Imperial College London

Professor Molly Stevens FREng

Faculty of EngineeringDepartment of Materials

Prof of Biomedical Materials&Regenerative Medicine



+44 (0)20 7594 6804m.stevens




208Royal School of MinesSouth Kensington Campus





Publication Type

351 results found

Loynachan C, Thomas MR, Gray ER, Richards DA, Kim J, MIller BS, Brookes JC, Chudasama V, McKendry RA, Stevens MMet al., 2017, Platinum Nanocatalyst Amplification: Redefining the Gold Standard for Lateral Flow Immunoassays with Ultra-broad Dynamic Range, ACS Nano, Vol: 12, Pages: 279-288, ISSN: 1936-0851

Paper-based lateral flow immunoassays (LFIAs) are one of the most widely used point-of-care (PoC) devices; however, their application in early disease diagnostics is often limited due to insufficient sensitivity for the requisite sample sizes and the short time frames of PoC testing. To address this, we developed a serum-stable, nanoparticle catalyst-labeled LFIA with a sensitivity surpassing that of both current commercial and published sensitivities for paper-based detection of p24, one of the earliest and most conserved biomarkers of HIV. We report the synthesis and characterization of porous platinum core–shell nanocatalysts (PtNCs), which show high catalytic activity when exposed to complex human blood serum samples. We explored the application of antibody-functionalized PtNCs with strategically and orthogonally modified nanobodies with high affinity and specificity toward p24 and established the key larger nanoparticle size regimes needed for efficient amplification and performance in LFIA. Harnessing the catalytic amplification of PtNCs enabled naked-eye detection of p24 spiked into sera in the low femtomolar range (ca. 0.8 pg·mL–1) and the detection of acute-phase HIV in clinical human plasma samples in under 20 min. This provides a versatile absorbance-based and rapid LFIA with sensitivity capable of significantly reducing the HIV acute phase detection window. This diagnostic may be readily adapted for detection of other biomolecules as an ultrasensitive screening tool for infectious and noncommunicable diseases and can be capitalized upon in PoC settings for early disease detection.

Journal article

You AYF, Bergholt MS, St-Pierre JP, Chester AH, Yacoub MH, Bertazzo S, Stevens MMet al., 2017, Raman spectroscopy imaging reveals interplay between atherosclerosis and medial calcification in human aorta, Science Advances, Vol: 3, ISSN: 2375-2548

Medial calcification in the human aorta accumulates during aging and is known to be aggravated in several diseases. Atherosclerosis, another major cause of cardiovascular calcification, shares some common aggravators. However, the mechanisms of cardiovascular calcification remain poorly understood. To elucidate the relationship between medial aortic calcification and atherosclerosis, we characterized the cross-sectional distributions of the predominant minerals in aortic tissue, apatite and whitlockite, and the associated extracellular matrix. We also compared the cellular changes between atherosclerotic and nonatherosclerotic human aortic tissues. This was achieved through the development of Raman spectroscopy imaging methods that adapted algorithms to distinguish between the major biomolecules present within these tissues. We present a relationship between apatite, cholesterol, and triglyceride in atherosclerosis, with the relative amount of all molecules concurrently increased in the atherosclerotic plaque. Further, the increase in apatite was disproportionately large in relation to whitlockite in the aortic media directly underlying a plaque, indicating that apatite is more pathologically significant in atherosclerosis-aggravated medial calcification. We also discovered a reduction of β-carotene in the whole aortic intima, including a plaque in atherosclerotic aortic tissues compared to nonatherosclerotic tissues. This unprecedented biomolecular characterization of the aortic tissue furthers our understanding of pathological and physiological cardiovascular calcification events in humans.

Journal article

Milner P, Parkes M, Puetzer J, Chapman R, Cann P, Stevens M, Jeffers Jet al., 2017, A Low Friction, Biphasic and Boundary Lubricating Hydrogel for Cartilage Replacement, Acta Biomaterialia, Vol: 65, Pages: 102-111, ISSN: 1742-7061

Partial joint repair is a surgical procedure where an artificial material is used to replace localised chondral damage. These artificial bearing surfaces must articulate against cartilage, but current materials do not replicate both the biphasic and boundary lubrication mechanisms of cartilage. A research challenge therefore exists to provide a material that mimics both boundary and biphasic lubrication mechanisms of cartilage.In this work a polymeric network of a biomimetic boundary lubricant, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), was incorporated into an ultra-tough double network (DN) biphasic (water phase + polymer phase) gel, to form a PMPC triple network (PMPC TN) hydrogel with boundary and biphasic lubrication capability. The presence of this third network of MPC was confirmed using ATR-FTIR. The PMPC TN hydrogel had a yield stress of 26 MPa, which is an order of magnitude higher than the peak stresses found in the native human knee. A preliminary pin on plate tribology study was performed where both the DN and PMPC TN hydrogels experienced a reduction in friction with increasing sliding speed which is consistent with biphasic lubrication. In the physiological sliding speed range, the PMPC TN hydrogel halved the friction compared to the DN hydrogel indicating the boundary lubricating PMPC network was working.A biocompatible, tough, strong and chondral lubrication imitating PMPC TN hydrogel was synthesised in this work. By complementing the biphasic and boundary lubrication mechanisms of cartilage, PMPC TN hydrogel could reduce the reported incidence of chondral damage opposite partial joint repair implants, and therefore increase the clinical efficacy of partial joint repair.Statement of SignificanceThis paper presents the synthesis, characterisation and preliminary tribological testing of a new biomaterial that aims to recreate the primary chondral lubrication mechanisms: boundary and biphasic lubrication. This work has demonstrated that the

Journal article

Luongo G, Perez JE, Kosel J, Georgiou TK, Regoutz A, Payne DJ, Stevens MM, Porter AE, Dunlop IEet al., 2017, Scalable high-affinity stabilization of magnetic iron oxide nanostructures by a biocompatible antifouling homopolymer, ACS Applied Materials and Interfaces, Vol: 9, Pages: 40059-40069, ISSN: 1944-8244

Iron oxide nanostructures have been widely developed for biomedical applications, due to their magnetic properties and biocompatibility. In clinical application, the stabilization of these nanostructures against aggregation and non-specific interactions is typically achieved using weakly anchored polysaccharides, with better-defined and more strongly anchored synthetic polymers not commercially adopted due to complexity of synthesis and use. Here, we show for the first time stabilization and biocompatibilization of iron oxide nanoparticles by a synthetic homopolymer with strong surface anchoring and a history of clinical use in other applications, poly(2-methacryloyloxyethy phosphorylcholine) (poly(MPC)). For the commercially important case of spherical particles, binding of poly(MPC) to iron oxide surfaces and highly effective individualization of magnetite nanoparticles (20 nm) are demonstrated. Next-generation high-aspect ratio nanowires (both magnetite/maghemite and core-shell iron/iron oxide) are furthermore stabilized by poly(MPC)-coating, with nanowire cytotoxicity at large concentrations significantly reduced. The synthesis approach is exploited to incorporate functionality into the poly(MPC) chain is demonstrated by random copolymerization with an alkyne-containing monomer for click-chemistry. Taking these results together, poly(MPC) homopolymers and random copolymers offer a significant improvement over current iron oxide nanoformulations, combining straightforward synthesis, strong surface-anchoring and well-defined molecular weight.

Journal article

Chow A, Stuckey DJ, Kidher E, Rocco M, Jabbour RJ, Mansfield CA, Darzi A, Harding SE, Stevens MM, Athanasiou Tet al., 2017, Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Encapsulating Bioactive Hydrogels Improve Rat Heart Function Post Myocardial Infarction., Stem Cell Reports, Vol: 9, Pages: 1415-1422, ISSN: 2213-6711

Tissue engineering offers an exciting possibility for cardiac repair post myocardial infarction. We assessed the effects of combined polyethylene glycol hydrogel (PEG), human induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM), and erythropoietin (EPO) therapy in a rat model of myocardial infarction. PEG with/out iPSC-CMs and EPO; iPSC-CMs in saline; or saline alone was injected into infarcted hearts shortly after infarction. Injection of almost any combination of the therapeutics limited acute elevations in chamber volumes. After 10 weeks, attenuation of ventricular remodeling was identified in all groups that received PEG injections, while ejection fractions were significantly increased in the gel-EPO, cell, and gel-cell-EPO groups. In all treatment groups, infarct thickness was increased and regions of muscle were identified within the scar. However, no grafted cells were detected. Hence, iPSC-CM-encapsulating bioactive hydrogel therapy can improve cardiac function post myocardial infarction and increase infarct thickness and muscle content despite a lack of sustained donor-cell engraftment.

Journal article

Lin Y, Thomas MR, Gelmi A, Leonardo V, Pashuck ET, Maynard SA, Wang Y, Stevens MMet al., 2017, Self-assembled 2D Free-Standing Janus Nanosheets with Single-Layer Thickness, Journal of the American Chemical Society, Vol: 139, Pages: 13592-13595, ISSN: 1520-5126

We report the thermodynamically controlled growth of solution-processable and free-standing nanosheets via peptide assembly in two dimensions. By taking advantage of self-sorting between peptide β-strands and hydrocarbon chains, we have demonstrated the formation of Janus 2D structures with single-layer thickness, which enable a predetermined surface heterofunctionalization. A controlled 2D-to-1D morphological transition was achieved by subtly adjusting the intermolecular forces. These nanosheets provide an ideal substrate for the engineering of guest components (e.g., proteins and nanoparticles), where enhanced enzyme activity was observed. We anticipate that sequence-specific programmed peptides will offer promise as design elements for 2D assemblies with face-selective functionalization.

Journal article

Rizzo R, Alvaro M, Danz N, Napione L, Descrovi E, Schmieder S, Sinibaldi A, Chandrawati R, Rana S, Munzert P, Schubert T, Maillart E, Anopchenko A, Rivolo P, Mascioletti A, Sonntag F, Stevens MM, Bussolino F, Michelotti Fet al., 2017, Bloch surface wave label-free and fluorescence platform for the detection of VEGF biomarker in biological matrices, Sensors and Actuators B: Chemical, Vol: 255, Pages: 2143-2150, ISSN: 0925-4005

We report on the detection of an angiogenic molecule Vascular Endothelial Growth Factor (VEGF) in different biological matrices by means of a new integrated biosensing platform exploiting the properties of Bloch surface waves. The new platform takes advantage of a tandem configuration, in which both label-free and enhanced fluorescence detection are implemented. Specifically designed one dimensional photonic crystals were deposited directly on disposable and low cost plastic biochips. A direct sandwich immunoassay was used to detect VEGF in buffer, cell culture supernatant and human plasma at low concentration (ng/mL). The platform enabled the detection of VEGF in all three matrices with high resolution, fast turnaround time (30 min) and in close agreement with the results of enzyme linked immunosorbent assays.

Journal article

Guex A, Puetzer J, Armgarth A, Littmann E, Stavrinidou E, Gianellis EP, Malliaras GG, Stevens MMet al., 2017, Highly porous scaffolds of PEDOT:PSS for bone tissue engineering, Acta Biomaterialia, Vol: 62, Pages: 91-101, ISSN: 1742-7061

Conjugated polymers have been increasingly considered for the design of conductive materials in the field of regenerative medicine. However, optimal scaffold properties addressing the complexity of the desired tissue still need to be developed. The focus of this study lies in the development and evaluation of a conductive scaffold for bone tissue engineering. In this study PEDOT:PSS scaffolds were designed and evaluated in vitro using MC3T3-E1 osteogenic precursor cells, and the cells were assessed for distinct differentiation stages and the expression of an osteogenic phenotype.Ice-templated PEDOT:PSS scaffolds presented high pore interconnectivity with a median pore diameter of 53.6 ± 5.9 µm and a total pore surface area of 7.72 ± 1.7 m2·g−1. The electrical conductivity, based on I-V curves, was measured to be 140 µS·cm−1 with a reduced, but stable conductivity of 6.1 µS·cm−1 after 28 days in cell culture media. MC3T3-E1 gene expression levels of ALPL, COL1A1 and RUNX2 were significantly enhanced after 4 weeks, in line with increased extracellular matrix mineralisation, and osteocalcin deposition. These results demonstrate that a porous material, based purely on PEDOT:PSS, is suitable as a scaffold for bone tissue engineering and thus represents a promising candidate for regenerative medicine.

Journal article

Wang ST, Lin Y, Spencer RK, Thomas MR, Nguyen AI, Amdursky N, Pashuck ET, Skaalure SC, Song C, Parmar PA, Morgan RM, Ercius P, Aloni S, Zuckermann RN, Stevens MMet al., 2017, Sequence-Dependent Self-Assembly and Structural Diversity of Islet Amyloid Polypeptide-Derived β-Sheet Fibrils, ACS Nano, Vol: 11, Pages: 8579-8589, ISSN: 1936-086X

Determining the structural origins of amyloid fibrillation is essential for understanding both the pathology of amyloidosis and the rational design of inhibitors to prevent or reverse amyloid formation. In this work, the decisive roles of peptide structures on amyloid self-assembly and morphological diversity were investigated by the design of eight amyloidogenic peptides derived from islet amyloid polypeptide. Among the segments, two distinct morphologies were highlighted in the form of twisted and planar (untwisted) ribbons with varied diameters, thicknesses, and lengths. In particular, transformation of amyloid fibrils from twisted ribbons into untwisted structures was triggered by substitution of the C-terminal serine with threonine, where the side chain methyl group was responsible for the distinct morphological change. This effect was confirmed following serine substitution with alanine and valine and was ascribed to the restriction of intersheet torsional strain through the increased hydrophobic interactions and hydrogen bonding. We also studied the variation of fibril morphology (i.e., association and helicity) and peptide aggregation propensity by increasing the hydrophobicity of the peptide side group, capping the N-terminus, and extending sequence length. We anticipate that our insights into sequence-dependent fibrillation and morphological diversity will shed light on the structural interpretation of amyloidogenesis and development of structure-specific imaging agents and aggregation inhibitors.

Journal article

Littmann E, Autefage H, Solanki AN, Kallepitis C, Jones JR, Alini M, Peroglio M, Stevens MMet al., 2017, Cobalt-containing bioactive glasses reduce humanmesenchymal stem cell chondrogenic differentiation despiteHIF-1α stabilisation, Journal of the European Ceramic Society, Vol: 38, Pages: 877-886, ISSN: 0955-2219

Bioactive glasses (BGs) are excellent delivery systems for the sustained release of therapeutic ions and have been extensively studied in the context of bone tissue engineering. More recently, due to their osteogenic properties and expanding application to soft tissue repair, BGs have been proposed as promising materials for use at the osteochondral interface. Since hypoxia plays a critical role during cartilage formation, we sought to investigate the influence of BGs releasing the hypoxia-mimicking agent cobalt (CoBGs) on human mesenchymal stem cell (hMSC) chondrogenesis, as a novel approach that may guide future osteochondral scaffold design. The CoBG dissolution products significantly increased the level of hypoxia-inducible factor-1 alpha in hMSCs in a cobalt dose-dependent manner. Continued exposure to the cobalt-containing BG extracts significantly reduced hMSC proliferation and metabolic activity, as well as chondrogenic differentiation. Overall, this study demonstrates that prolonged exposure to cobalt warrants careful consideration for cartilage repair applications.

Journal article

Chan WCW, Chhowalla M, Glotzer S, Gogotsi Y, Hafner JH, Hammond PT, Hersam MC, Javey A, Kagan CR, Kataoka K, Khademhosseini A, Kotov NA, Lee S-T, Li Y, Moehwald H, Mulvaney P, Nel AE, Nordlander PJ, Parak WJ, Penner RM, Rogach AL, Schaak RE, Stevens MM, Wee ATS, Willson CG, Fernandez LEE, Weiss PSet al., 2017, Our First and Next Decades at ACS Nano, Publisher: AMER CHEMICAL SOC


Chung JJ, Sum BST, Li S, Stevens MM, Georgiou TK, Jones JRet al., 2017, Effect of comonomers on physical properties and cell attachment to silica-methacrylate/acrylate hybrids for bone substitution, Macromolecular Rapid Communications, Vol: 38, ISSN: 1022-1336

Hybrids with a silica network covalently bonded to a polymer are promising materials for bone repair. Previous work on synthesizing methyl methacrylate (MMA) based copolymers by reversible addition‐fragmentation chain transfer (RAFT) polymerization gives high tailorability of mechanical properties since sophisticated polymer structures can be designed. However, more flexible hybrids would be beneficial. Here, n‐butyl methacrylate (BMA) and methyl acrylate (MA) based hybrids are produced. Unlike MMA, BMA and MA hybrids do not show plastic deformation, and BMA hybrid has strain to failure of 33%. Although the new hybrids are more flexible, preosteoblast cells do not adhere on their surfaces, due to higher hydrophobicity and lower stiffness. Comonomer choice is crucial for bone regenerative hybrids.

Journal article

Amdursky N, Rashid MH, Stevens MM, Yarovsky Iet al., 2017, Exploring the binding sites and proton diffusion on insulin amyloid fibril surface by naphthol-based photoacid fluorescence and molecular simulations, Scientific Reports, Vol: 7, ISSN: 2045-2322

The diffusion of protons along biological surfaces and the interaction of biological structures with water are fundamental areas of interest in biology and chemistry. Here, we examine the surface of insulin amyloid fibrils and follow the binding of small molecules (photoacids) that differ according to the number and location of their sulfonic groups. We use transient fluorescence combined with a spherically-symmetric diffusion theory to show that the binding mode of different photoacids determines the efficiency of proton dissociation from the photoacid and the dimensionality of the proton’s diffusion. We use molecular dynamics simulations to examine the binding mode and mechanism of the photoacids and its influence on the unique kinetic rates and diffusion properties of the photoacid’s dissociated proton, where we also suggest a proton transfer process between one of the photoacids to proximal histidine residues. We show that the photoacids can be used as fluorescent markers for following the progression of amyloidogenic processes. The detailed characterisation of different binding modes to the surface of amyloid fibrils paves the way for better understanding of the binding mechanism of small molecules to amyloid fibrils.

Journal article

Chandrawati R, Olesen MTJ, Marini TCC, Bisra G, Guex AG, De Oliveira MG, Zelikin AN, Stevens MMet al., 2017, Enzyme prodrug therapy engineered into electrospun fibers with embedded liposomes for controlled, localized synthesis of therapeutics, Advanced Healthcare Materials, Vol: 6, ISSN: 2192-2640

Enzyme prodrug therapy (EPT) enables localized conversion of inert prodrugs to active drugs by enzymes. Performance of EPT necessitates that the enzyme remains active throughout the time frame of the envisioned therapeutic application. β-glucuronidase is an enzyme with historically validated performance in EPT, however it retains its activity in biomaterials for an insufficiently long period of time, typically not exceeding 7 d. Herein, the encapsulation of β-glucuronidase in liposomal subcompartments within poly(vinyl alcohol) electrospun fibers is reported, leading to the assembly of biocatalytically active materials with activity of the enzyme sustained over at least seven weeks. It is further shown that liposomes provide the highly beneficial stabilization of the enzyme when incubated in cell culture media. The assembled biocatalytic materials successfully produce antiproliferative drugs (SN-38) using externally administered prodrugs (SN-38-glucuronide) and effectively suppress cell proliferation, with envisioned utility in the design of cardiovascular grafts.

Journal article

Guex AG, Spicer CD, Armgarth A, Gelmi A, Humphrey EJ, Terracciano CM, Harding S, Stevens MMet al., 2017, Electrospun aniline-tetramer-co-polycaprolactone fibres for conductive, biodegradable scaffolds, MRS Communications, Vol: 7, Pages: 375-382, ISSN: 2159-6867

Conjugated polymers have been proposed as promising materials for scaffolds in tissue engineering applications. However, the restricted processability and biodegradability of conjugated polymers limit their use for biomedical applications. Here we synthesized a block-co-polymer of aniline tetramer and PCL (AT–PCL), and processed it into fibrous non-woven scaffolds by electrospinning. We showed that fibronectin (Fn) adhesion was dependent on the AT–PCL oxidative state, with a reduced Fn unfolding length on doped membranes. Furthermore, we demonstrated the cytocompatibility and potential of these membranes to support the growth and osteogenic differentiation of MC3T3-E1 cells over 21 days.

Journal article

Reznikov N, Phillips C, Cooke M, Garbout A, Ahmed F, Stevens MMet al., 2017, Functional adaptation of the calcaneus in historical foot binding, Journal of Bone and Mineral Research, Vol: 32, Pages: 1915-1925, ISSN: 1523-4681

The normal structure of human feet is optimized for shock dampening during walking and running. Foot binding was a historical practice in China aimed at restricting the growth of female feet for aesthetic reasons. In a bound foot the shock-dampening function normally facilitated by the foot arches is withdrawn, resulting in the foot functioning as a rigid extension of the lower leg. An interesting question inspiring this study regards the nature of adaptation of the heel bone to this nonphysiological function using the parameters of cancellous bone anisotropy and 3D fabric topology and a novel intertrabecular angle (ITA) analysis. We found that the trabecular microarchitecture of the normal heel bone, but not of the bound foot, adapts to function by increased anisotropy and preferred orientation of trabeculae. The anisotropic texture in the normal heel bone consistently follows the physiological stress trajectories. However, in the bound foot heel bone the characteristic anisotropy pattern fails to develop, reflecting the lack of a normal biomechanical input. Moreover, the basic topological blueprint of cancellous bone investigated by the ITA method is nearly invariant in both normal and bound foot. These findings suggest that the anisotropic cancellous bone texture is an acquired characteristic that reflects recurrent loading conditions; conversely, an inadequate biomechanical input precludes the formation of anisotropic texture. This opens a long-sought-after possibility to reconstruct bone function from its form. The conserved topological parameters characterize the generic 3D fabric of cancellous bone, which is to a large extent independent of its adaptation to recurrent loading and perhaps determines the mechanical competence of trabecular bone regardless of its functional adaptation. © 2017 American Society for Bone and Mineral Research.

Journal article

Li S, Macon ALB, Jacquemin M, Stevens MM, Jones JRet al., 2017, Sol-gel derived lithium-releasing glass for cartilage regeneration, Journal of Biomaterials Applications, Vol: 32, Pages: 104-113, ISSN: 0885-3282

Wnt-signalling cascadeis one of the crucial pathways involved in the development and homeostasis of cartilage. Influencing this pathway can potentially contribute to improved cartilage repair or regeneration. One key molecular regulator of the Wnt pathway is the glycogen synthase kinase-3 (GSK-3) enzyme, the inhibition of which allows initiation of the signalling pathway. This study aims to utilise a binary SiO2-Li2O sol-gel derived glass for controlled delivery of lithium, a known GSK-3 antagonist. The effect of the dissolution products of the glass on chondrogenic differentiation inanin vitro3-D pellet culture modelis reported. Dissolution productsthat contained5 mM lithium and 3.5 mM silicon, were capable of inducing chondrogenic differentiation and hyaline cartilaginous matrix formation without the presence of growth factors such as TGF-β3. The results suggest that sol-gel derived glass has the potential to be used as a delivery vehicle for therapeutic lithium ions in cartilage regeneration applications.

Journal article

Soh JH, Lin Y, Thomas MR, Todorova N, Kallepitis C, Ying JY, Yarovsky I, Stevens MMet al., 2017, Distinct bimodal roles of aromatic molecules in controlling gold nanorod growth for biosensing, Advanced Functional Materials, Vol: 27, ISSN: 1616-3028

New aromatic molecule–seed particle interactions are examined and exploited to control and guide seed-mediated gold nanorod (Au NR) growth. This new approach enables better understanding of how small molecules impact the synthesis of metallic nanostructures, catalysing their use in various biomedical applications, such as plasmonic biosensing. We perform experimental studies and theoretical molecular simulations using a library of aromatic molecules where we take advantage of the chemical versatility of the molecules with varied spatial arrangements of electron donating/withdrawing groups, charge, and Au-binding propensity. Au NR growth is regulated by two principal mechanisms, producing either a red or blue shift in the longitudinal localized surface plasmon resonance (LLSPR) peaks. Aromatic molecules with high redox potentials produced an increase in NR aspect ratio and red shift of LLSPR peaks. In contrast, molecules that strongly bind gold surfaces resulted in blue shifts, demonstrating a strong correlation between their binding energy and blue shifts produced. Through enzymatic conversion of selected molecules, 4-aminophenylphosphate to 4-aminophenol, we obtained opposing growth mechanisms at opposite extremes of target concentration, and established a chemical pathway for performing plasmonic ELISA. This unlocks new strategies for tailoring substrate design and enzymatic mechanisms for controlling plasmonic response to target detection in biosensing applications.

Journal article

Bergholt M, Albro M, Stevens MM, 2017, Online quantitative monitoring of live cell engineered cartilage growth using diffuse fiber-optic Raman spectroscopy, Biomaterials, Vol: 140, Pages: 128-137, ISSN: 1878-5905

Tissue engineering (TE) has the potential to improve the outcome for patients with osteoarthritis (OA). The successful clinical translation of this technique as part of a therapy requires the ability to measure extracellular matrix (ECM) production of engineered tissues in vitro, in order to ensure quality control and improve the likelihood of tissue survival upon implantation. Conventional techniques for assessing the ECM content of engineered cartilage, such as biochemical assays and histological staining are inherently destructive. Raman spectroscopy, on the other hand, represents a non-invasive technique for in situ biochemical characterization. Here, we outline current roadblocks in translational Raman spectroscopy in TE and introduce a comprehensive workflow designed to non-destructively monitor and quantify ECM biomolecules in large (>3 mm), live cell TE constructs online. Diffuse near-infrared fiber-optic Raman spectra were measured from live cell cartilaginous TE constructs over a 56-day culturing period. We developed a multivariate curve resolution model that enabled quantitative biochemical analysis of the TE constructs. Raman spectroscopy was able to non-invasively quantify the ECM components and showed an excellent correlation with biochemical assays for measurement of collagen (R2 = 0.84) and glycosaminoglycans (GAGs) (R2 = 0.86). We further demonstrated the robustness of this technique for online prospective analysis of live cell TE constructs. The fiber-optic Raman spectroscopy strategy developed in this work offers the ability to non-destructively monitor construct growth online and can be adapted to a broad range of TE applications in regenerative medicine toward controlled clinical translation.

Journal article

Horejs CM, St-Pierre JP, Ojala JRM, Steele JAM, Barros da Silva P, Rynne-Vidal A, Maynard SA, Hansel CS, Rodriguez-Fernandez C, Mazo MM, You AYF, Wang AJ, von Erlach T, Tryggvason K, Lopez-Cabrera M, Stevens MMet al., 2017, Preventing tissue fibrosis by local biomaterials interfacing of specific cryptic extracellular matrix information, Nature Communications, Vol: 8, ISSN: 2041-1723

Matrix metalloproteinases (MMPs) contribute to the breakdown of tissue structures such as the basement membrane, promoting tissue fibrosis. Here we developed an electrospun membrane biofunctionalized with a fragment of the laminin β1-chain to modulate the expression of MMP2 in this context. We demonstrate that interfacing of the β1-fragment with the mesothelium of the peritoneal membrane via a biomaterial abrogates the release of active MMP2 in response to transforming growth factor β1 and rescues tissue integrity ex vivo and in vivo in a mouse model of peritoneal fibrosis. Importantly, our data demonstrate that the membrane inhibits MMP2 expression. Changes in the expression of epithelial-to-mesenchymal transition (EMT)-related molecules further point towards a contribution of the modulation of EMT. Biomaterial-based presentation of regulatory basement membrane signals directly addresses limitations of current therapeutic approaches by enabling a localized and specific method to counteract MMP2 release applicable to a broad range of therapeutic targets.

Journal article

Amdursky N, Wang X, Meredith P, Riley DJ, Payne DJ, Bradley D, Stevens MMet al., 2017, Electron Hopping Across Hemin-Doped Serum Albumin Mats on Centimetre-Length Scales, Advanced Materials, Vol: 29, ISSN: 1521-4095

Exploring long-range electron transport across protein assemblies is a central interest in both the fundamental research of biological processes and the emerging field of bioelectronics. This work examines the use of serum-albumin-based freestanding mats as macroscopic electron mediators in bioelectronic devices. In particular, this study focuses on how doping the protein mat with hemin improves charge-transport. It is demonstrated that doping can increase conductivity 40-fold via electron hopping between adjacent hemin molecules, resulting in the highest measured conductance for a protein-based material yet reported, and transport over centimeter length scales. The use of distance-dependent AC impedance and DC current–voltage measurements allows the contribution from electron hopping between adjacent hemin molecules to be isolated. Because the hemin-doped serum albumin mats have both biocompatibility and fabrication simplicity, they should be applicable to a range of bioelectronic devices of varying sizes, configurations, and applications.

Journal article

Hall CE, Yao Z, Choi M, Tyzack GE, Serio A, Luisier R, Harley J, Preza E, Arber C, Crisp SJ, Watson PMD, Kullmann DM, Abramov AY, Wray S, Burley R, Loh SHY, Martins LM, Stevens MM, Luscombe NM, Sibley CR, Lakatos A, Ule J, Gandhi S, Patani Ret al., 2017, Progressive motor neuron pathology and the role of astrocytes in a human stem cell model of VCP-related ALS, Cell Reports, Vol: 19, Pages: 1739-1749, ISSN: 2211-1247

Motor neurons (MNs) and astrocytes (ACs) are implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), but their interaction and the sequence of molecular events leading to MN death remain unresolved. Here, we optimized directed differentiation of induced pluripotent stem cells (iPSCs) into highly enriched (> 85%) functional populations of spinal cord MNs and ACs. We identify significantly increased cytoplasmic TDP-43 and ER stress as primary pathogenic events in patient-specific valosin-containing protein (VCP)-mutant MNs, with secondary mitochondrial dysfunction and oxidative stress. Cumulatively, these cellular stresses result in synaptic pathology and cell death in VCP-mutant MNs. We additionally identify a cell-autonomous VCP-mutant AC survival phenotype, which is not attributable to the same molecular pathology occurring in VCP-mutant MNs. Finally, through iterative co-culture experiments, we uncover non-cell-autonomous effects of VCP-mutant ACs on both control and mutant MNs. This work elucidates molecular events and cellular interplay that could guide future therapeutic strategies in ALS.

Journal article

Clarke D, Pashuck ET, Bertazzo S, Weaver JVM, Stevens MMet al., 2017, Self-healing self-assembled B-sheet peptide poly(G-glutamic acid) hybrid hydrogels, Journal of the American Chemical Society, Vol: 139, Pages: 7250-7255, ISSN: 1520-5126

Self-assembled biomaterials are an important class of materials that can be injected and formed in situ. However, they often are not able to meet the mechanical properties necessary for many biological applications, losing mechanical properties at low strains. We synthesized hybrid hydrogels consisting of a poly(γ-glutamic acid) polymer network physically cross-linked via grafted self-assembling β-sheet peptides to provide non-covalent cross-linking through β-sheet assembly, reinforced with a polymer backbone to improve strain stability. By altering the β-sheet peptide graft density and concentration, we can tailor the mechanical properties of the hydrogels over an order of magnitude range of 10–200 kPa, which is in the region of many soft tissues. Also, due to the ability of the non-covalent β-sheet cross-links to reassemble, the hydrogels can self-heal after being strained to failure, in most cases recovering all of their original storage moduli. Using a combination of spectroscopic techniques, we were able to probe the secondary structure of the materials and verify the presence of β-sheets within the hybrid hydrogels. Since the polymer backbone requires less than a 15% functionalization of its repeating units with β-sheet peptides to form a hydrogel, it can easily be modified further to incorporate specific biological epitopes. This self-healing polymer−β-sheet peptide hybrid hydrogel with tailorable mechanical properties is a promising platform for future tissue-engineering scaffolds and biomedical applications.

Journal article

Chang JYH, Chow LW, Dismuke WM, Ethier CR, Stevens MM, Stamer WD, Overby Det al., 2017, Peptide-functionalized fluorescent particles for in situ detection of nitric oxide via peroxynitrite-mediated nitration, Advanced Healthcare Materials, Vol: 6, ISSN: 2192-2640

Nitric oxide (NO) is a free radical signaling molecule that plays a crucial role in modulating physiological homeostasis across multiple biological systems. NO dysregulation is linked to the pathogenesis of multiple diseases; therefore, its quantification is important for understanding pathophysiological processes. The detection of NO is challenging, typically limited by its reactive nature and short half-life. Additionally, the presence of interfering analytes and accessibility to biological fluids in the native tissues make the measurement technically challenging and often unreliable. Here, a bio-inspired peptide-based NO sensor is developed, which detects NO-derived oxidants, predominately peroxynitrite-mediated nitration of tyrosine residues. It is demonstrated that these peptide-based NO sensors can detect peroxynitrite-mediated nitration in response to physiological shear stress by endothelial cells in vitro. Using the peptide-conjugated fluorescent particle immunoassay, peroxynitrite-mediated nitration activity with a detection limit of ≈100 × 10−9m is detected. This study envisions that the NO detection platform can be applied to a multitude of applications including monitoring of NO activity in healthy and diseased tissues, localized detection of NO production of specific cells, and cell-based/therapeutic screening of peroxynitrite levels to monitor pronitroxidative stress in biological samples.

Journal article

Wang C, Lin Y, Hsu C, Amdursky N, Spicer C, Stevens MMet al., 2017, Probing amylin fibrillation at an early stage via a tetracysteine-recognising fluorophore, Talanta, Vol: 173, Pages: 44-50, ISSN: 1873-3573

Amyloid fibrillation is a nucleation-dependent process known be involved in the development of more than 20 progressive and chronic diseases. The detection of amyloid formation at the nucleation stage can greatly advance early diagnoses and treatment of diseases. In this work, we developed a new assay for the early detection of amylin fibrillation using the biarsenical dye 4,5-bis(1,3,2-dithiarsolan-2-yl)fluorescein (FlAsH), which could recognise tetracysteine motifs and transform from non-fluorescent form into strongly fluorescent complexes. Due to the close proximity of two cysteine residues within the hydrophilic domain of amylin, a non-contiguous tetracysteine motif can form upon amylin dimerisation or oligomerisation, which can be recognised by FlAsH and emit strong fluorescence. This enables us to report the nucleation-growth process of amylin without modification of the protein sequence. We showed that the use of this assay not only allowed the tracking of initial nucleation events, but also enabled imaging of amyloid fibrils and investigation of the effects of amyloid inhibitor/modulator toward amylin fibrillation.

Journal article

Ember KJI, Hoeve MA, McAughtrie SL, Bergholt MS, Dwyer BJ, Stevens MM, Faulds K, Forbes SJ, Campbell CJet al., 2017, Raman spectroscopy and regenerative medicine: a review, npj Regenerative Medicine, Vol: 2, ISSN: 2057-3995

The field of regenerative medicine spans a wide area of the biomedical landscape—from single cell culture in laboratories to human whole-organ transplantation. To ensure that research is transferrable from bench to bedside, it is critical that we are able to assess regenerative processes in cells, tissues, organs and patients at a biochemical level. Regeneration relies on a large number of biological factors, which can be perturbed using conventional bioanalytical techniques. A versatile, non-invasive, non-destructive technique for biochemical analysis would be invaluable for the study of regeneration; and Raman spectroscopy is a potential solution. Raman spectroscopy is an analytical method by which chemical data are obtained through the inelastic scattering of light. Since its discovery in the 1920s, physicists and chemists have used Raman scattering to investigate the chemical composition of a vast range of both liquid and solid materials. However, only in the last two decades has this form of spectroscopy been employed in biomedical research. Particularly relevant to regenerative medicine are recent studies illustrating its ability to characterise and discriminate between healthy and disease states in cells, tissue biopsies and in patients. This review will briefly outline the principles behind Raman spectroscopy and its variants, describe key examples of its applications to biomedicine, and consider areas of regenerative medicine that would benefit from this non-invasive bioanalytical tool.

Journal article

Kim E, Zwi Dantsis L, Reznikov N, Hansel CS, Agarwal S, Stevens MMet al., 2017, One-pot synthesis of multiple protein-encapsulated DNA flowers and their application in intracellular protein delivery, Advanced Materials, Vol: 29, ISSN: 1521-4095

Inspired by biological systems, many biomimetic methods suggest fabrication of functional materials with unique physicochemical properties. Such methods frequently generate organic–inorganic composites that feature highly ordered hierarchical structures with intriguing properties, distinct from their individual components. A striking example is that of DNA–inorganic hybrid micro/nanostructures, fabricated by the rolling circle technique. Here, a novel concept for the encapsulation of bioactive proteins in DNA flowers (DNF) while maintaining the activity of protein payloads is reported. A wide range of proteins, including enzymes, can be simultaneously associated with the growing DNA strands and Mg2PPi crystals during the rolling circle process, ultimately leading to the direct immobilization of proteins into DNF. The unique porous structure of this construct, along with the abundance of Mg ions and DNA molecules present, provides many interaction sites for proteins, enabling high loading efficiency and enhanced stability. Further, as a proof of concept, it is demonstrated that the DNF can deliver payloads of cytotoxic protein (i.e., RNase A) to the cells without a loss in its biological function and structural integrity, resulting in highly increased cell death compared to the free protein.

Journal article

Higgins SG, Stevens MM, 2017, Extracting the contents of living cells, SCIENCE, Vol: 356, Pages: 379-380, ISSN: 0036-8075

Being able to monitor cells at different times is key to tracking fundamental cellular processes such as differentiation and cellular senescence, as well as disease progression and the effectiveness of drugs. However, most approaches are destructive and involve lysing the cells. Different time points can be studied by using parallel cell cultures, but the inferred changes could also be the result of cell heterogeneity (1, 2). Techniques for extracting small quantities of the cytosol for long-term tracking of a single cell's response must manipulate picoliter-scale volumes, maintain high cell viability, and give an accurate reflection of the cell's multiple biological components, as well as avoid influencing the ongoing development of the cell (see the figure) (1, 3). Cao et al. approached this problem by culturing cells on top of a random arrangement of hollow cylinders, which they call nanostraws (2). These 150-nm-diameter alumina tubes can sample 5 to 10% of proteins, messenger RNA (mRNA), and small molecules from the cells but only reduce cell viability by ∼5%. Their approach allows intracellular sampling and characterization at multiple time points from the same cells to track changes.

Journal article

Speidel AT, Stuckey DJ, Chow LW, Jackson LH, Noseda M, Abreu Pavia M, Schneider MD, Stevens MMet al., 2017, Multi-modal hydrogel-based platform to deliver and monitor cardiac progenitor/stem cell engraftment, ACS Central Science, Vol: 3, Pages: 338-348, ISSN: 2374-7951

Retention and survival of transplanted cells are major limitations to the efficacy of regenerative medicine, with short-term paracrine signals being the principal mechanism underlying current cell therapies for heart repair. Consequently, even improvements in short-term durability may have a potential impact on cardiac cell grafting. We have developed a multimodal hydrogel-based platform comprised of a poly(ethylene glycol) network cross-linked with bioactive peptides functionalized with Gd(III) in order to monitor the localization and retention of the hydrogel in vivo by magnetic resonance imaging. In this study, we have tailored the material for cardiac applications through the inclusion of a heparin-binding peptide (HBP) sequence in the cross-linker design and formulated the gel to display mechanical properties resembling those of cardiac tissue. Luciferase-expressing cardiac stem cells (CSC-Luc2) encapsulated within these gels maintained their metabolic activity for up to 14 days in vitro. Encapsulation in the HBP hydrogels improved CSC-Luc2 retention in the mouse myocardium and hind limbs at 3 days by 6.5- and 12- fold, respectively. Thus, this novel heparin-binding based, Gd(III)-tagged hydrogel and CSC-Luc2 platform system demonstrates a tailored, in vivo detectable theranostic cell delivery system that can be implemented to monitor and assess the transplanted material and cell retention.

Journal article

Kallepitis C, Bergholt MS, Mazo MM, Leonardo V, Skaalure SC, Maynard SA, Stevens MMet al., 2017, Quantitative volumetric Raman imaging of three dimensional cell cultures, Nature Communications, Vol: 8, ISSN: 2041-1723

The ability to simultaneously image multiple biomolecules in biologically relevant three-dimensional (3D) cell culture environments would contribute greatly to the understanding of complex cellular mechanisms and cell-material interactions. Here, we present a computational framework for label-free quantitative volumetric Raman imaging (qVRI). We apply qVRI to a selection of biological systems: human pluripotent stem cells with their cardiac derivatives, monocytes and monocyte-derived macrophages in conventional cell culture systems and mesenchymal stem cells inside biomimetic hydrogels that supplied a 3D cell culture environment. We demonstrate visualization and quantification of fine details in 3D cell shape, cytoplasm, nucleus, lipid bodies and cytoskeletal structures in 3D with unprecedented biomolecular specificity for vibrational microspectroscopy.

Journal article

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