Imperial College London

Professor Molly Stevens FREng

Faculty of EngineeringDepartment of Materials

Prof of Biomedical Materials&Regenerative Medicine
 
 
 
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Contact

 

+44 (0)20 7594 6804m.stevens

 
 
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Location

 

208Royal School of MinesSouth Kensington Campus

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Summary

 

Publications

Publication Type
Year
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350 results found

Hansel C, Holme M, Gopal S, Stevens Met al., Advances in High-Resolution Microscopy for the Study of Intracellular Interactions with Biomaterials, Biomaterials

Journal article

Armstrong J, Stevens M, Using remote fields for complex tissue engineering, Trends in Biotechnology, ISSN: 0167-7799

Great strides have been taken towards the in vitro engineering of clinically-relevant tissue constructsusing the classic triad of cells, materials and biochemical factors. In this perspective, we highlight ways in which these elements can be manipulated or stimulated using a fourth component: the application of remote fields.This arena has gained great momentum over the last few years, with a recent surge of interest in using magnetic, optical and acoustic fields to guide the organization of cells, materials and growth factors. We summarize recent developments and trends in this arena and then lay out a series of challenges that we believe, if met, could enable the widespread adoption of remote fields in mainstream tissue engineering.

Journal article

Loynachan C, Soleimany AP, Dudani JS, Lin Y, Najer A, Bekdemir A, Chen Q, Bhatia SN, Stevens Met al., Renal clearable catalytic gold nanoclusters for in vivo disease monitoring, Nature Nanotechnology, ISSN: 1748-3387

Ultra-small gold nanoclusters (AuNCs) have emerged as agile probes for in vivo imaging, asthey exhibit exceptional tumour accumulation and efficient renal clearance properties.However, their intrinsic catalytic activity, which can enable increased detection sensitivity, hasyet to be explored for in vivo sensing. By exploiting the peroxidase-mimicking activity of AuNCsand the precise nanometer size filtration of the kidney, we designed multifunctional proteasenanosensors that respond to disease microenvironments to produce a direct colorimetricurinary readout of disease state in less than 1 h. We monitored the catalytic activity of AuNCsin collected urine of a mouse model of colorectal cancer where tumour-bearing mice showeda 13-fold increase in colorimetric signal compared to healthy mice. Nanosensors wereeliminated completely through hepatic and renal excretion within 4 weeks after injection withno evidence of toxicity. We envision that this modular approach will enable rapid detection ofa diverse range of diseases by exploiting their specific enzymatic signatures.

Journal article

Autefage H, Allen F, Tang HM, Kallepitis C, Gentleman E, Reznikov N, Nitiputri K, Nommeots-Nomm A, Young G, Lee PD, Pierce BF, Wagermaier W, Fratzl P, Goodship A, Jones JR, Blunn G, Stevens Met al., 2019, Multiscale analyses reveal native-like lamellar bone repair and near perfect bone-contact with porous strontium-loaded bioactive glass, Biomaterials, Vol: 209, Pages: 152-162, ISSN: 0142-9612

The efficient healing of critical-sized bone defects using synthetic biomaterial-based strategies is promising but remains challenging as it requires the development of biomaterials that combine a 3D porous architecture and a robust biological activity. Bioactive glasses (BGs) are attractive candidates as they stimulate a biological response that favors osteogenesis and vascularization, but amorphous 3D porous BGs are difficult to produce because conventional compositions crystallize during processing. Here, we rationally designed a porous, strontium-releasing, bioactive glass-based scaffold (pSrBG) whose composition was tailored to deliver strontium and whose properties were optimized to retain an amorphous phase, induce tissue infiltration and encourage bone formation. The hypothesis was that it would allow the repair of a critical-sized defect in an ovine model with newly-formed bone exhibiting physiological matrix composition and structural architecture. Histological and histomorphometric analyses combined with indentation testing showed pSrBG encouraged near perfect bone-to-material contact and the formation of well-organized lamellar bone. Analysis of bone quality by a combination of Raman spectral imaging, small-angle X-ray scattering, X-ray fluorescence and focused ion beam-scanning electron microscopy demonstrated that the repaired tissue was akin to that of normal, healthy bone, and incorporated small amounts of strontium in the newly formed bone mineral. These data show the potential of pSrBG to induce an efficient repair of critical-sized bone defects and establish the importance of thorough multi-scale characterization in assessing biomaterial outcomes in large animal models.

Journal article

Barrioni BR, Naruphontjirakul P, Norris E, Li S, Kelly NL, Hanna J, Stevens MM, Jones JR, Pereira MDMet al., 2019, Effects of manganese incorporation on the morphology, structure and cytotoxicity of spherical bioactive glass nanoparticles, Journal of Colloid and Interface Science, Vol: 547, Pages: 382-392, ISSN: 0021-9797

Bioactive glass nanoparticles (BGNPs) are of great interest in tissue engineering as they possess high dissolution rate and capability of being internalized by cells, releasing their dissolution products with therapeutic benefits intracellularly. A modified Stöber process can be applied to obtain different BGNPs compositions containing therapeutic ions while maintaining controllable particle morphology, monodispersity and reduce agglomeration. Here, BGNPs containing Mn, an ion that has been shown to influence the osteoblast proliferation and bone mineralization, were evaluated. Particles with up to 142.3 ± 10.8 nm and spherical morphology were obtained after MnO incorporation in the SiO2 – CaO system. X-ray photoelectron spectroscopy (XPS) indicated the presence of Mn2+ species and also a reduction in the number of bridging oxygen bonds due to the Ca and Mn. The Ca and Mn network modifier role on the silica network was also confirmed by magic-angle spinning 29Si solid-state nuclear magnetic resonance (MAS NMR). MTT evaluation showed no reduction in the mitochondrial metabolic activity of human mesenchymal stem cells exposed to the glass ionic products. Thus, evaluation showed that Mn could be incorporated into BGNPs by the modified Stöber method while maintaining their spherical morphology and features as a promising strategy for tissue regeneration.

Journal article

Belessiotis-Richards A, Higgins SG, Butterworth B, Stevens MM, Alexander-Katz Aet al., 2019, Single-nanometer changes in nanopore geometry influence curvature, local properties, and protein localization in membrane simulations, Nano Letters, ISSN: 1530-6984

Nanoporous surfaces are used in many applications in intracellular sensing and drug delivery. However, the effects of such nanostructures on cell membrane properties are still far from understood. Here, we use coarse-grained molecular dynamics simulations to show that nanoporous substrates can stimulate membrane-curvature effects and that this curvature-sensing effect is much more sensitive than previously thought. We define a series of design parameters for inducing a nanoscale membrane curvature and show that nanopore taper plays a key role in membrane deformation, elucidating a previously unexplored fabrication parameter applicable to many nanostructured biomaterials. We report significant changes in the membrane area per lipid and thickness at regions of curvature. Finally, we demonstrate that regions of the nanopore-induced membrane curvature act as local hotspots for an increased bioactivity. We show spontaneous binding and localization of the epsin N-terminal homology (ENTH) domain to the regions of curvature. Understanding this interplay between the membrane curvature and nanoporosity at the biointerface helps both explain recent biological results and suggests a pathway for developing the next generation of cell-active substrates.

Journal article

Wang Y, Kim E, Lin Y, Kim N, Kit-Anan W, Gopal S, Agarwal S, Howes P, Stevens Met al., 2019, Rolling circle transcription-amplified hierarchically structured organic-inorganic hybrid RNA flowers for enzyme immobilization, ACS Applied Materials and Interfaces, ISSN: 1944-8244

Programmable nucleic acids have emerged as powerful building blocks for the bottom-up fabrication of two- or three-dimensional nano- and microsized constructs. Here we describe the construction of organic–inorganic hybrid RNA flowers (hRNFs) via rolling circle transcription (RCT), an enzyme-catalyzed nucleic acid amplification reaction. These hRNFs are highly adaptive structures with controlled sizes, specific nucleic acid sequences, and a highly porous nature. We demonstrated that hRNFs are applicable as potential biological platforms, where the hRNF scaffold can be engineered for versatile surface functionalization and the inorganic component (magnesium ions) can serve as an enzyme cofactor. For surface functionalization, we proposed robust and straightforward approaches including in situ synthesis of functional hRNFs and postfunctionalization of hRNFs that enable facile conjugation with various biomolecules and nanomaterials (i.e., proteins, enzymes, organic dyes, inorganic nanoparticles) using selective chemistries (i.e., avidin–biotin interaction, copper-free click reaction). In particular, we showed that hRNFs can serve as soft scaffolds for β-galactosidase immobilization and greatly enhance enzymatic activity and stability. Therefore, the proposed concepts and methodologies are not only fundamentally interesting when designing RNA scaffolds or RNA bionanomaterials assembled with enzymes but also have significant implications on their future utilization in biomedical applications ranging from enzyme cascades to biosensing and drug delivery.

Journal article

Gopal S, Chiappini C, Armstrong J, Chen Q, Serio A, Hsu C, Meinert C, Klein TJ, Hutmacher DW, Stevens Met al., 2019, Immunogold FIB-SEM: combining volumetric ultrastructure visualization with 3D biomolecular analysis to dissect cell-environment interactions, Advanced Materials, ISSN: 0935-9648

Volumetric imaging techniques capable of correlating structural and functional information with nanoscale resolution are necessary to broaden the insight into cellular processes within complex biological systems. The recent emergence of focused ion beam scanning electron microscopy (FIB‐SEM) has provided unparalleled insight through the volumetric investigation of ultrastructure; however, it does not provide biomolecular information at equivalent resolution. Here, immunogold FIB‐SEM, which combines antigen labeling with in situ FIB‐SEM imaging, is developed in order to spatially map ultrastructural and biomolecular information simultaneously. This method is applied to investigate two different cell–material systems: the localization of histone epigenetic modifications in neural stem cells cultured on microstructured substrates and the distribution of nuclear pore complexes in myoblasts differentiated on a soft hydrogel surface. Immunogold FIB‐SEM offers the potential for broad applicability to correlate structure and function with nanoscale resolution when addressing questions across cell biology, biomaterials, and regenerative medicine.

Journal article

Kim H, Park Y, Stevens MM, Kwon W, Hahn SKet al., 2019, Multifunctional hyaluronate - nanoparticle hybrid systems for diagnostic, therapeutic and theranostic applications, Journal of Controlled Release, Vol: 303, Pages: 55-66, ISSN: 0168-3659

Diagnostic and therapeutic nanoparticles have been actively investigated for the last few decades as new platforms for biomedical applications. Despite their great versatility and potency, nanoparticles have generally required further modification with biocompatible materials such as biopolymers and synthetic polymers for in vivo administration to improve their biological functions, stability, and biocompatibility. Among a variety of natural and synthetic biomaterials, hyaluronate (HA) has been considered a promising biomolecule with which to construct nanohybrid systems, as it can enable long-term and efficient delivery of nanoparticles to target sites as well as physiological stabilization of nanoparticles by forming hydrophilic shells. In this review, we first describe various kinds of HA derivatives and their interactions with nanoparticles, and discuss how to design and develop optimal HA-nanoparticle hybrid systems for biomedical applications. Furthermore, we show several exemplary applications of HA-nanoparticle hybrid systems and provide our perspectives to their futuristic translational applications.

Journal article

Ghouse S, Reznikov N, Boughton O, Babu S, Ng KCG, Blunn G, Cobb J, Stevens M, Jeffers Jet al., 2019, The design and in vivo testing of a locally stiffness-matched porous scaffold, Applied Materials Today, Vol: 15, Pages: 377-388, ISSN: 2352-9407

An increasing volume of work supports utilising the mechanobiology of bone for bone ingrowth into a porous scaffold. However, typically during in vivo testing of implants, the mechanical properties of the bone being replaced are not quantified. Consequently there remains inconsistencies in the literature regarding ‘optimum’ pore size and porosity for bone ingrowth. It is also difficult to compare ingrowth results between studies and to translate in vivo animal testing to human subjects without understanding the mechanical environment. This study presents a clinically applicable approach to determining local bone mechanical properties and design of a scaffold with similar properties. The performance of the scaffold was investigated in vivo in an ovine model.The density, modulus and strength of trabecular bone from the medial femoral condyle from ovine bones was characterised and power-law relationships were established. A porous titanium scaffold, intended to maintain bone mechanical homeostasis, was additively manufactured and implanted into the medial femoral condyle of 6 ewes. The stiffness of the scaffold varied throughout the heterogeneous structure and matched the stiffness variation of bone at the surgical site. Bone ingrowth into the scaffold was 10.73 ± 2.97% after 6 weeks. Fine woven bone, in the interior of the scaffold, and intense formations of more developed woven bone overlaid with lamellar bone at the implant periphery were observed. The workflow presented will allow future in vivo testing to test specific bone strains on bone ingrowth in response to a scaffold and allow for better translation from in vivo testing to commercial implants.

Journal article

Zhao S, Caruso F, Dähne L, Decher G, De Geest BG, Fan J, Feliu N, Gogotsi Y, Hammond PT, Hersam MC, Khademhosseini A, Kotov N, Leporatti S, Li Y, Lisdat F, Liz-Marzán LM, Moya S, Mulvaney P, Rogach AL, Roy S, Shchukin DG, Skirtach AG, Stevens MM, Sukhorukov GB, Weiss PS, Yue Z, Zhu D, Parak WJet al., 2019, The future of layer-by-layer assembly: A tribute to ACS Nano associate editor Helmuth Möhwald, ACS Nano, ISSN: 1936-0851

Layer-by-layer (LbL) assembly is a widely used tool for engineering materials and coatings. In this Perspective, dedicated to the memory of ACS Nano associate editor Prof. Dr. Helmuth Möhwald, we discuss the developments and applications that are to come in LbL assembly, focusing on coatings, bulk materials, membranes, nanocomposites, and delivery vehicles.

Journal article

Mohammed AA, Aviles Milan J, Li S, Chung JJ, Stevens MM, Georgiou TK, Jones JRet al., 2019, Open vessel free radical photopolymerization of double network gels for biomaterial applications using glucose oxidase, Journal of Materials Chemistry B, ISSN: 2050-750X

Polymerization of certain gels in the presence of oxygen can lead to hindered reaction rates and low conversion rates, limiting the use of open vessel polymerization and material synthesis. Here, the oxido-reductase enzyme glucose oxidase (GOx) was used to enable open vessel free radical photopolymerization (FRP) of neutral polyacrylamide (PAAm), and polyelectrolyte poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS) under ambient conditions. GOx successfully blocks the inhibition pathways created by O2 in FRP, dramatically increasing the polymer conversion rate for both polymers. In the presence of GOx, PAAm and PAMPS achieved conversion of 78% and 100% respectively at a photoinitiator (PI) concentration of 0.05 wt% with GOx, compared to 0% without GOx at the same PI concentration. Cytotoxicity studies of these polymers show high cell viability after GOx is denatured. Double network hydrogels (DNHGs) were successfully produced using the polymers and use of GOx improved compressive fracture stress by a factor of ten. Vinyl functionalized silica nanoparticles (VSNPs) were used as cross linkers of the first network to further enhance the mechanical properties.

Journal article

Nele V, Holme M, Kauscher U, Thomas MR, Doutch JJ, Stevens Met al., 2019, Effect of formulation method, lipid composition and PEGylation on vesicle lamellarity: a small-angle neutron scattering study, Langmuir, Vol: 35, Pages: 6064-6074, ISSN: 0743-7463

Liposomes are well-established systems for drug delivery and biosensing applications. The design of a liposomal carrier requires careful choice of lipid composition and formulation method. These determine many vesicle properties including lamellarity, which can have a strong effect on both encapsulation efficiency and the efflux rate of encapsulated active compounds. Despite this, a comprehensive study on how the lipid composition and formulation method affect vesicle lamellarity is still lacking. Here, we combine small-angle neutron scattering and cryogenic transmission electron microscopy to study the effect of three different well-established formulation methods followed by extrusion through 100 nm polycarbonate membranes on the resulting vesicle membrane structure. Specifically, we examine vesicles formulated from the commonly used phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) via film hydration followed by (i) agitation on a shaker or (ii) freeze–thawing, or (iii) the reverse-phase evaporation vesicle method. After extrusion, up to half of the total lipid content is still assembled into multilamellar structures. However, we achieved unilamellar vesicle populations when as little as 0.1 mol % PEG-modified lipid was included in the vesicle formulation. Interestingly, DPPC with 5 mol % PEGylated lipid produces a combination of cylindrical micelles and vesicles. In conclusion, our results provide important insights into the effect of the formulation method and lipid composition on producing liposomes with a defined membrane structure.

Journal article

Paxton NC, Ren J, Ainsworth MJ, Solanki AK, Jones JR, Allenby MC, Stevens M, Woodruff MAet al., 2019, Rheological characterization of biomaterials directs additive manufacturing of strontium-substituted bioactive Gglass/polycaprolactone microfibers, Macromolecular Rapid Communications, ISSN: 1022-1336

Additive manufacturing via melt electrowriting (MEW) can create ordered microfiber scaffolds relevant for bone tissue engineering; however, there remain limitations in the adoption of new printing materials, especially in MEW of biomaterials. For example, while promising composite formulations of polycaprolactone with strontium‐substituted bioactive glass have been processed into large or disordered fibres, from what is known, biologically‐relevant concentrations (>10 wt%) have never been printed into ordered microfibers using MEW. In this study, rheological characterization is used in combination with a predictive mathematical model to optimize biomaterial formulations and MEW conditions required to extrude various PCL and PCL/SrBG biomaterials to create ordered scaffolds. Previously, MEW printing of PCL/SrBG composites with 33 wt% glass required unachievable extrusion pressures. The composite formulation is modified using an evaporable solvent to reduce viscosity 100‐fold to fall within the predicted MEW pressure, temperature, and voltage tolerances, which enabled printing. This study reports the first fabrication of reproducible, ordered high‐content bioactive glass microfiber scaffolds by applying predictive modeling.

Journal article

Hansel C, Crowder S, Cooper S, Gopal S, Pardelha da Cruz J, De Oliveira Martins L, Keller D, Rothery S, Becce M, Cass A, Bakal C, Chiappini C, Stevens Met al., 2019, Nanoneedle-mediated stimulation of cell mechanotransduction machinery, ACS Nano, Vol: 13, Pages: 2913-2019, ISSN: 1936-0851

Biomaterial substrates can be engineered to present topographical signals to cells which, through interactions between the material and active components of the cell membrane, regulate key cellular processes and guide cell fate decisions. However, targeting mechanoresponsive elements that reside within the intracellular domain is a concept that has only recently emerged. Here, we show that mesoporous silicon nanoneedle arrays interact simultaneously with the cell membrane, cytoskeleton, and nucleus of primary human cells, generating distinct responses at each of these cellular compartments. Specifically, nanoneedles inhibit focal adhesion maturation at the membrane, reduce tension in the cytoskeleton, and lead to remodeling of the nuclear envelope at sites of impingement. The combined changes in actin cytoskeleton assembly, expression and segregation of the nuclear lamina, and localization of Yes-associated protein (YAP) correlate differently from what is canonically observed upon stimulation at the cell membrane, revealing that biophysical cues directed to the intracellular space can generate heretofore unobserved mechanosensory responses. These findings highlight the ability of nanoneedles to study and direct the phenotype of large cell populations simultaneously, through biophysical interactions with multiple mechanoresponsive components.

Journal article

Ferrini A, Stevens MM, Sattler S, Rosenthal Net al., 2019, Toward regeneration of the heart: Bioengineering strategies for immunomodulation, Frontiers in Cardiovascular Medicine, Vol: 6, ISSN: 2297-055X

Myocardial Infarction (MI) is the most common cardiovascular disease. An average-sized MI causes the loss of up to 1 billion cardiomyocytes and the adult heart lacks the capacity to replace them. Although post-MI treatment has dramatically improved survival rates over the last few decades, more than 20% of patients affected by MI will subsequently develop heart failure (HF), an incurable condition where the contracting myocardium is transformed into an akinetic, fibrotic scar, unable to meet the body's need for blood supply. Excessive inflammation and persistent immune auto-reactivity have been suggested to contribute to post-MI tissue damage and exacerbate HF development. Two newly emerging fields of biomedical research, immunomodulatory therapies and cardiac bioengineering, provide potential options to target the causative mechanisms underlying HF development. Combining these two fields to develop biomaterials for delivery of immunomodulatory bioactive molecules holds great promise for HF therapy. Specifically, minimally invasive delivery of injectable hydrogels, loaded with bioactive factors with angiogenic, proliferative, anti-apoptotic and immunomodulatory functions, is a promising route for influencing the cascade of immune events post-MI, preventing adverse left ventricular remodeling, and offering protection from early inflammation to fibrosis. Here we provide an updated overview on the main injectable hydrogel systems and bioactive factors that have been tested in animal models with promising results and discuss the challenges to be addressed for accelerating the development of these novel therapeutic strategies.

Journal article

Li C, Ouyang L, Pence I, Moore A, Lin Y, Winter C, Armstrong J, Stevens Met al., 2019, Buoyancy-driven Gradients for Biomaterial Fabrication and Tissue Engineering, Advanced Materials, ISSN: 0935-9648

The controlled fabrication of gradient materials is becoming increasingly important as the next generation of tissue engineering seeks to produce inhomogeneous constructs with physiological complexity. Current strategies for fabricating gradient materials can require highly specialized materials or equipment and cannot be generally applied to the wide range of systems used for tissue engineering. Here, the fundamental physical principle of buoyancy is exploited as a generalized approach for generating materials bearing well‐defined compositional, mechanical, or biochemical gradients. Gradient formation is demonstrated across a range of different materials (e.g., polymers and hydrogels) and cargos (e.g., liposomes, nanoparticles, extracellular vesicles, macromolecules, and small molecules). As well as providing versatility, this buoyancy‐driven gradient approach also offers speed (<1 min) and simplicity (a single injection) using standard laboratory apparatus. Moreover, this technique is readily applied to a major target in complex tissue engineering: the osteochondral interface. A bone morphogenetic protein 2 gradient, presented across a gelatin methacryloyl hydrogel laden with human mesenchymal stem cells, is used to locally stimulate osteogenesis and mineralization in order to produce integrated osteochondral tissue constructs. The versatility and accessibility of this fabrication platform should ensure widespread applicability and provide opportunities to generate other gradient materials or interfacial tissues.

Journal article

Wood CS, Thomas M, Budd J, Mashamba-Thompson TP, Herbst K, Pillay D, Peeling RW, Johnson AM, McKendry RA, Stevens Met al., Taking connected mobile-health diagnostics of infectious diseases to the field., Nature, ISSN: 0028-0836

Mobile Health or mHealth - The application of mobile devices, their componentsand related technologies to healthcare is improving patients’ access to treatment andadvice. Now, in combination with connected diagnostic devices it offers new possibilitiesto diagnose, track and control infectious diseases and improve health systemefficiencies. In this context we look at these technologies and highlight their promise butalso the challenges in realising their potential to increase patient access to testing, aid intheir treatment and improve the capability of public health authorities to monitoroutbreaks, implement responses, and assess the impact of interventions across theworld.

Journal article

Lin Y, Penna M, Thomas MR, Wojciechowski J, Leonardo V, Wang Y, Pashuck ET, Yarovsky I, Stevens Met al., 2019, Residue-specific solvation directed thermodynamic and kinetic control over peptide self-assembly with 1D/2D structure selection, ACS Nano, Vol: 13, Pages: 1900-1909, ISSN: 1936-0851

Understanding the self-organization and structural transformations of molecular ensembles is important to explore the complexity of biological systems. Here, we illustrate the crucial role of cosolvents and solvation effects in thermodynamic and kinetic control over peptide association into ultrathin Janus nanosheets, elongated nanobelts, and amyloid-like fibrils. We gained further insight into the solvation-directed self-assembly (SDSA) by investigating residue-specific peptide solvation using molecular dynamics modeling. We proposed the preferential solvation of the aromatic and alkyl domains on the peptide backbone and protofibril surface, which results in volume exclusion effects and restricts the peptide association between hydrophobic walls. We explored the SDSA phenomenon in a library of cosolvents (protic and aprotic), where less polar cosolvents were found to exert a stronger influence on the energetic balance at play during peptide propagation. By tailoring cosolvent polarity, we were able to achieve precise control of the peptide nanostructures with 1D/2D shape selection. We also illustrated the complexity of the SDSA system with pathway-dependent peptide aggregation, where two self-assembly states (i.e., thermodynamic equilibrium state and kinetically trapped state) from different sample preparation methods were obtained.

Journal article

Armstrong J, Stevens M, 2019, Emerging technologies for tissue engineering: from gene editing to personalized medicine, Tissue Engineering: Parts A, B, and C, ISSN: 1937-3341

Technological innovation has been integral to the development of tissue engineering over the last 25 years. Future advances will require the next-generation of tissue engineers to embrace emerging technologies. Here, we discuss four key areas of opportunity in which technology can play a role: biological manipulation, advanced characterization, process automation and personalized medicine. This encompasses key developments in transdifferentiation, gene editing, spatially-resolved -omics and 3D bioprinting. Taken together, we can imagine an idealized future in which computational predictions made by machine learning algorithms are used to programme cells and materials to create personalized tissue constructs within an automated culture system.

Journal article

Armstrong J, Maynard S, Pence I, Franklin AC, Drinkwater BW, Stevens Met al., 2019, Spatiotemporal quantification of acoustic cell patterning using Voronoi Tessellation, Lab on a Chip, Vol: 19, Pages: 562-573, ISSN: 1473-0189

Acoustic patterning using ultrasound standing waves has recently emerged as a potent biotechnology enabling the remote generation of ordered cell systems. This capability has opened up exciting opportunities, for example, in guiding the development of organoid cultures or the organization of complex tissues. The success of these studies is often contingent on the formation of tightly-packed and uniform cell arrays; however, a number of factors can act to disrupt or prevent acoustic patterning. Yet, to the best of our knowledge, there has been no comprehensive assessment of the quality of acoustically-patterned cell populations. In this report we use a mathematical approach, known as Voronoï tessellation, to generate a series of metrics that can be used to measure the effect of cell concentration, pressure amplitude, ultrasound frequency and biomaterial viscosity upon the quality of acoustically-patterned cell systems. Moreover, we extend this approach towards the characterization of spatiotemporal processes, namely, the acoustic patterning of cell suspensions and the migration of patterned, adherent cell clusters. This strategy is simple, unbiased and highly informative, and we anticipate that the methods described here will provide a systematic framework for all stages of acoustic patterning, including the robust quality control of devices, statistical comparison of patterning conditions, the quantitative exploration of parameter limits and the ability to track patterned tissue formation over time.

Journal article

Kim E, Agarwal S, Kim N, Hage FS, Gelmi A, Stevens Met al., 2019, Bio-inspired fabrication of DNA-inorganic hybrid composites using synthetic DNA, ACS Nano, ISSN: 1936-0851

Nucleic acid nanostructures have attracted significant interest as potential therapeutic and diagnostic platforms due to their intrinsic biocompatibility and biodegradability, structural and functional diversity, and compatibility with various chemistries for modification and stabilization. Among the fabrication approaches for such structures, the rolling circle techniques have emerged as particularly promising, producing morphologically round, flower-shaped nucleic acid particles: typically hybrid composites of long nucleic acid strands and inorganic magnesium pyrophosphate (Mg2PPi). These constructs are known to form via anisotropic nucleic acid-driven crystallization in a sequence-independent manner, rendering monodisperse and densely packed RNA or DNA–inorganic composites. However, it still remains to fully explore how flexible polymer-like RNA or DNA strands (acting as biomolecular additives) mediate the crystallization process of Mg2PPi and affect the structure and properties of the product crystals. To address this, we closely examined nanoscale details to mesoscopic features of Mg2PPi/DNA hybrid composites fabricated by two approaches, namely rolling circle amplification (RCA)-based in situ synthesis and long synthetic DNA-mediated crystallization. Similar to the DNA constructs fabricated by RCA, the rapid crystallization of Mg2PPi was retarded on a short-range order when we precipitated the crystals in the presence of presynthesized long DNA, which resulted in effective incorporation of biomolecular additives such as DNA and enzymes. These findings further provide a more feasible way to encapsulate bioactive enzymes within DNA constructs compared to in situ RCA-mediated synthesis, i.e., by not only protecting them from possible denaturation under the reaction conditions but also preventing nonselective association of proteins arising from the RCA reaction mixtures.

Journal article

Reznikov N, Boughton O, Ghouse S, Weston AE, Collinson L, Blunn GW, Jeffers J, Cobb J, Stevens Met al., 2019, Individual response variations in scaffold-guided bone regeneration are determined by independent strain- and injury-induced mechanisms, Biomaterials, Vol: 194, Pages: 183-194, ISSN: 0142-9612

This study explored the regenerative osteogenic response in the distal femur of sheep using scaffolds having stiffness values within, and above and below, the range of trabecular bone apparent modulus. Scaffolds 3D-printed from stiff titanium and compliant polyamide were implanted into a cylindrical metaphyseal defect 15 × 15 mm. After six weeks, bone ingrowth varied between 7 and 21% of the scaffold pore volume and this was generally inversely proportional to scaffold stiffness. The individual reparative response considerably varied among the animals, which could be divided into weak and strong responders. Notably, bone regeneration specifically within the interior of the scaffold was inversely proportional to scaffold stiffness and was strain-driven in strongly-responding animals. Conversely, bone regeneration at the periphery of the defect was injury-driven and equal in all scaffolds and in all strongly- and weakly-responding animals. The observation of the strain-driven response in some, but not all, animals highlights that scaffold compliance is desirable for triggering host bone regeneration, but scaffold permanence is important for the load-bearing, structural role of the bone-replacing device. Indeed, scaffolds may benefit from being nonresorbable and mechanically reliable for those unforeseeable cases of weakly responding recipients.

Journal article

Lin Y, Mazo M, Skaalure S, Thomas MR, Schultz SR, Stevens Met al., 2019, Activatable cell-biomaterial interfacing with photo-caged peptides, Chemical Science, Vol: 10, Pages: 1158-1167, ISSN: 2041-6520

Spatio-temporally tailoring cell–material interactions is essential for developing smart delivery systems and intelligent biointerfaces. Here we report new photo-activatable cell–material interfacing systems that trigger cellular uptake of various cargoes and cell adhesion towards surfaces. To achieve this, we designed a novel photo-caged peptide which undergoes a structural transition from an antifouling ligand to a cell-penetrating peptide upon photo-irradiation. When the peptide is conjugated to ligands of interest, we demonstrate the photo-activated cellular uptake of a wide range of cargoes, including small fluorophores, proteins, inorganic (e.g., quantum dots and gold nanostars) and organic nanomaterials (e.g., polymeric particles), and liposomes. Using this system, we can remotely regulate drug administration into cancer cells by functionalizing camptothecin-loaded polymeric nanoparticles with our synthetic peptide ligands. Furthermore, we show light-controlled cell adhesion on a peptide-modified surface and 3D spatiotemporal control over cellular uptake of nanoparticles using two-photon excitation. We anticipate that the innovative approach proposed in this work will help to establish new stimuli-responsive delivery systems and biomaterials.

Journal article

Gopal S, Chiappini C, Penders J, Leonardo V, Seong H, Rothery S, Korchev Y, Shevchuk A, Stevens MMet al., 2019, Porous silicon nanoneedles modulate endocytosis to deliver biological payloads, Advanced Materials, ISSN: 0935-9648

Owing to their ability to efficiently deliver biological cargo and sense the intracellular milieu, vertical arrays of high aspect ratio nanostructures, known as nanoneedles, are being developed as minimally invasive tools for cell manipulation. However, little is known of the mechanisms of cargo transfer across the cell membrane‐nanoneedle interface. In particular, the contributions of membrane piercing, modulation of membrane permeability and endocytosis to cargo transfer remain largely unexplored. Here, combining state‐of‐the‐art electron and scanning ion conductance microscopy with molecular biology techniques, it is shown that porous silicon nanoneedle arrays concurrently stimulate independent endocytic pathways which contribute to enhanced biomolecule delivery into human mesenchymal stem cells. Electron microscopy of the cell membrane at nanoneedle sites shows an intact lipid bilayer, accompanied by an accumulation of clathrin‐coated pits and caveolae. Nanoneedles enhance the internalization of biomolecular markers of endocytosis, highlighting the concurrent activation of caveolae‐ and clathrin‐mediated endocytosis, alongside macropinocytosis. These events contribute to the nanoneedle‐mediated delivery (nanoinjection) of nucleic acids into human stem cells, which distribute across the cytosol and the endolysosomal system. This data extends the understanding of how nanoneedles modulate biological processes to mediate interaction with the intracellular space, providing indications for the rational design of improved cell‐manipulation technologies.

Journal article

Lin Y, Charchar P, Christofferson AJ, Thomas MR, Todorova N, Mazo MM, Chen Q, Doutch J, Richardson R, Yarovsky I, Stevens Met al., 2018, Surface dynamics and ligand-core interactions of quantum sized photoluminescent gold nanoclusters, Journal of the American Chemical Society, Vol: 140, Pages: 18217-18226, ISSN: 1520-5126

Quantum-sized metallic clusters protected by biological ligands represent a new class of luminescent materials; yet the understanding of structural information and photoluminescence origin of these ultrasmall clusters remains a challenge. Herein we systematically study the surface ligand dynamics and ligand–metal core interactions of peptide-protected gold nanoclusters (AuNCs) with combined experimental characterizations and theoretical molecular simulations. We show that the peptide sequence plays an important role in determining the surface peptide structuring, interfacial water dynamics and ligand–Au core interaction, which can be tailored by controlling peptide acetylation, constituent amino acid electron donating/withdrawing capacity, aromaticity/hydrophobicity and by adjusting environmental pH. Specifically, emission enhancement is achieved through increasing the electron density of surface ligands in proximity to the Au core, discouraging photoinduced quenching, and by reducing the amount of surface-bound water molecules. These findings provide key design principles for understanding the surface dynamics of peptide-protected nanoparticles and maximizing the photoluminescence of metallic clusters through the exploitation of biologically relevant ligand properties.

Journal article

Pujari-Palmer M, Guo H, Wenner D, Autefage H, Spicer CD, Stevens MM, Omar O, Thomsen P, Eden M, Insley G, Procter P, Engqvist Het al., 2018, A novel class of injectable bioceramics that glue tissues and biomaterials, Materials, Vol: 11, ISSN: 1996-1944

Calcium phosphate cements (CPCs) are clinically effective void fillers that are capable of bridging calcified tissue defects and facilitating regeneration. However, CPCs are completely synthetic/inorganic, unlike the calcium phosphate that is found in calcified tissues, and they lack an architectural organization, controlled assembly mechanisms, and have moderate biomechanical strength, which limits their clinical effectiveness. Herein, we describe a new class of bioinspired CPCs that can glue tissues together and bond tissues to metallic and polymeric biomaterials. Surprisingly, alpha tricalcium phosphate cements that are modified with simple phosphorylated amino acid monomers of phosphoserine (PM-CPCs) bond tissues up to 40-fold stronger (2.5–4 MPa) than commercial cyanoacrylates (0.1 MPa), and 100-fold stronger than surgical fibrin glue (0.04 MPa), when cured in wet-field conditions. In addition to adhesion, phosphoserine creates other novel properties in bioceramics, including a nanoscale organic/inorganic composite microstructure, and templating of nanoscale amorphous calcium phosphate nucleation. PM-CPCs are made of the biocompatible precursors calcium, phosphate, and amino acid, and these represent the first amorphous nano-ceramic composites that are stable in liquids.

Journal article

Jumeaux C, Kim E, Howes P, Kim H, Chandrawati R, Stevens Met al., 2018, Detection of microRNA biomarkers via inhibition of DNA-mediated liposome fusion, Nanoscale Advances, ISSN: 2516-0230

We report the specific and sensitive detection of microRNA using an inverse DNA-mediated liposome fusion assay. This assay is homogeneous, and does not require washing, separation, or enzyme-associated amplification steps. By fine-tuning the surface functionalisation of the liposomes, liposome concentration, and assay temperature, we demonstrated a sub-nanomolar limit of detection for the target.

Journal article

Stevens M, Kauscher U, Holme M, Björnmalm Met al., Physical stimuli-responsive vesicles in drug delivery: Beyond liposomes and polymersomes, Advanced Drug Delivery Reviews, ISSN: 0169-409X

Over the past few decades, a range of vesicle-based drug delivery systems have entered clinical practice and several others are in various stages of clinical translation. While most of these vesicle constructs are lipid-based (liposomes), or polymer-based (polymersomes), recently new classes of vesicles have emerged that defy easy classification. Examples include assemblies with small molecule amphiphiles, biologically derived membranes, hybrid vesicles with two or more classes of amphiphiles, or more complex hierarchical structures such as vesicles incorporating gas bubbles or nanoparticulates in the lumen or membrane. In this review, we explore these recent advances and emerging trends at the edge and just beyond the research fields of conventional liposomes and polymersomes. A focus of this review is the distinct behaviors observed for these classes of vesicles when exposed to physical stimuli - such as ultrasound, heat, light and mechanical triggers - and we discuss the resulting potential for new types of drug delivery, with a special emphasis on current challenges and opportunities.

Journal article

Armstrong J, Puetzer JL, Serio A, Guex AG, Kapnisi K, Breant A, Zong Y, Assal V, Skaalure S, King O, Murty T, Meinert C, Franklin AC, Bassindale PG, Nichols MK, Terracciano C, Hutmacher DW, Drinkwater BW, Klein TJ, Perriman AW, Stevens MMet al., 2018, Engineering anisotropic muscle tissue using acoustic cell patterning, Advanced Materials, Vol: 30, ISSN: 0935-9648

Tissue engineering has offered unique opportunities for disease modeling and regenerative medicine; however, the success of these strategies is dependent on faithful reproduction of native cellular organization. Here, it is reported that ultrasound standing waves can be used to organize myoblast populations in material systems for the engineering of aligned muscle tissue constructs. Patterned muscle engineered using type I collagen hydrogels exhibits significant anisotropy in tensile strength, and under mechanical constraint, produced microscale alignment on a cell and fiber level. Moreover, acoustic patterning of myoblasts in gelatin methacryloyl hydrogels significantly enhances myofibrillogenesis and promotes the formation of muscle fibers containing aligned bundles of myotubes, with a width of 120–150 µm and a spacing of 180–220 µm. The ability to remotely pattern fibers of aligned myotubes without any material cues or complex fabrication procedures represents a significant advance in the field of muscle tissue engineering. In general, these results are the first instance of engineered cell fibers formed from the differentiation of acoustically patterned cells. It is anticipated that this versatile methodology can be applied to many complex tissue morphologies, with broader relevance for spatially organized cell cultures, organoid development, and bioelectronics.

Journal article

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