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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Multifunctional Surface Modification: Facile and Flexible Reactivity toward a Precisely Controlled Biointerface.

A multicomponent functional polymer is synthesized to support specific reactivity for successful conjugation with the vast array of functionality present in biological systems and the flexibility to conjugate biomolecules without requiring additional modification to install a terminal functional group. The multifunctional surface is realized using a novel coating composed of distinct N-hydroxysuccinimide (NHS) ester and benzoyl functionalities, which can provide accessibility to both the NHS ester-amine coupling reaction and the photochemically induced benzophenone crosslinking reaction, respectively. In addition, the multifunctional polymer is fabricated and transformed to form nanoscale colloids through the solvent displacement of a water/DMF system due to solubility characteristics of the resulting polymer with high polarity. A facile and efficient fabrication approach using the multifunctional nanocolloid is thus demonstrated to create a drug carrier by installing paclitaxel and folic acid for targeted cancer therapy.

Enhanced bone morphogenic property of parylene-C.

The ability to induce osteointegration was introduced to a parylene-C surface via the simple and intuitive process of protein adsorption mediated by hydrophobic interactions. In this way, bone morphogenetic protein (BMP)-2, fibronectin, and platelet-rich plasma (PRP) could be immobilized on parylene-C surfaces. This approach alleviates concerns related to the use of potentially harmful substances in parylene-C modification processes. The adsorbed protein molecules were quantitatively characterized with respect to adsorption efficacy and binding affinity, and the important biological activities of the proteins were also examined using both early and late markers of osteogenetic activity, including alkaline phosphatase expression, calcium mineralization and marker gene expression. Additionally, the adsorbed PRP exhibited potential as a substitute for expensive recombinant growth factors by effectively inducing comparable osteogenetic activity. In addition to the excellent biocompatibility of parylene-C and its ability to coat a wide variety of substrate materials, the modification of parylene-C via protein adsorption provides unlimited possibilities for installing specific biological functions, expanding the potential applications of this material to include various biointerface platforms.

Vapor-based coatings for antibacterial and osteogenic functionalization and the immunological compatibility.

The immobilization of biofunctional molecules to biomaterial surfaces has enabled and expanded the versatility of currently available biomaterials to a wider range of applications. In addition, immobilized biomolecules offer modified surfaces that allow the use of smaller amounts of potentially harmful substances or prevent overdose, while the exhibited biological functions remain persistently effective. Surface concentrations of chlorhexidine (CHX) (1.40±0.08×10(-9)mol·cm(-2)) and bone morphogenetic protein 2 (BMP-2) (1.51±0.08×10(-11)mol·cm(-2)) immobilized molecules were determined in this study, and their specific biological functions in terms of antibacterial activity and osteogenesis potency, respectively, were demonstrated to be unambiguously effective. Immobilization exploits the use of vapor-based poly-p-xylylenes, which exhibit excellent biocompatibility and wide applicability for various substrate materials. This technique represents a practical and economical approach for the manufacture of certain industrial products. Furthermore, a minimal degree of macrophage activation was indicated on the modified surfaces via insignificant morphological changes and low levels of adverse inflammatory signals, including suppressed production of the pro-inflammatory cytokines IL-1ß and TNF-α as well as nitric oxide (NO). The results and the modification strategy illustrate a concept for designing prospective biomaterial surfaces such that the manipulation employed to elicit targeted biological responses does not compromise immunological compatibility.

Multifunctional and Continuous Gradients of Biointerfaces Based on Dual Reverse Click Reactions.

Chemical or biological gradients that are composed of multifunctional and/or multidirectional guidance cues are of fundamental importance for prospective biomaterials and biointerfaces. As a proof of concept, a general modification approach for generating multifunctional and continuous gradients was realized via two controlled and reversed click reactions, namely, thermo-activated thiol-yne and copper-free alkyne and azide click reactions. The cell adhesion property of fibroblasts was guided in a gradient with an enhancement, showing that the PEG molecule and RGD peptide were countercurrently immobilized to form such reversed gradients (with negating of the cell adhesion property). Using the gradient modification protocol to also create countercurrent distributions of FGF-2 and BMP-2 gradients, the demonstration of not only multifunctional but also gradient biointerfacial properties was resolved in time latencies on one surface by showing the manipulation in gradients toward proliferation and osteogenic differentiation for adipose-derived stem cells.

Fabrication of multipotent poly-para-xylylene particles in controlled nanoscopic dimensions.

In this study, poly-para-xylylene-based multifunctional nanoparticles (PPX-NPs) were fabricated. Based on the solubility characteristics determined for asymmetrically substituted poly-para-xylylenes in polar solvents, well-dispersed nanocolloids with a controllable size ranging from 50 to 800nm were produced in solution by the displacement of the solvent (water). These size ranges were found to have acceptable cellular compatibility through examinations of cultured 3T3 fibroblasts and adipose-derived stem cells treated with the PPX-NPs. In addition, these nanoscale PPX-NPs exhibited versatile bioconjugation properties in that a variety of available functional groups can be adopted from their counterpart, thin-film poly-para-xylylenes, during the production of these nanoparticles. For instance, bifunctional PPX-NPs with maleimide and benzoyl moieties were produced to enable immobilization via a maleimide-thiol reaction concurrent with a photochemical reaction. A cleavable PPX-NP was also produced with a thiol-exchangeable surface property. Additionally, by performing electrohydrodynamic jetting of parallel polymer solutions of selected poly-para-xylylenes, Janus-type or multicompartment PPX-NPs were created. The PPX-NPs can potentially be used for various biomedical applications such as combined diagnostics and drug delivery, multiplexing of detection, multiple-drug loading, and the targeted delivery of biomolecules or drugs.