Site-Directed Differentiation of Human Adipose-Derived Mesenchymal Stem Cells to Nucleus Pulposus Cells Using an Injectable Hydroxyl-Functional Diblock Copolymer Worm Gel.

Adipose-derived mesenchymal stem cells (ASCs) have been identified for their promising therapeutic potential to regenerate and repopulate the degenerate intervertebral disk (IVD), which is a major cause of lower back pain. The optimal cell delivery system remains elusive but encapsulation of cells within scaffolds is likely to offer a decisive advantage over the delivery of cells in solution by ensuring successful retention within the tissue. Herein, we evaluate the use of a fully synthetic, thermoresponsive poly(glycerol monomethacrylate)-poly(2-hydroxypropyl methacrylate) (PGMA-PHPMA) diblock copolymer worm gel that mimics the structure of hydrophilic glycosaminoglycans. The objective was to use this gel to direct differentiation of human ASCs toward a nucleus pulposus (NP) phenotype, with or without the addition of discogenic growth factors TGFß or GDF6. Accordingly, human ASCs were incorporated into a cold, free-flowing aqueous dispersion of the diblock copolymer, gelation induced by warming to 37 °C and cell culture was conducted for 14 days with or without such growth factors to assess the expression of characteristic NP markers compared to those produced when using collagen gels. In principle, the shear-thinning nature of the biocompatible worm gel enables encapsulated human ASCs to be injected into the IVD using a 21G needle. Moreover, we find significantly higher gene expression levels of ACAN, SOX-9, KRT8, and KR18 for ASCs encapsulated within worm gels compared to collagen scaffolds, regardless of the growth factors employed. In summary, such wholly synthetic worm gels offer considerable potential as an injectable cell delivery scaffold for the treatment of degenerate disk disease by promoting the transition of ASCs toward an NP-phenotype.

A Single Thermoresponsive Diblock Copolymer Can Form Spheres, Worms or Vesicles in Aqueous Solution.

It is well-known that the self-assembly of AB diblock copolymers in solution can produce various morphologies depending on the relative volume fraction of each block. Recently, polymerization-induced self-assembly (PISA) has become widely recognized as a powerful platform technology for the rational design and efficient synthesis of a wide range of block copolymer nano-objects. In this study, PISA is used to prepare a new thermoresponsive poly(N-(2-hydroxypropyl) methacrylamide)-poly(2-hydroxypropyl methacrylate) [PHPMAC-PHPMA] diblock copolymer. Remarkably, TEM, rheology and SAXS studies indicate that a single copolymer composition can form well-defined spheres (4 °C), worms (22 °C) or vesicles (50 °C) in aqueous solution. Given that the two monomer repeat units have almost identical chemical structures, this system is particularly well-suited to theoretical analysis. Self-consistent mean field theory suggests this rich self-assembly behavior is the result of the greater degree of hydration of the PHPMA block at lower temperature, which is in agreement with variable temperature 1 H NMR studies.

Synthesis of polyacid nanogels: pH-responsive sub-100 nm particles for functionalisation and fluorescent hydrogel assembly.

Nanogels are crosslinked polymer particles with a swollen size between 1 and 100 nm. They are of major interest for advanced surface coatings, drug delivery, diagnostics and biomaterials. Synthesising polyacid nanogels that show triggered swelling using a scalable approach is a key objective of polymer colloid chemistry. Inspired by the ability of polar surfaces to enhance nanoparticle stabilisation, we report the first examples of pH-responsive polyacid nanogels containing high -COOH contents prepared by a simple, scalable, aqueous method. To demonstrate their functionalisation potential, glycidyl methacrylate was reacted with the -COOH chemical handles and the nanogels were converted to macro-crosslinkers. The concentrated (functionalised) nanogel dispersions retained their pH-responsiveness, were shear-thinning and formed physical gels at pH 7.4. The nanogels were covalently interlinked via free-radical coupling at 37 °C to form transparent, ductile, hydrogels. Mixing of the functionalised nanogels with polymer dots enabled covalent assembly of fluorescent hydrogels.

Occlusion of Sulfate-Based Diblock Copolymer Nanoparticles within Calcite: Effect of Varying the Surface Density of Anionic Stabilizer Chains.

Polymerization-induced self-assembly (PISA) offers a highly versatile and efficient route to a wide range of organic nanoparticles. In this article, we demonstrate for the first time that poly(ammonium 2-sulfatoethyl methacrylate)-poly(benzyl methacrylate) [PSEM-PBzMA] diblock copolymer nanoparticles can be prepared with either a high or low PSEM stabilizer surface density using either RAFT dispersion polymerization in a 2:1 v/v ethanol/water mixture or RAFT aqueous emulsion polymerization, respectively. We then use these model nanoparticles to gain new insight into a key topic in materials chemistry: the occlusion of organic additives into inorganic crystals. Substantial differences are observed for the extent of occlusion of these two types of anionic nanoparticles into calcite (CaCO3), which serves as a suitable model host crystal. A low PSEM stabilizer surface density leads to uniform nanoparticle occlusion within calcite at up to 7.5% w/w (16% v/v), while minimal occlusion occurs when using nanoparticles with a high PSEM stabilizer surface density. This counter-intuitive observation suggests that an optimum anionic surface density is required for efficient occlusion, which provides a hitherto unexpected design rule for the incorporation of nanoparticles within crystals.

Inducing an Order-Order Morphological Transition via Chemical Degradation of Amphiphilic Diblock Copolymer Nano-Objects.

The disulfide-based cyclic monomer, 3-methylidene-1,9-dioxa-5,12,13-trithiacyclopentadecane-2,8-dione (MTC), is statistically copolymerized with 2-hydroxypropyl methacrylate to form a range of diblock copolymer nano-objects via reversible addition-fragmentation chain transfer (RAFT) polymerization. Poly(glycerol monomethacrylate) (PGMA) is employed as the hydrophilic stabilizer block in this aqueous polymerization-induced self-assembly (PISA) formulation, which affords pure spheres, worms or vesicles depending on the target degree of polymerization for the core-forming block. When relatively low levels (<1 mol %) of MTC are incorporated, high monomer conversions (>99%) are achieved and high blocking efficiencies are observed, as judged by (1)H NMR spectroscopy and gel permeation chromatography (GPC), respectively. However, the side reactions that are known to occur when cyclic allylic sulfides such as MTC are statistically copolymerized with methacrylic comonomers lead to relatively broad molecular weight distributions. Nevertheless, the worm-like nanoparticles obtained via PISA can be successfully transformed into spherical nanoparticles by addition of excess tris(2-carboxyethyl)phosphine (TCEP) at pH 8-9. Surprisingly, DLS and TEM studies indicate that the time scale needed for this order-order transition is significantly longer than that required for cleavage of the disulfide bonds located in the worm cores indicated by GPC analysis. This reductive degradation pathway may enable the use of these chemically degradable nanoparticles in biomedical applications, such as drug delivery systems and responsive biomaterials.

pH-responsive non-ionic diblock copolymers: ionization of carboxylic acid end-groups induces an order-order morphological transition.

A carboxylic acid based reversible additionfragmentation transfer (RAFT) agent is used to prepare gels composed of worm-like diblock copolymers using two non-ionic monomers, glycerol monomethacrylate (GMA) and 2-hydroxypropyl methacrylate (HPMA). Ionization of the carboxylic acid end-group on the PGMA stabilizer block induces a worm-to-sphere transition, which in turn causes immediate degelation. This morphological transition is fully reversible as determined by TEM and rheology studies and occurs because of a subtle change in the packing parameter for the copolymer chains. A control experiment where the methyl ester derivative of the RAFT agent is used to prepare the same diblock copolymer confirms that no pH-responsive behavior occurs in this case. This end-group ionization approach is important for the design of new pH-responsive copolymer nano-objects as, unlike polyacids or polybases, only a minimal amount of added base (or acid) is required to drive the morphological transition.

Importance of Graphical User Interface in the design of P300 based Brain-Computer Interface systems.

OBJECTIVES: Develop an effective and intuitive Graphical User Interface (GUI) for a Brain-Computer Interface (BCI) system, that achieves high classification accuracy and Information Transfer Rates (ITRs), while using a simple classification technique. Objectives also include the development of an output device, that is capable of real time execution of the selected commands. METHODS: A region based T9 BCI system with familiar face presentation cues capable of eliciting strong P300 responses was developed. Electroencephalogram (EEG) signals were collected from the Oz, POz, CPz and Cz electrode locations on the scalp and subsequently filtered, averaged and used to extract two features. These feature sets were classified using the Nearest Neighbour Approach (NNA). To complement the developed BCI system, a 'drone prototype' capable of simulating six different movements, each over a range of eight distinct selectable distances, was also developed. This was achieved through the construction of a body with 4 movable legs, capable of tilting the main body forward, backward, up and down, as well as a pointer capable of turning left and right. RESULTS: From ten participants, with normal or corrected to normal vision, an average accuracy of 91.3 ± 4.8% and an ITR of 2.2 ± 1.1 commands/minute (12.2 ± 6.0 bits/minute) was achieved. CONCLUSION: The proposed system was shown to elicit strong P300 responses. When compared to similar P300 BCI systems, which utilise a variety of more complex classifiers, competitive accuracy and ITR results were achieved, implying the superiority of the proposed GUI. SIGNIFICANCE: This study supports the hypothesis that more research, time and care should be taken when developing GUIs for BCI systems.

Critical Dependence of Molecular Weight on Thermoresponsive Behavior of Diblock Copolymer Worm Gels in Aqueous Solution.

Reversible addition-fragmentation chain transfer (RAFT) aqueous dispersion polymerization of 2-hydroxypropyl methacrylate was used to prepare three poly(glycerol monomethacrylate) x -poly(2-hydroxypropyl methacrylate) y (denoted G x -H y  or PGMA-PHPMA) diblock copolymers, namely G37-H80, G54-H140, and G71-H200. A master phase diagram was used to select each copolymer composition to ensure that a pure worm phase was obtained in each case, as confirmed by transmission electron microscopy (TEM) and small-angle x-ray scattering (SAXS) studies. The latter technique indicated a mean worm cross-sectional diameter (or worm width) ranging from 11 to 20 nm as the mean degree of polymerization (DP) of the hydrophobic PHPMA block was increased from 80 to 200. These copolymer worms form soft hydrogels at 20 °C that undergo degelation on cooling. This thermoresponsive behavior was examined using variable temperature DLS, oscillatory rheology, and SAXS. A 10% w/w G37-H80 worm dispersion dissociated to afford an aqueous solution of molecularly dissolved copolymer chains at 2 °C; on returning to ambient temperature, these chains aggregated to form first spheres and then worms, with the original gel strength being recovered. In contrast, the G54-H140 and G71-H200 worms each only formed spheres on cooling to 2 °C, with thermoreversible (de)gelation being observed in the former case. The sphere-to-worm transition for G54-H140 was monitored by variable temperature SAXS: these experiments indicated the gradual formation of longer worms at higher temperature, with a concomitant reduction in the number of spheres, suggesting worm growth via multiple 1D sphere-sphere fusion events. DLS studies indicated that a 0.1% w/w aqueous dispersion of G71-H200 worms underwent an irreversible worm-to-sphere transition on cooling to 2 °C. Furthermore, irreversible degelation over the time scale of the experiment was also observed during rheological studies of a 10% w/w G71-H200 worm dispersion. Shear-induced polarized light imaging (SIPLI) studies revealed qualitatively different thermoreversible behavior for these three copolymer worm dispersions, although worm alignment was observed at a shear rate of 10 s-1 in each case. Subsequently conducting this technique at a lower shear rate of 1 s-1 combined with ultra small-angle x-ray scattering (USAXS) also indicated that worm branching occurred at a certain critical temperature since an upturn in viscosity, distortion in the birefringence, and a characteristic feature in the USAXS pattern were observed. Finally, SIPLI studies indicated that the characteristic relaxation times required for loss of worm alignment after cessation of shear depended markedly on the copolymer molecular weight.

Bespoke contrast-matched diblock copolymer nanoparticles enable the rational design of highly transparent Pickering double emulsions.

We report the preparation of highly transparent oil-in-water Pickering emulsions using contrast-matched organic nanoparticles. This is achieved via addition of judicious amounts of either sucrose or glycerol to an aqueous dispersion of poly(glycerol monomethacrylate)56-poly(2,2,2-trifluoroethyl methacrylate)500 [PGMA-PTFEMA] diblock copolymer nanoparticles prior to high shear homogenization with an equal volume of n-dodecane. The resulting Pickering emulsions comprise polydisperse n-dodecane droplets of 20-100 µm diameter and exhibit up to 96% transmittance across the visible spectrum. In contrast, control experiments using non-contrast-matched poly(glycerol monomethacrylate)56-poly(benzyl methacrylate)300 [PGMA56-PBzMA300] diblock copolymer nanoparticles as a Pickering emulsifier only produced conventional highly turbid emulsions. Thus contrast-matching of the two immiscible phases is a necessary but not sufficient condition for the preparation of highly transparent Pickering emulsions: it is essential to use isorefractive nanoparticles in order to minimize light scattering. Furthermore, highly transparent oil-in-water-in-oil Pickering double emulsions can be obtained by homogenizing the contrast-matched oil-in-water Pickering emulsion prepared using the PGMA56-PTFEMA500 nanoparticles with a contrast-matched dispersion of hydrophobic poly(lauryl methacrylate)39-poly(2,2,2-trifluoroethyl methacrylate)800 [PLMA39-PTFEMA800] diblock copolymer nanoparticles in n-dodecane. Finally, we show that an isorefractive oil-in-water Pickering emulsion enables fluorescence spectroscopy to be used to monitor the transport of water-insoluble small molecules (pyrene and benzophenone) between n-dodecane droplets. Such transport is significantly less efficient than that observed for the equivalent isorefractive surfactant-stabilized emulsion. Conventional turbid emulsions do not enable such a comparison to be made because the intense light scattering leads to substantial spectral attenuation.