Molly M Stevens

Imperial College London, Londinium, England, United Kingdom

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Publications (198)1769.17 Total impact

  • Martin A. B. Hedegaard · Mads S. Bergholt · Molly M. Stevens
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    ABSTRACT: Imaging by Raman spectroscopy enables unparalleled label-free insights into cell and tissue composition at the molecular level. With established approaches limited to single image analysis, there are currently no general guidelines or consensus on how to quantify biochemical components across multiple Raman images. Here, we describe a broadly applicable methodology for the combination of multiple Raman images into a single image for analysis. This is achieved by removing image specific background interference, unfolding the series of Raman images into a single dataset, and normalisation of each Raman spectrum to render comparable Raman images. Multivariate image analysis is finally applied to derive the contributing 'pure' biochemical spectra for relative quantification. We present our methodology using four independently measured Raman images of control cells and four images of cells treated with strontium ions from substituted bioactive glass. We show that the relative biochemical distribution per area of the cells can be quantified. In addition, using k-means clustering, we are able to discriminate between the two cell types over multiple Raman images. This study shows a streamlined quantitative multi-image analysis tool for improving cell/tissue characterisation and opens new avenues in biomedical Raman spectroscopic imaging.
    No preview · Article · Mar 2016 · Journal of Biophotonics
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    Full-text · Dataset · Feb 2016
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    ABSTRACT: Free-standing serum-albumin mats can transport protons over millimetre length-scales. The results of photoinduced proton transfer and voltage-driven proton conductivity measurements, together with temperature dependent and isotope effect studies, suggest that oxo-amino-acids of the protein serum albumin play a major role in the translocation of protons via an "over-the-barrier" hopping mechanism. The use of proton-conducting protein mats opens new possibilities for bioelectronic interfaces.
    No preview · Article · Feb 2016 · Advanced Materials
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    ABSTRACT: Secretory phospholipase A2 group IIA (sPLA2-IIA) was examined as a point of care marker for determining disease activity in rheumatoid (RA) and psoriatic (PsA) arthritis. Serum concentration and activity of sPLA2-IIA were measured using in-house antibodies and a novel point of care lateral flow device assay in patients diagnosed with varying severities of RA (n = 30) and PsA (n = 25) and found to correlate strongly with C-reactive protein (CRP). Levels of all markers were elevated in patients with active RA over those with inactive RA as well as both active and inactive PsA, indicating that sPLA2-IIA can be used as an analogue to CRP for RA diagnosis at point of care.
    No preview · Article · Feb 2016 · Nanoscale
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    ABSTRACT: Biophysical signals act as potent regulators of stem cell function, lineage commitment, and epigenetic status. In recent years, synthetic biomaterials have been used to study a wide range of outside-in signaling events, and it is now well appreciated that material cues modulate the epigenome. Here, we review the role of extracellular signals in guiding stem cell behavior via epigenetic regulation, and we stress the role of physicochemical material properties as an often-overlooked modulator of intracellular signaling. We also highlight promising new research tools for ongoing interrogation of the stem cell-material interface.
    No preview · Article · Jan 2016 · Cell stem cell
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    Preview · Article · Dec 2015 · ACS Nano
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    ABSTRACT: The current study has investigated the use of decellularised, demineralised bone extracellular matrix (ECM) hydrogel constructs for in vivo tissue mineralisation and bone formation. Stro-1-enriched human bone marrow stromal cells were incorporated together with select growth factors including VEGF, TGF-β3, BMP-2, PTHrP and VitD3, to augment bone formation, and mixed with alginate for structural support. Growth factors were delivered through fast (non-osteogenic factors) and slow (osteogenic factors) release PLGA microparticles. Constructs of 5 mm length were implanted in vivo for 28 days within mice. Dense tissue assessed by micro-CT correlated with histologically assessed mineralised bone formation in all constructs. Exogenous growth factor addition did not enhance bone formation further compared to alginate/bone ECM (ALG/ECM) hydrogels alone. UV irradiation reduced bone formation through degradation of intrinsic growth factors within the bone ECM component and possibly also ECM cross-linking. BMP-2 and VitD3 rescued osteogenic induction. ALG/ECM hydrogels appeared highly osteoinductive and delivery of angiogenic or chondrogenic growth factors led to altered bone formation. All constructs demonstrated extensive host tissue invasion and vascularisation aiding integration and implant longevity. The proposed hydrogel system functioned without the need for growth factor incorporation or an exogenous inducible cell source. Optimal growth factor concentrations and spatiotemporal release profiles require further assessment, as the bone ECM component may suffer batch variability between donor materials. In summary, ALG/ECM hydrogels provide a versatile biomaterial scaffold for utilisation within regenerative medicine which may be tailored, ultimately, to form the tissue of choice through incorporation of select growth factors.
    Full-text · Article · Dec 2015 · PLoS ONE
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    ABSTRACT: A microvalve-based bioprinting system for the manufacturing of high-resolution, multimaterial 3D-structures is reported. Applying a straightforward fluid-dynamics model, the shear stress at the nozzle site can precisely be controlled. Using this system, a broad study on how cell viability and proliferation potential are affected by different levels of shear stress is conducted. Complex, multimaterial 3D structures are printed with high resolution. This work pioneers the investigation of shear stress-induced cell damage in 3D bioprinting and might help to comprehend and improve the outcome of cell-printing studies in the future.
    No preview · Article · Dec 2015 · Advanced Healthcare Materials
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    ABSTRACT: Statement of significance: This novelty of this research work is that it provide a comprehensive comparison, both in vitro and in vivo, between 3 different composite materials widely used in the field of bone tissue engineering for their bone regeneration capabilities. The materials used in this study include polycaprolactone, 45S5 Bioglass, strontium-substituted bioactive glass and calcium phosphate. Additionally, the composite materials were fabricated into the form of 3D scaffolds using additive manufacturing technique, a widely used technique in tissue engineering.
    No preview · Article · Nov 2015 · Acta biomaterialia
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    ABSTRACT: HIV-1 protease is a key enzyme in the life cycle of HIV/AIDS, as it is responsible for the formation of the mature virus particle. We demonstrate here that phage-display peptides raised against this enzyme can be used as peptide sensors for the detection of HIV-1 protease in a simple, one-pot assay. The presence of the enzyme is detected through an energy transfer between two peptide sensors when simultaneously complexed with the target protein. The multivalent nature of this assay increases the specificity of the detection by requiring all molecules to be interacting in order for there to be a FRET signal. We also perform molecular dynamics simulations to explore the interaction between the protease and the peptides in order to guide the design of these peptide sensors and to understand the mechanisms which cause these simultaneous binding events. This approach aims to facilitate the development of new assays for enzymes that are not dependent on the cleavage of a substrate and do not require multiple washing steps.
    No preview · Article · Oct 2015 · Chemistry of Materials
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    ABSTRACT: Sol–gel hybrids are inorganic/organic co-networks with nanoscale interactions between the components leading to unique synergistic mechanical properties, which can be tailored, via a selection of the organic moiety. Methacrylate based polymers present several benefits for class II hybrids (which exhibit formal covalent bonding between the networks) as they introduce great versatility and can be designed with a variety of chemical side-groups, structures and morphologies. In this study, the effect of high cross-linking density polymers on the structure–property relationships of hybrids generated using poly(3-trimethoxysilylpropyl methacrylate) (pTMSPMA) and tetraethyl orthosilicate (TEOS) was investigated. The complexity and fine scale of the co-network interactions requires the development of new analytical methods to understand how network evolution dictates the wide-ranging mechanical properties. Within this work we developed data manipulation techniques of acoustic-AFM and solid state NMR output that provide new approaches to understand the influence of the network/structure on the macroscopic elasticity. The concentration of pTMSPMA in the silica sol affected the gelation time, ranging from 2 h for a hybrid made with 75 wt% inorganic with pTMSPMA at 2.5 kDa, to 1 minute for pTMSPMA with molecular weight of 30 kDa without any TEOS. A new mechanism of gelation was proposed based on the different morphologies derived by AC-AFM observations. We established that the volumetric density of bridging oxygen bonds is an important parameter in structure property relationships in SiO2 hybrids and developed a method for determining it from solid state NMR data. The variation in the elasticity of pTMSPMA/SiO2 hybrids originated from pTMSPMA acting as a molecular spacer, thus decreasing the volumetric density of bridging oxygen bonds as the inorganic to organic ratio decreased.
    Full-text · Article · Oct 2015 · Physical Chemistry Chemical Physics
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    ABSTRACT: Conjugated polymers possess excellent qualities as fluorescent probes for biomedical applications, because of their extremely high brightness, extinction coefficients, and photostability. Encapsulating these hydrophobic polymers in nanoparticulate form allows transfer to aqueous environments and construction of high-performance fluorescent nanoparticle constructs, and several surface capping strategies have been demonstrated to date. Here, we describe the development of a new class of multifunctional capping ligands for conjugated polymer nanoparticles based on custom-designed amphiphilic peptides. These versatile peptide ligands provide a protective hydrophilic capping layer, chemical handles for further conjugation, and directed biological activity tuned by altering the specific amino acid sequence. We show that (i) cellular uptake can be regulated as a function of peptide composition, and (ii) the nanoparticles show no signs of toxicity under the conditions used, which is a vital health and environmental issue when developing these technologies for clinical use. Finally, we demonstrate that this one-pot method can be applied can be applied to three classes of conjugated polymers and demonstrate potential for multicolor imaging.
    No preview · Article · Sep 2015 · Chemistry of Materials
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    ABSTRACT: The development of decellularised scaffolds for small diameter vascular grafts is hampered by their limited patency, due to the lack of luminal cell coverage by endothelial cells (EC) and to the low tone of the vessel due to absence of a contractile smooth muscle cells (SMC). In this study, we identify a population of vascular progenitor c-Kit+/Sca-1- cells available in large numbers and derived from immuno-privileged embryonic stem cells (ESCs). We also define an efficient and controlled differentiation protocol yielding fully to differentiated ECs and SMCs in sufficient numbers to allow the repopulation of a tissue engineered vascular graft. When seeded ex vivo on a decellularised vessel, c-Kit+/Sca-1-derived cells recapitulated the native vessel structure and upon in vivo implantation in the mouse, markedly reduced neointima formation and mortality, restoring functional vascularisation. We showed that Krüppel-like transcription factor 4 (Klf4) regulates the choice of differentiation pathway of these cells through β-catenin activation and was itself regulated by the canonical Wnt pathway activator lithium chloride. Our data show that ESC-derived c-Kit+/Sca-1-cells can be differentiated through a Klf4/β-catenin dependent pathway and are a suitable source of vascular progenitors for the creation of superior tissue-engineered vessels from decellularised scaffolds. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Aug 2015 · Biomaterials
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    ABSTRACT: The layer-by-layer (LbL) technique is a simple and robust process for fabricating functional multilayer thin films. Here, we report the use of de novo designed polypeptides that self-assemble into coiled-coil structures (four-helix bundles) as a driving force for specific multilayer assembly. These pH- (sensitive between pH 4 and 7) and enzyme-responsive polypeptides were conjugated to polymers, and the LbL assembly of the polymer–peptide conjugates allowed the deposition of up to four polymer–peptide layers on planar surfaces and colloidal substrates. Stable hollow capsules were obtained, and by taking advantage of the peptide’s susceptibility to specific enzymatic cleavage, release of encapsulated cargo within the carriers can be triggered within 2 h in the presence of matrix metalloproteinase-7. The enormous diversity of materials that can form highly controllable and programmable coiled-coil interactions creates new opportunities and allows further exploration of the multilayer assembly and the formation of carrier capsules with unique functional properties.
    Full-text · Article · Aug 2015 · Chemistry of Materials
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    ABSTRACT: Angiogenesis, the formation of blood vessels from pre-existing ones, is of vital importance during the early stages of bone healing. Extracellular stiffness plays an important role in regulating endothelial cell behavior and angiogenesis, but how this mechanical cue affects proliferation kinetics, gene regulation, and the expression of proteins implicated in angiogenesis and bone regeneration remains unclear. Using collagen-coated polyacrylamide (PAAm) hydrogels, human umbilical vein endothelial cells (HUVECs) are exposed to an environment that mimics the elastic properties of collagenous bone, and cellular proliferation and gene and protein expressions are assessed. The proliferation and gene expression of HUVECs are not differentially affected by culture on 3 or 30 kPa PAAm hydrogels, henceforth referred to as low and high stiffness gels, respectively. Although the proliferation and gene transcript levels remain unchanged, significant differences are found in the expressions of functional proteins and growth factors implicated both in the angiogenic and osteogenic processes. The down-regulation of the vascular endothelial growth factor receptor-2 protein with concomitant over-expression of caveolin-1, wingless-type 2, bone morphogenic protein 2, and basic fibroblast growth factor on the high stiffness PAAm hydrogel suggests that rigidity has a pro-angiogenic effect with inherent benefits for bone regeneration. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    No preview · Article · Aug 2015 · Advanced Healthcare Materials
  • Nadav Amdursky · Molly M. Stevens
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    ABSTRACT: Circular dichroism (CD) is frequently used to assess the secondary structure of peptides and proteins, whereas less attention has been given to their building blocks, that is, single amino acids, as they do not possess a secondary structure. Here, we follow the CD signal of amino acids and reveal that several acids exhibit a unique CD pattern as a function of their concentration. Accordingly, we propose an eight-level classification of the CD signal of the various amino acids. Special focus is given to the CD pattern of phenylalanine (Phe), for which we observe the formation of an ultra-narrow CD peak (full width at high maximum of only 5 nm). This CD peak can be attributed to the formation of Phe-based chiral structural features. Further support for the formation of an ordered structure is given by using NMR, and the additional self-assembly process of Phe to tubular structures. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    No preview · Article · Aug 2015 · ChemPhysChem
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    ABSTRACT: Generation of de novo cardiomyocytes through viral over-expression of key transcription factors represents a highly promising strategy for cardiac muscle tissue regeneration. Although the feasibility of cell reprogramming has been proven possible both in vitro and in vivo, the efficiency of the process remains extremely low. Here, we report a chemical-free technique in which topographical cues, more specifically parallel microgrooves, enhance the directed differentiation of cardiac progenitors into cardiomyocyte-like cells. Using a lentivirus-mediated direct reprogramming strategy for expression of Myocardin, Tbx5, and Mef2c, we showed that the microgrooved substrate provokes an increase in histone H3 acetylation (AcH3), known to be a permissive environment for reprogramming by "stemness" factors, as well as stimulation of myocardin sumoylation, a post-translational modification essential to the transcriptional function of this key co-activator. These biochemical effects mimicked those of a pharmacological histone deacetylase inhibitor, valproic acid (VPA), and like VPA markedly augmented the expression of cardiomyocyte-specific proteins by the genetically engineered cells. No instructive effect was seen in cells unresponsive to VPA. In addition, the anisotropy resulting from parallel microgrooves induced cellular alignment, mimicking the native ventricular myocardium and augmenting sarcomere organization. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.
    No preview · Article · Aug 2015 · Biomaterials
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    ABSTRACT: Native tissues are typically heterogeneous and hierarchically organized, and generating scaffolds that can mimic these properties is critical for tissue engineering applications. By uniquely combining controlled radical polymerization (CRP), end-functionalization of polymers, and advanced electrospinning techniques, a modular and versatile approach is introduced to generate scaffolds with spatially organized functionality. Poly-ε-caprolactone is end functionalized with either a polymerization-initiating group or a cell-binding peptide motif cyclic Arg-Gly-Asp-Ser (cRGDS), and are each sequentially electrospun to produce zonally discrete bilayers within a continuous fiber scaffold. The polymerization-initiating group is then used to graft an antifouling polymer bottlebrush based on poly(ethylene glycol) from the fiber surface using CRP exclusively within one bilayer of the scaffold. The ability to include additional multifunctionality during CRP is showcased by integrating a biotinylated monomer unit into the polymerization step allowing postmodification of the scaffold with streptavidin-coupled moieties. These combined processing techniques result in an effective bilayered and dual-functionality scaffold with a cell-adhesive surface and an opposing antifouling non-cell-adhesive surface in zonally specific regions across the thickness of the scaffold, demonstrated through fluorescent labelling and cell adhesion studies. This modular and versatile approach combines strategies to produce scaffolds with tailorable properties for many applications in tissue engineering and regenerative medicine.
    Full-text · Article · Aug 2015 · Advanced Functional Materials
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    ABSTRACT: Polymer-drug conjugates have significantly influenced polymer therapeutics over the last decade via controlled pharmacokinetics. Dapsone (4,4'-diamino diphenylsulphone) is not only widely used in the treatment of leprosy but forms an essential component in the treatment of autoimmune inflammatory diseases and malaria. However, its low bioavailability and non-specific distribution in the body leads to absorption throughout organs including skin, liver, and kidneys that can cause serious side effects. Thus, in this study we report the synthesis of polymer-drug conjugates of dapsone covalently bonded to macromolecular chains towards the development of new bioactive polymeric formulations with anti-inflammatory properties. Dapsone was functionalised with an acrylic moiety in which the acrylamide residue was directly bonded to one of the aromatic rings of dapsone. This functionalization yielded an unsymmetrical dapsone methacrylamide (DapMA) structure, which on free radical polymerisation and co-polymerisation with HEMA yielded polymers of hydrocarbon macromolecules with pendant dapsone units. Thermal and size-exclusion chromatographic analysis revealed an increase in thermal stabilisation of the homopolymer (p(DapMA)) in comparison to the copolymer (p(Dap-co-HEMA)) with relatively high average molecular weight. The polymer conjugates exhibited high stability with low dapsone release from the polymeric backbone due to hydrolysis. However, a significant anti-inflammatory activity in a nitric oxide inhibition assay confirmed that this property was the consequence of only the macromolecular composition and not related to the release of low molecular weight compounds. Thus, the conjugation of dapsone to macromolecular systems provides a synthetic route to incorporate this drug into polymeric systems, facilitating their development into new anti-inflammatory therapies. Copyright © 2015. Published by Elsevier Ltd.
    Full-text · Article · Aug 2015 · Acta biomaterialia
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    ABSTRACT: We describe the design and fabrication of biochips based on one dimensional photonic crystals supporting Bloch surface waves for label-free optical bio-sensing. The planar stacks of the biochips are composed of silica, tantala and titania that were deposited using plasma ion assisted evaporation under high vacuum conditions. The biochip surfaces were functionalized by silanization and appropriate fluidic cells were designed to operate in an automated platform. An angularly resolved optical sensing apparatus was assembled to carry out the sensing studies. The angular operation is obtained by a focused laser beam at a fixed wavelength and detection of the angular reflectance spectrum by means of an array detector. Practical application of the sensor was demonstrated by detecting a specific glycoprotein, Angiopoietin 2, that is involved in angiogenesis and inflammation processes. The protocol used for the label-free detection of Angiopoietin 2 is described and the results of an exemplary assay are given, confirming that an efficient detection can be achieved.
    No preview · Conference Paper · Jul 2015

Publication Stats

8k Citations
1,769.17 Total Impact Points

Institutions

  • 1970-2015
    • Imperial College London
      • • Department of Materials
      • • Institute of Biomedical Engineering (IBME)
      Londinium, England, United Kingdom
  • 2007
    • The University of Manchester
      Manchester, England, United Kingdom
  • 2004-2006
    • Massachusetts Institute of Technology
      • Department of Chemical Engineering
      Cambridge, Massachusetts, United States