linked to the composition of the protein mixture in the solution phase. Although the mechanism of this complex competing adsorption process is not fully understood, the adsorbed proteins were irreversibly adsorbed and were unaffected by the further adsorption of homologous or heterologous proteins. Moreover, synergistic biological activities, including cell osteogenesis and proliferation independently and specifically induced by BMP-2 or fibronectin, were observed on the modified surface, and these biological activities were positively correlated with the surface composition of the multiple adsorbed proteins. These results provide insights and important design parameters for prospective biomaterials and biointerfaces for (multi)functional modifications. The ability to control protein/interface properties will be beneficial for the processing of biomaterials for clinical applications and industrial products.