Fabrication of polymer microstructures for MEMS: sacrificial layer micromolding and patterned substrate micromolding

Two soft lithography based fabrication techniques are employed for fabricating mechanically independent, freely suspended polymer microstructure from poly(n-propyl methacrylate) (PPMA), poly(methyl methacrylate) (PMMA), and polystyrene. Both methods involve a micromolding process followed by thermal bonding to the substrate. The first method, sacrificial layer micromolding, uses a water soluble sacrificial layer, allowing functional structures to be released by immersion in water. The second method, patterned substrate micromolding, uses a permanent substrate patterned via photolithography. Functional regions of the polymer MEMS are suspended over the voids in the photoresist pattern. The processes have been applied to the fabrication of polymer microstructures with a variety of geometries for specific applications. Devices have included microcantilevers, beams, and other more complicated microstuctures. The thermal molding process is conceivably applicable to the fabrication of microstructures from a wide variety of thermoplastic polymers, allowing material selection to be tailored based on application.     

A novel method to fabricate thermoresponsive microstructures with improved cell attachment/detachment properties

A novel, simple, and rapid method to fabricate thermoresponsive micropatterned substrate for cell adhesion, growth, and thermally induced detachment was developed. Thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAAm), was grafted onto the surface of polystyrene (PS) film with microstructure by plasma-induced graft polymerization technique. The thermoresponsive micropatterned films were characterized by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, hydrogen nuclear magnetic resonance ((1) H NMR) spectroscopy, X-ray photoelectron spectroscopy (XPS) and scanning electron microscope (SEM). These results indicated that the grafting ratio of PNIPAAm increased with increasing roughness of PS film. However, the microstructures on the substrate were not affected by grafted PNIPAAm. The optimal grafting conditions, such as plasma treatment time, monomer concentration, graft polymerization time, and graft medium were investigated. The thermoresponsive micropatterned films were investigated with the fibroblast cell (L929) adhesion, proliferation, and thermally induced detachment assay. The microstructure on the thermoresponsive micropatterned substrate facilitated cell adhesion above the lower critical solution temperature (LCST) of PNIPAAm and cell detachment below the LCST. Moreover, it can be used to regulate cell organization and tissue growth.     

Integrating novel technologies to fabricate smart scaffolds

Tissue engineering aims at restoring or regenerating a damaged tissue by combining cells, derived from a patient biopsy, with a 3D porous matrix functioning as a scaffold. After isolation and eventual in vitro expansion, cells are seeded on the 3D scaffolds and implanted directly or at a later stage in the patient's body. 3D scaffolds need to satisfy a number of requirements: (i) biocompatibility, (ii) biodegradability and/or bioresorbability, (iii) suitable mechanical properties, (iv) adequate physicochemical properties to direct cell-material interactions matching the tissue to be replaced and (v) ease in regaining the original shape of the damaged tissue and the integration with the surrounding environment. Still, it appears to be a challenge to satisfy all the aforementioned requisites with the biomaterials and the scaffold fabrication technologies nowadays available. 3D scaffolds can be fabricated with various techniques, among which rapid prototyping and electrospinning seem to be the most promising. Rapid prototyping technologies allow manufacturing scaffolds with a controlled, completely accessible pore network--determinant for nutrient supply and diffusion--in a CAD/CAM fashion. Electrospinning (ESP) allows mimicking the extracellular matrix (ECM) environment of the cells and can provide fibrous scaffolds with instructive surface properties to direct cell faith into the proper lineage. Yet, these fabrication methods have some disadvantages if considered alone. This review aims at summarizing conventional and novel scaffold fabrication techniques and the biomaterials used for tissue engineering and drug-delivery applications. A new trend seems to emerge in the field of scaffold design where different scaffolds fabrication technologies and different biomaterials are combined to provide cells with mechanical, physicochemical and biological cues at the macro-, micro- and nano-scale. If merged together, these integrated technologies may lead to the generation of a new set of 3D scaffolds that satisfies all of the scaffolds' requirements for tissue-engineering applications and may contribute to their success in a long-term scenario.     

A novel wet extrusion technique to fabricate self-assembled microfiber scaffolds for controlled drug delivery

We have developed a novel wet extrusion process to fabricate nonwoven self-assembled microfiber scaffolds with uniform diameters less than 5 μm and without any postmanipulation. In this method, a poly(L-lactic acid) solution flows dropwise into a stirring nonsolvent bath, deforming into liquid polymer streams that self-assemble into a nonwoven microfiber scaffold. The ability to tune fiber diameter was achieved by decreasing polymer spin dope concentration and increasing the silicon oil to petroleum ether ratio of the nonsolvent spin bath. To demonstrate the drug delivery capabilities of scaffolds, heparin was encapsulated using a conventional water-in-oil (W/O) emulsion technique and a cryogenic emulsion technique developed in our laboratory. Spin dope preparation was found to significantly effect the release kinetics of self-assembled scaffolds by altering the interconnectivity of pores within the precipitating filaments. After 35 days, scaffolds prepared from W/O emulsions released up to 45% encapsulated heparin, whereas nearly 80% release of heparin was observed from cryogenic emulsion formulations. The versatility of our system, combined with the prolonged release of small molecules and the ability to control the homogeneity of self-assembling scaffolds, could be beneficial for many tissue regeneration and engineering applications. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A: 2793-2802, 2012.     

Non-bioengineered silk gland fibroin micromolded matrices to study cell-surface interactions

Micropatterning/micromolding of protein molecules has played a significant role in developing biosensors, micro arrays, and tissue engineering devices for cellular investigations. Relevantly, there have been ample scopes for silk to be used as natural biomaterial in tissue engineering applications due to its attractive properties such as slow-controllable degradation, mechanical robustness, and inherent biocompatibility. In this paper, we report the fabrication of micromolded silk fibroin matrices, which have essentially been utilized to study cell-surface interactions. Fibroin protein has been isolated from the silk glands of nonmulberry Indian tropical tasar silkworms, Antheraea mylitta. The surface uniformity has been investigated using atomic force microscopy following the fabrication of silk micromolds. Subsequently, cellular interactions in terms of cell attachment, spreading, mitochondrial activity and proliferation have been studied in vitro using feline fibroblasts. Results have indicated a long term stability of patterns in micromolded silk matrices and negligible swelling. The versatility of described silk dissolution method coupled with ability to process large amount of silk protein into micromolded matrices and controllable surface topology may augment the desirability of silk fibroin as a natural biomaterial for bioengineering and biotechnological applications.     

A novel rotating electrochemically anodizing process to fabricate titanium oxide surface nanostructures enhancing the bioactivity of osteoblastic cells

Titanium oxide (TiO(2) ) surface layers with various surface nanostructures (nanotubes and nanowires) have been developed using an anodizing technique. The pore size and length of TiO(2) nanotubes can be tailored by changing the anodizing time and applied voltage. We developed a novel method to transform the upper part of the formed TiO(2) nanotubes into a nanowire-like structure by rotating the titanium anode during anodizing process. The transformation of nanotubes contributed to the preferential chemical dissolution of TiO(2) on the areas with intense interface tension stress. Furthermore, we further compared the effect of various TiO(2) surface nanostructures including flat, nanotubes, and nanowires on bioactive applications. The MG-63 osteoblastic cells cultured on the TiO(2) nanowires exhibited a polygonal shape with extending filopodia and showed highest levels of cell viability and alkaline phosphatase activity (ALP). The TiO(2) nanowire structure formed by our novel method can provide beneficial effects for MG-63 osteoblastic cells in attachment, proliferation, and secretion of ALP on the TiO(2) surface layer.     

Development of novel fluorescent particles applicable for phagocytosis assays with human macrophages

Phagocytosis is a fundamental process for removal of pathogens and for clearance of apoptotic cells. The objective of this work was the preparation of fluorescent microspheres by a simple method and the evaluation of its applicability in phagocytosis assays by using different human derived cells, differentiated THP-1 cell line and blood monocytes, with flow cytometry measurements for functionality assays. Our results show that microparticles are efficiently internalised in a non-opsonised form and in dose-dependent manner by both cellular types. Concerning mechanism we determined that tTG-β3 integrin signaling could be involved in the uptake of these particles.     

Novel approach to fabricate porous gelatin-siloxane hybrids for bone tissue engineering

Porous and bioactive gelatin–siloxane hybrids were successfully synthesized by using a combined sol–gel processing, post-gelation soaking, and freeze-drying process to provide a novel kind of materials in the developments and optimization of bone tissue engineering. The pore sizes of the hybrids can be well controlled by varying the freezing temperature. The scaffolds were soaked in a simulated body fluid (SBF) up to 14 days to evaluate the in vitro bioactivity. The Ca²⁺-containing scaffolds showed in vitro bioactivity as they biomimetically deposited apatite, but the Ca²⁺-free scaffolds failed. Cytotoxicity and cytocompatibility of those scaffolds and their extracts were monitored by the MC3T3-E1 cell responses, including the cell proliferation and the alkaline phosphatase activity. It was demonstrated that appropriate incorporation of Ca²⁺ ions stimulated osteoblast proliferation and differentiation in vitro.     

Systematic engineering of 3D pluripotent stem cell niches to guide blood development

Pluripotent stem cells (PSC) provide insight into development and may underpin new cell therapies, yet controlling PSC differentiation to generate functional cells remains a significant challenge. In this study we explored the concept that mimicking the local in vivo microenvironment during mesoderm specification could promote the emergence of hematopoietic progenitor cells from embryonic stem cells (ESCs). First, we assessed the expression of early phenotypic markers of mesoderm differentiation (E-cadherin, brachyury (T-GFP), PDGFRα, and Flk1: +/−ETPF) to reveal that E−T+P+F+ cells have the highest capacity for hematopoiesis. Second, we determined how initial aggregate size influences the emergence of mesodermal phenotypes (E−T+P+F+, E−T−P+/−F+, and E−T−P+F−) and discovered that colony forming cell (CFC) output was maximal with ∼100 cells per PSC aggregate. Finally, we introduced these 100-cell PSC aggregates into a low oxygen environment (5%; to upregulate endogenous VEGF secretion) and delivered two potent blood-inductive molecules, BMP4 and TPO (bone morphogenetic protein-4 and thrombopoietin), locally from microparticles to obtain a more robust differentiation response than soluble delivery methods alone. Approximately 1.7-fold more CFCs were generated with localized delivery in comparison to exogenous delivery, while combined growth factor use was reduced ∼14.2-fold. By systematically engineering the complex and dynamic environmental signals associated with the in vivo blood developmental niche we demonstrate a significant role for inductive endogenous signaling and introduce a tunable platform for enhancing PSC differentiation efficiency to specific lineages.     

Dendritic cells in the host response to implanted materials

The role of dendritic cells (DCs) and their targeted manipulation in the body’s response to implanted materials is an important and developing area of investigation, and a large component of the emerging field of biomaterials-based immune engineering. The key position of DCs in the immune system, serving to bridge innate and adaptive immunity, is facilitated by rich diversity in type and function and places DCs as a critical mediator to biomaterials of both synthetic and natural origins. This review presents current views regarding DC biology and summarizes recent findings in DC responses to implanted biomaterials. Based on these findings, there is promise that the directed programming of application-specific DC responses to biomaterials can become a reality, enabling and enhancing applications almost as diverse as the larger field of biomaterials itself.     

In vivo biocompatibility of porous silicon biomaterials for drug delivery to the heart

Myocardial infarction (MI), commonly known as a heart attack, is the irreversible necrosis of heart muscle secondary to prolonged ischemia, which is an increasing problem in terms of morbidity, mortality and healthcare costs worldwide. Along with the idea to develop nanocarriers that efficiently deliver therapeutic agents to target the heart, in this study, we aimed to test the in vivo biocompatibility of different sizes of thermally hydrocarbonized porous silicon (THCPSi) microparticles and thermally oxidized porous silicon (TOPSi) micro and nanoparticles in the heart tissue. Despite the absence or low cytotoxicity, both particle types showed good in vivo biocompatibility, with no influence on hematological parameters and no considerable changes in cardiac function before and after MI. The local injection of THCPSi microparticles into the myocardium led to significant higher activation of inflammatory cytokine and fibrosis promoting genes compared to TOPSi micro and nanoparticles; however, both particles showed no significant effect on myocardial fibrosis at one week post-injection. Our results suggest that THCPSi and TOPSi micro and nanoparticles could be applied for cardiac delivery of therapeutic agents in the future, and the PSi biomaterials might serve as a promising platform for the specific treatment of heart diseases.     

Attuning hydroxypropyl methylcellulose phthalate to oral delivery vehicle for effective and selective delivery of protein vaccine in ileum

Orally delivered proteins or antigens are taken up by epithelial microfold cells (M cells) in Peyer's patches, especially abundant in the ileum of small intestine. However, several barriers including gastric pH, enzymatic degradation, rapid transit and lack of specificity of proteins towards M cells, has made the goal of oral delivery of proteins very challenging. To overcome the problems, we developed an ileum targeted protein delivery system using hydroxypropyl methylcellulose phthalate (HPMCP). Initially, we attuned pH-sensitive property of HPMCP for controlled dissolution at ileum pH (≥7.4) by thiolation. Thiolation also improved mucoadhesive property of HPMCP to prolong the particles transit time through the gastrointestinal tract. Typically, thiolated HPMCP (T-HPMCP) prevented protein release in acidic pH in stomach and duodenum but released the proteins at ileal pH in a controlled manner. To evaluate the effectiveness of an oral delivery vehicle, T-HPMCP was used to deliver an M cell targeting protein antigen to mice through oral route. The antigens were mostly delivered and located in Peyer's patches in the ileum demonstrating the higher uptake of antigens through M-cells. Importantly, oral delivery of the antigen with T-HPMCP not only induced strong antibody mediated immune responses but also generated memory T cells in the spleen as adaptive immunity indicating a direct evidence of an effective delivery system. Thus, this study represents the first demonstration of HPMCP for ileum-specific delivery of protein vaccine through oral route.     

Induction of Cell-Mediated Immune Responses to Peptide Antigens of P. vivax in Microparticles Using Intranasal Immunization

T-cells play a critical role in resistance to malaria, not only because they function as helper cells for an antibody response, but also because they serve as effector cells. Such cellular immunity is directly implicated in protection from sporozoites as well as from blood stage parasites. The aim of this study was to induce cell mediated immune responses to peptide antigens of Plasmodium vivax co-encapsulated with CpG oligodeoxynucleotide (ODN) in microparticles. In the present study, we have investigated the immunomodulatory effects of two CpG adjuvants, CpG 1826 and CpG 2006 to the five peptide antigens of Plasmodium vivax derived from circumsporozoite protein, merozoite surface protein-1, apical membrane antigen-1 and gametocyte surface antigen (Pvs24) in microparticle delivery. The T-cell proliferation response study of the cells collected from spleen, lamina propria and peyer's patches showed significantly high (p<0.001) stimulation index when primed with peptide antigens in microparticles co-encapsulating CpG ODN adjuvant as compared to peptide alone primed mice. The cytokine measurement profile of IFN-γ, TNF-α, IL-2, IL-4 and IL-10 in culture supernatants of cells primed with peptide antigens in microparticles co-encapsulating CpG ODN showed higher levels of IFN- γ followed by TNF-α and IL-2, with relatively low levels of IL-4 and IL-10.     

The storage lesions: From past to future

Red blood cell (RBC) concentrates are stored in additive solutions at 4 ^oC for up to 42 days, whereas platelets concentrates (PCs) are stored at 22 ^oC with continuous agitation for up to 5 to 7 days, according national regulations, and the use or not of pathogen inactivation procedures. Storage induces cellular lesion and alters either RBC or platelet metabolism, and is associated with protein alterations. Some age-related alterations prove reversible, while other changes are irreversible, notably following protein oxidation. It is likely that any irreversible damage affects the blood component quality and thus the transfusion efficiency. Nevertheless, there still exists a debate surrounding the impact of storage lesions, for both RBCs and PCs. Uncertainty is not completely resolved. Several studies show a tendency for poorer outcomes to occur in patients receiving older blood products; however, no clear significant association has yet been demonstrated. The present short review aims to promote a better understanding of the occurrence of storage lesions, with particular emphasis on biochemical modifications opening discussions of the future advancement of blood transfusion processes. The paper is also an advocacy for the implementation of an independent international organization in charge of planning and controlling clinical studies in transfusion medicine, in order to base transfusion medicine practices both on security principles, but also on clinical evidences.     

Peripheral blood mononuclear cell proliferative responses to soluble and particulate heat shock protein 65 in health and inflammatory bowel disease

Objectives: The aims of this study were to determine, in peripheral blood mononuclear cells (PBMC), whether particulate antigen triggers (i) an amplified cell proliferative response compared to soluble antigen and (ii) a dysfunctional response in cells derived from patients with chronic inflammation and specifically in those with inflammatory bowel disease (IBD). Subjects: Healthy volunteers (n = 17), inflammatory controls (n = 8) and patients with IBD (n = 17) were recruited from St Thomas’ and Guys’ Hospital, London, UK. Methods: Following optimisation of experimental conditions (0.1–10.0 μg/ml antigen), PBMC were stimulated with (i) 10.0 μg/ml recombinant soluble heat shock protein 65 (hsp 65) and (ii) 1.0 and 10.0 μg/ml hsp 65 conjugated to microparticles (0.5 μm diameter). PBMC proliferative responses were measured by ³H-Thymidine incorporation at day 5 and results compared between groups using unpaired t-test. Results: Conjugation to microparticles of low dose hsp 65 significantly increased overall proliferative responses by 2–11 fold compared to soluble antigen alone (p < 0.05). However, no specific PBMC proliferative dysregulation was noted in cells from subjects with IBD. Conclusions: Low dose antigen, in microparticulate form, leads to amplified cell proliferation in primary human cells, as showed previously in cell lines and animal studies. However there is no abnormal proliferative response in cells from subjects with IBD. Received 8 February 2006; returned for revision 7 March 2006; accepted by G. Wallace 25 October 2006     

Dendritic processing: using microstructures to solve a hitherto intractable neurobiological problem

In vivo, intracellular recordings of mammalian brain stem motoneurones, followed by peroxidase staining and tridimensional reconstruction, suggest that the shape of the dendritic tree plays an important role in the processing of neural information. To test this hypothesis attempts were made to guide, in culture, the growth of neuritic branches of neurones dissociated from the hypoglossal nucleus of rat brain stem. This was performed using topographical and adhesive microstructures which were designed to control the shape of the neuritic tree. Guidance of the neuritic processes can be observed with small grooves engraved on quartz and plastic substrates, and simple shapes with few processes and bifurcations on each neurite could be obtained using adhesive microstructures. These procedures, which allow the shape of a neurone to be controlled, are very promising in the study, by means of classical electrophysiological methods as well as optical recordings, of the involvement of dendritic architecture in the processing of neural information.     

Electrosprayed Hydroxyapatite on Polymer Nanofibers to Differentiate Mesenchymal Stem Cells to Osteogenesis

Abstract

Electrospraying of hydroxyapatite (HA) nanoparticles onto the surface of polymer nanofibers provides a potentially novel substrate for the adhesion, proliferation and differentiation of mesenchymal stem cells (MSCs) into bone tissue regeneration. HA nanoparticles (4%) were electrosprayed on the surface of electrospun polycaprolactone (PCL) nanofibers (420 ± 15 nm) for bone tissue engineering. PCL/HA nanofibers were comparatively characterized with PCL/Collagen (275 ± 56 nm) nanofibers by FT-IR analysis to confirm the presence of HA. Fabricated PCL/HA and PCL/Collagen nanofibers and TCP (control) were used for the differentiation of equine MSC into osteogenic lineages in the presence of DMEM/F12 medium supplemented with β-glycerophosphate, ascorbic acid and dexamethasone. Cell proliferation and differentiation into an osteogenic lineage was evaluated by MTS assay, SEM observation, ALP activity, ARS staining, quantification of mineral deposition and expression of osteocalcin. Proliferation of MSCs increased significantly (P ? 0.05) up to 12% in PCL/Collagen (day 15) compared to PCL/HA nanofibrous substrate. ALP activity was increased 20% in PCL/HA by day 10 confirming the direction of osteogenic lineage from MSCs differentiation. PCL/HA stimulated an increased mineral secretion up to 26% by day 15 on ARS staining compared to PCL/Collagen nanofibers and showing cuboidal morphology by expressing osteocalcin. These results confirmed that the specifically fabricated PCL/HA composite nanofibrous substrate enhanced the differentiation of MSCs into osteogenesis.     

Polymer Melting Kinetics Appears to be Driven by Heterogeneous Nucleation

A recently proposed method is used to parameterize the nucleation‐driven kinetics of poly(ε‐caprolactone) melting. The method is based on fitting a theoretically derived temperature dependence of the effective activation energy to the experimental dependence obtained from the Kissinger plot. Although the theoretical dependence fits the experimental one accurately, the fit gives rise to a surface free energy that is significantly smaller than the one obtained from crystallization data. A similar result is reported in other publications that make use of a homogeneous nucleation model for poly­mer melting. It is argued that a heterogeneous nucleation model is a more appropriate representation of the melting process and that its use resolves the problem of estimating the unusually small surface free energies.

     

In Situ Emulsion Polymerization to Multifunctional Polymer Nanocomposites: A Review

Polymer nanocomposites are a class of composite materials containing polymer continuous phase as matrix and inorganic nanoparticles as fillers. Combining the advantages of both polymer and nanofillers, polymer nanocomposites can yield some desirable functionalities that cannot be achieved by the individual components, thus exhibiting tremendous potential for a variety of applications. However, their performances markedly depend on the dispersion and distribution of nanofillers in the polymer matrix. Recently, in situ emulsion polymerization has been demonstrated to be a powerful approach for the preparation of multifunctional polymer nanocomposites with good dispersion and homogeneous distribution of nanofillers. In this review, the fundamentals of polymer nanocomposites and their preparation strategies are briefly discussed, and then are comprehensively summarized recent advances in the design, preparation, functionalization, and applications of polymer nanocomposites via in situ emulsion polymerization. The particular focus is placed on the strategies for achieving homogeneous and good dispersion of nanofillers within polymer matrixes. Moreover, the current challenges and future opportunities of in situ emulsion polymerization to polymer nanocomposites are discussed to motivate future contributions and explore new possibilities.