3D mesenchymal stem/stromal cell osteogenesis and autocrine signalling

Mesenchymal stem/stromal cells (MSC) are rapidly becoming a leading candidate for use in tissue regeneration, with first generation of therapies being approved for use in orthopaedic repair applications. Capturing the full potential of MSC will likely require the development of novel in vitro culture techniques and devices. Herein we describe the development of a straightforward surface modification of an existing commercial product to enable the efficient study of three dimensional (3D) human bone marrow-derived MSC osteogenic differentiation. Hundreds of 3D microaggregates, of either 42 or 168 cells each, were cultured in osteogenic induction medium and their differentiation was compared with that occurring in traditional two dimensional (2D) monolayer cultures. Osteogenic gene expression and matrix composition was significantly enhanced in the 3D microaggregate cultures. Additionally, BMP-2 gene expression was significantly up-regulated in 3D cultures at day 3 and 7 by approximately 25- and 30-fold, respectively. The difference in BMP-2 gene expression between 2D and 3D cultures was negligible in the more mature day 14 osteogenic cultures. These data support the notion that BMP-2 autocrine signalling is up-regulated in 3D MSC cultures, enhancing osteogenic differentiation. This study provides both mechanistic insight into MSC differentiation, as well as a platform for the efficient generation of microtissue units for further investigation or use in tissue engineering applications.

The interplay between chondrocyte redifferentiation pellet size and oxygen concentration

Chondrocytes dedifferentiate during ex vivo expansion on 2-dimensional surfaces. Aggregation of the expanded cells into 3-dimensional pellets, in the presence of induction factors, facilitates their redifferentiation and restoration of the chondrogenic phenotype. Typically 1×105–5×105 chondrocytes are aggregated, resulting in “macro” pellets having diameters ranging from 1–2 mm. These macropellets are commonly used to study redifferentiation, and recently macropellets of autologous chondrocytes have been implanted directly into articular cartilage defects to facilitate their repair. However, diffusion of metabolites over the 1–2 mm pellet length-scales is inefficient, resulting in radial tissue heterogeneity. Herein we demonstrate that the aggregation of 2×105 human chondrocytes into micropellets of 166 cells each, rather than into larger single macropellets, enhances chondrogenic redifferentiation. In this study, we describe the development of a cost effective fabrication strategy to manufacture a microwell surface for the large-scale production of micropellets. The thousands of micropellets were manufactured using the microwell platform, which is an array of 360×360 µm microwells cast into polydimethylsiloxane (PDMS), that has been surface modified with an electrostatic multilayer of hyaluronic acid and chitosan to enhance micropellet formation. Such surface modification was essential to prevent chondrocyte spreading on the PDMS. Sulfated glycosaminoglycan (sGAG) production and collagen II gene expression in chondrocyte micropellets increased significantly relative to macropellet controls, and redifferentiation was enhanced in both macro and micropellets with the provision of a hypoxic atmosphere (2% O2). Once micropellet formation had been optimized, we demonstrated that micropellets could be assembled into larger cartilage tissues. Our results indicate that micropellet amalgamation efficiency is inversely related to the time cultured as discreet microtissues. In summary, we describe a micropellet production platform that represents an efficient tool for studying chondrocyte redifferentiation and demonstrate that the micropellets could be assembled into larger tissues, potentially useful in cartilage defect repair.

Supported gold nanoparticles as photocatalysts utilising the full solar spectrum for organic synthesis

This thesis is an innovative study for organic synthesis using supported gold nanoparticles as photocatalysts under visible light irradiation. It especially examines a novel green process for efficient hydroamination of alkynes with amines. The investigation of other traditional reduction and oxidation reactions also adds significantly to the knowledge of gold nanoparticles and titania nanofibres as photocatalysts for organic synthesis.

Modified alumina nanofiber membranes for protein separation

Large-scale purification/separation of bio-substances is a key technology required for rapid production of biological substances in bioengineering. Membrane filtration is a new separation process and has potential to be used for concentration (removal of solvent), desalting (removal of low molecular weight compounds), clarification (removal of particles), and fractionation (protein-protein separation). In this study, we developed an efficient membrane for protein separation based on ceramic nanofibers. Alumina nanofibers were prepared on a porous support and formed large flow passages. The radical changes in membrane structure provided new ceramic membranes with a large porosity (more than 70%) due to the replacement of bulk particles with fine fibers as building components. The pore size had an average of 11 nm and pure water flux was approximately 360 L•h-1•m-2•bar-1. Further surface modification with a self-assembled monolayer of (3-aminopropyl) triethoxysilane enhanced the membrane filtration properties. Characterization with SEM, FTIR, contact angle, and proteins separation tests indicated that the fibril layers uniformly spread on the surface of the porous support. Moreover, the membrane surface was changed from hydrophilic to hydrophobic after silane groups were grafted. It demonstrated that the silane-grafted alumina fiber membrane can reject 100% BSA protein and 92% cellulase protein. It was also able to retain 75% trypsin protein while maintaining a permeation flux of 48 L•h-1•m-2•bar-1.

A study of localised galvanic replacement of copper and silver films with gold using scanning electrochemical microscopy

Galvanic replacement represents a highly significant process for the fabrication of bimetallic materials, but to date its application has been limited to either modification of large area metal surfaces or nanoparticles in solution. Here, the localised surface modification of copper and silver substrates with gold through the galvanic replacement process is reported. This was achieved by generation of a localised flux of AuCl4− ions from a gold ultramicroelectrode tip which interacts with the unbiased substrate of interest. The extent of modification with gold can be controlled through the tip–substrate distance and electrolysis time.

The facile formation of silver dendritic structures in the absence of surfactants and their electrochemical and SERS properties

In this work a simple approach to the creation of highly dispersed electrocatalytically active silver microstructured dendrites on indium tin oxide in the absence of any surface modification or surfactant is presented. It is found that the addition of low concentrations of supporting electrolyte to the AgNO3 solution dramatically influences the morphology of electrodeposited silver which is independent of both the anion and the cation employed. The silver dendrites are characterized by SEM, XRD, XPS as well as by cyclic voltammetry under alkaline conditions. It is found that the surface oxide formation and removal processes are significantly influenced by the microstructured morphology of the silver electrodeposits compared to a smooth macrosized silver electrode. The facile formation of dendritic silver microstructures is also shown to be beneficial for the electrocatalytic oxidation of both formaldehyde and hydrazine and oxygen reduction. The formation of a continuous film of dendritic silver is also investigated for its SERS activity where the connectivity between the individual dendrites is found to be particularly important.

Design, characterisation and in vivo testing of a new, adjustable stiffness, external fixator for the rat femur

Very little is known about the infl uence of the mechanical environment on the healing of large segmental defects. This partly reflects the lack of standardised, well characterised technologies to enable such studies. Here we report the design, construction and characterisation of a novel external fixator for use in conjunction with rat femoral defects. This device not only imposes a predetermined axial stiffness on the lesion, but also enables the stiffness to be changed during the healing process. The main frame of the fi xator consists of polyethylethylketone with titanium alloy mounting pins. The stiffness of the fi xator is determined by interchangeable connection elements of different thicknesses. Fixators were shown to stabilise 5 mm femoral defects in rats in vivo for at least 8 weeks during unrestricted cage activity. No distortion or infections, including pin infections, were noted. The healing process was simulated in vitro by inserting into a 5 mm femoral defect, materials whose Young’s moduli approximated those of the different tissues present in regenerating bone. These studies confirmed that, although the external fixator is the major determinant of axial stiffness during the early phase of healing, the regenerate within the lesion subsequently dominates this property. There is much clinical interest in altering the mechanics of the defect to enhance bone healing. Our data suggest that, if alteration of the mechanical environment is to be used to modulate the healing of large segmental defects, this needs to be performed before the tissue properties become dominant.

Simulation and visualization of self-assembled nanodevice networks synthesized via plasma–surface interaction

Using advanced visualization techniques, a comprehensive visualization of all the stages of the self-organized growth of internetworked nanostructures on plasma-exposed surface has been made. Atomistic kinetic Monte Carlo simulation for the initial stage of deposition, with 3-D visualization of the whole system and half-tone visualization of the density field of the adsorbed atoms, makes it possible to implement a multiscale predictive modeling of the development of the nanoscale system.

Two-dimensional simulation of nanoparticle deposition from high-density plasmas on microstructured surfaces

Selective and controlled deposition of plasma-grown nanoparticles is one of the pressing problems of plasma-aided nanofabrication. The results of advanced numerical simulations of motion of charge-variable nanoparticles in the plasma presheath and sheath areas and in localized microscopic electric fields created by surface microstructures are reported. Conditions for site-selective deposition of such nanoparticles onto individual microstructures and open surface areas within a periodic micropattern are formulated. The effects of plasma parameters, surface potential, and micropattern features on nanoparticle deposition are investigated and explained using particle charging and plasma force arguments. The results are generic and applicable to a broad range of nanoparticle-generating plasmas and practical problems ranging from management of nanoparticle contamination in microelectronics to site-selective nanoparticle deposition into specified device locations, and synthesis of advanced microporous materials and nanoparticle superlattices. © 2007 American Institute of Physics.

Self-assembly of uniform carbon nanotip structures in chemically active inductively coupled plasmas

Self-assembly of carbon nanotip (CNTP) structures on Ni-based catalyst in chemically active inductively coupled plasmas of CH 4 + H 2 + Ar gas mixtures is reported. By varying the process conditions, it appears possible to control the shape, size, and density of CNTPs, content of the nanocrystalline phase in the films, as well as to achieve excellent crystallinity, graphitization, uniformity and vertical alignment of the resulting nanostructures at substrate temperatures 300-500°C and low gas pressures (below 13.2 Pa). This study provides a simple and efficient plasma-enhanced chemical vapor deposition (PECVD) technique for the fabrication of vertically aligned CNTP arrays for electron field emitters.

Flammability and thermal properties of novel semi aromatic polyamide/organoclay nanocomposite

The development of semi aromatic polyamide/organoclays nanocomposites (PANC) is reported in this communication. New polyamide (PA) was successfully synthesized through direct polycondensation reaction between bio-based diacid and aromatic diamine. PA exhibited strong UV vis absorption band at 412 nm. Its photoluminescence spectrum showed maximum band at 511 nm in the green region. The surface modification of montmorillonite was carried out through ion-exchange reaction using 1,4-bis[4-aminophenoxy]butane (APB) as a modifier. Then PANCs containing 3 and 6 wt.% of the modified montmorillonite (MMT-APB) were prepared. Flammability and thermal properties of PA and the nanocomposites were studied by microscale combustion calorimeter (MCC), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). TGA results in both air and nitrogen atmospheres indicated improving in thermal properties of PANCs compared to the neat PA. According to MCC analysis, a 31.6% reduction in pHRR value has been achieved by introducing 6 wt.% of the organoclay in PA matrix.

3D cultures of prostate cancer cells cultured in a novel high-throughput culture platform are more resistant to chemotherapeutics compared to cells cultured in monolayer

Despite monolayer cultures being widely used for cancer drug development and testing, 2D cultures tend to be hypersensitive to chemotherapy and are relatively poor predictors of whether a drug will provide clinical benefit. Whilst generally more complicated, three dimensional (3D) culture systems often better recapitulate true cancer architecture and provide a more accurate drug response. As a step towards making 3D cancer cultures more accessible, we have developed a microwell platform and surface modification protocol to enable high throughput manufacture of 3D cancer aggregates. Herein we use this novel system to characterize prostate cancer cell microaggregates, including growth kinetics and drug sensitivity. Our results indicate that prostate cancer cells are viable in this system, however some non-cancerous prostate cell lines are not. This system allows us to consistently control for the presence or absence of an apoptotic core in the 3D cancer microaggregates. Similar to tumor tissues, the 3D microaggregates display poor polarity. Critically the response of 3D microaggregates to the chemotherapeutic drug, docetaxel, is more consistent with in vivo results than the equivalent 2D controls. Cumulatively, our results demonstrate that these prostate cancer microaggregates better recapitulate the morphology of prostate tumors compared to 2D and can be used for high-throughput drug testing.

Reproducible and label-free biosensor for the selective extraction and rapid detection of proteins in biological fluids

Erythropoietin (EPO), a glycoprotein hormone of ∼34 kDa, is an important hematopoietic growth factor, mainly produced in the kidney and controls the number of red blood cells circulating in the blood stream. Sensitive and rapid recombinant human EPO (rHuEPO) detection tools that improve on the current laborious EPO detection techniques are in high demand for both clinical and sports industry. A sensitive aptamer-functionalized biosensor (aptasensor) has been developed by controlled growth of gold nanostructures (AuNS) over a gold substrate (pAu/AuNS). The aptasensor selectively binds to rHuEPO and, therefore, was used to extract and detect the drug from horse plasma by surface enhanced Raman spectroscopy (SERS). Due to the nanogap separation between the nanostructures, the high population and distribution of hot spots on the pAu/AuNS substrate surface, strong signal enhancement was acquired. By using wide area illumination (WAI) setting for the Raman detection, a low RSD of 4.92% over 150 SERS measurements was achieved. The significant reproducibility of the new biosensor addresses the serious problem of SERS signal inconsistency that hampers the use of the technique in the field. The WAI setting is compatible with handheld Raman devices. Therefore, the new aptasensor can be used for the selective extraction of rHuEPO from biological fluids and subsequently screened with handheld Raman spectrometer for SERS based in-field protein detection.

Cytotoxic Effects and Biocompatibility of Antimicrobial Materials

The rising demand for medical implants for ageing populations and ongoing advancements in medical technology continue to drive the use of implantable devices. Higher implant usage has a consequent increased incidence of implant-related infections, and associated prolonged patient care, pain and loss of limb and other organ function. Numerous antibacterial surfaces have been designed that prevent the onset of biofilm formation, thus reducing or preventing implant-associated infections through inhibiting bacterial adhesion or by killing the organisms that successfully attach to the surface of the implant. Other surfaces have been designed to stimulate a local immune response, promoting the natural clearing of the invading pathogen. The desired antibacterial effects are typically achieved by modulating the surface chemistry and morphology of the implant material, by means of the controlled release of pharmacological agents and bioactive compounds from the surface of the material, or by a combination of both processes. An important issue for any type of antibacterial surface modification lies in balancing the non-fouling, bacteriostatic or bactericidal effects against local and systemic biocompatibility. In this chapter, we will first describe the concept of biocompatibility and its evolution, from devices that do not evoke a negative host response to those that actively drive host regeneration. We will then review the challenges associated with merging the need for an implant material to withstand a bacterial load with those associated with supporting function restoration and tissue healing.