Biomaterials (v.29, #17)
Editorial board (IFC).
A thermoresponsive, microtextured substrate for cell sheet engineering with defined structural organization by Brett C. Isenberg; Yukiko Tsuda; Corin Williams; Tatsuya Shimizu; Masayuki Yamato; Teruo Okano; Joyce Y. Wong (2565-2572).
The proper function of many tissues depends critically on the structural organization of the cells and matrix of which they are comprised. Therefore, in order to engineer functional tissue equivalents that closely mimic the unique properties of native tissues it is necessary to develop strategies for reproducing the complex, highly organized structure of these tissues. To this end, we sought to develop a simple method for generating cell sheets that have defined ECM/cell organization using microtextured, thermoresponsive polystyrene substrates to guide cell organization and tissue growth. The patterns consisted of large arrays of alternating grooves and ridges (50 μm wide, 5 μm deep). Vascular smooth muscle cells cultured on these substrates produced intact sheets consisting of cells that exhibited strong alignment in the direction of the micropattern. These sheets could be readily transferred from patterned substrates to non-patterned substrates without the loss of tissue organization. Ultimately, such sheets will be layered to form larger tissues with defined ECM/cell organization that spans multiple length scales.
Keywords: Thermoresponsive surfaces; Surface topography; Embossing; Cell sheet; Tissue engineering; Smooth muscle cell;
Patterned growth of neuronal cells on modified diamond-like carbon substrates by Stephen Kelly; Edward M. Regan; James B. Uney; Andrew D. Dick; Joseph P. McGeehan; Eric J. Mayer; Frederik Claeyssens (2573-2580).
Diamond-like carbon (DLC) has been explored as a biomaterial with potential use for coating implantable devices and surgical instruments. In this study the interaction of DLC with mammalian neuronal cells has been studied along with its modifications to improve its function as a biomaterial. We describe the use of DLC, oxidised DLC and phosphorus-doped DLC to support the growth and survival of primary central nervous system neurones and neuroblastoma cells. None of these substrates were cytotoxic and primary neurones adhered better to phosphorus-doped DLC than unmodified DLC. This property was used to culture cortical neurones in a predetermined micropattern. This raises the potential of DLC as a biomaterial for central nervous system (CNS) implantation. Furthermore, patterned DLC and phosphorus-doped DLC can direct neuronal growth, generating a powerful tool to study neuronal networks in a spatially distinct way. This study reports the generation of nerve cell patterns via patterned deposition of DLC.
Keywords: Laser ablation; Neural cell; Carbon; Diamond; Micropatterning;
Extracellular matrix remodelling during cell adhesion monitored by the quartz crystal microbalance by Megan S. Lord; Charlotte Modin; Morten Foss; Mogens Duch; Anne Simmons; Finn S. Pedersen; Flemming Besenbacher; Bruce K. Milthorpe (2581-2587).
A cell's ability to remodel adsorbed protein layers on surfaces is influenced by the nature of the protein layer itself. Remodelling is often required to accomplish cellular adhesion and extracellular matrix formation which forms the basis for cell spreading, increased adhesion and expression of different phenotypes. The adhesion of NIH3T3 (EGFP) fibroblasts to serum protein (albumin or fibronectin) precoated tantalum (Ta) and oxidised polystyrene (PSox) surfaces was examined using the quartz crystal microbalance with dissipation (QCM-D) monitoring and fluorescence microscopy. The cells were either untreated or treated with cycloheximide to examine the contribution of endogenous protein production during cell adhesion to the QCM-D response over a period of 2 h. Following adsorption of albumin onto Ta and PSox there was no difference detected between the response to seeding untreated and cycloheximide treated cells. The QCM-D was able to detect differences in the untreated cellular responses to fibronectin versus serum precoated Ta and PSox substrates, while cycloheximide treatment of the cells produced the same QCM-D response for fibronectin and serum precoatings on each of the materials. This confirmed that the process of matrix remodelling by the cells is dependent on the underlying substrate and the preadsorbed proteins and that the QCM-D response is dominated by changes in the underlying protein layer. Changes in dissipation correspond to the development of the actin cytoskeleton as visualised by actin staining.
Keywords: Cell adhesion; Quartz crystal microbalance; Fibroblast; Protein adsorption; Polystyrene; Tantalum;
Reconstruction of calvarial defect of rabbits using porous calcium silicate bioactive ceramics by Songfeng Xu; Kaili Lin; Zhen Wang; Jiang Chang; Lin Wang; Jianxi Lu; Congqin Ning (2588-2596).
In this study, the in vivo bone-regenerative capacity and resorption of the porous β-calcium silicate (β-CaSiO3, β-CS) bioactive ceramics were investigated in a rabbit calvarial defect model, and the results were compared with porous β-tricalcium phosphate (β-Ca3(PO4)2, β-TCP) bioceramics. The porous β-CS and β-TCP ceramics were implanted in rabbit calvarial defects and the specimens were harvested after 4, 8 and 16 weeks, and evaluated by Micro-CT and histomorphometric analysis. The Micro-CT and histomorphometric analysis showed that the resorption of β-CS was much higher than that of β-TCP. The TRAP-positive multinucleated cells were observed on the surface of β-CS, suggesting a cell-mediated process involved in the degradation of β-CS in vivo. The amount of newly formed bone was also measured and more bone formation was observed with β-CS as compared with β-TCP (p < 0.05). Histological observation demonstrated that newly formed bone tissue grew into the porous β-CS, and a bone-like apatite layer was identified between the bone tissue and β-CS materials. The present studies showed that the porous β-CS ceramics could stimulate bone regeneration and may be used as bioactive and biodegradable materials for hard tissue repair and tissue engineering applications.
Keywords: Calcium silicate; Porous ceramics; Bone regeneration; Resorption; In vivo;
The effects of matrix stiffness and RhoA on the phenotypic plasticity of smooth muscle cells in a 3-D biosynthetic hydrogel system by Shelly R. Peyton; Peter D. Kim; Cyrus M. Ghajar; Dror Seliktar; Andrew J. Putnam (2597-2607).
Studies using 2-D cultures have shown that the mechanical properties of the extracellular matrix (ECM) influence cell migration, spreading, proliferation, and differentiation; however, cellular mechanosensing in 3-D remains under-explored. To investigate this topic, a unique biomaterial system based on poly(ethylene glycol)-conjugated fibrinogen was adapted to study phenotypic plasticity in smooth muscle cells (SMCs) as a function of ECM mechanics in 3-D. Tuning the compressive modulus between 448 and 5804 Pa modestly regulated SMC cytoskeletal assembly in 3-D, with spread cells in stiff matrices having a slightly higher degree of F-actin bundling after prolonged culture. However, vinculin expression in all 3-D conditions was qualitatively low and was not assembled into the classic focal adhesions typically seen in 2-D cultures. Given the evidence that RhoA-mediated cytoskeletal contractility represents a critical node in mechanosensing, we molecularly upregulated contractility by inducing SMCs to express constitutively active RhoA. In these cells, F-actin bundling and total vinculin expression increased, and focal adhesion-like structures began to emerge, consistent with RhoA's mechanism of action in cells cultured on 2-D substrates. Furthermore, SMC proliferation in 3-D did not depend significantly on matrix stiffness, and was reduced by constitutive activation of RhoA irrespective of ECM mechanical properties. Conversely, the expression of contractile markers globally increased with constitutive RhoA activation and depended on 3-D matrix stiffness only in cells with heightened RhoA activity. Combined, these data suggest that the synergistic effects of ECM mechanics and RhoA activity on SMC phenotype in 3-D are distinct from those in 2-D, and highlight the importance of studying the mechanical role of cell–matrix interactions in tunable 3-D environments.
Keywords: ECM (extracellular matrix); Hydrogel; Mechanical properties; Polyethylene oxide; Smooth muscle cell;
Rapid prototyped porous titanium coated with calcium phosphate as a scaffold for bone tissue engineering by Marco A. Lopez-Heredia; Jerome Sohier; Cedric Gaillard; Sophie Quillard; Michel Dorget; Pierre Layrolle (2608-2615).
High strength porous scaffolds and mesenchymal stem cells are required for bone tissue engineering applications. Porous titanium scaffolds (TiS) with a regular array of interconnected pores of 1000 μm in diameter and a porosity of 50% were produced using a rapid prototyping technique. A calcium phosphate (CaP) coating was applied to these titanium (Ti) scaffolds with an electrodeposition method. Raman spectroscopy and energy dispersive X-ray analysis showed that the coating consisted of carbonated hydroxyapatite. Cross-sectioned observations by scanning electron microscopy indicated that the coating evenly covered the entire structure with a thickness of approximately 25 μm. The bonding strength of the coating to the substrate was evaluated to be around 25 MPa. Rat bone marrow cells (RBMC) were seeded and cultured on the Ti scaffolds with or without coating. The Alamar Blue assay provided a low initial cell attachment (40%) and cell numbers were similar on both the uncoated and coated Ti scaffolds after 3 days. The Ti scaffolds were subsequently implanted subcutaneously for 4 weeks in syngenic rats. Histology revealed the presence of a mineralized collagen tissue in contact with the implants, but no bone formation. This study demonstrated that porous Ti scaffolds with high strength and defined geometry may be evenly coated with CaP layers and cultured mesenchymal stem cells for bone tissue engineering.
Keywords: Titanium; Rapid prototyping; Scaffold; Electrodeposition; CaP coating;
Flow perfusion culture of human mesenchymal stem cells on silicate-substituted tricalcium phosphate scaffolds by Lea Bjerre; Cody E. Bünger; Moustapha Kassem; Tina Mygind (2616-2627).
Autologous bone grafts are currently the gold standard for treatment of large bone defects, but their availability is limited due to donor site morbidity. Different substitutes have been suggested to replace these grafts, and this study presents a bone tissue engineered alternative using silicate-substituted tricalcium phosphate (Si-TCP) scaffolds seeded with human bone marrow-derived mesenchymal stem cells (hMSC). The cells were seeded onto the scaffolds and cultured either statically or in a perfusion bioreactor for up to 21 days and assessed for osteogenic differentiation by alkaline phosphatase activity assays and by quantitative real-time RT-PCR on bone markers. During culture, cells from the flow cultured constructs demonstrated improved proliferation and osteogenic differentiation verified by a more pronounced expression of several bone markers, e.g. alkaline phosphatase, osteopontin, Runx2, bone sialoprotein II, and bone morphogenetic protein 2. Cells and matrix were distributed homogenously throughout the entire scaffold in flow culture, whereas only a peripheral layer was obtained after static culture. A viable and homogenous ex vivo bone construct with superior osteogenic properties was produced in dynamic culture and may provide a replacement for autologous grafts.
Keywords: Bioreactor; Stem cells; Silicate-substituted tricalcium phosphate; Scaffold; Osteogenesis; Real-time RT-PCR;
Development of anti-atherosclerotic tissue-engineered blood vessel by A20-regulated endothelial progenitor cells seeding decellularized vascular matrix by Chuhong Zhu; Dajun Ying; Jianhong Mi; Li Li; Wen Zeng; Chunli Hou; Jiansen Sun; Wei Yuan; Can Wen; Wei Zhang (2628-2636).
To investigate whether decellularized vascular tissues and A20-regulated endothelial progenitor cells can be used for constructing a transgenic tissue-engineered blood vessel with anti-atherosclerotic vascular stenotic properties. A20 gene-transfected endothelial progenitor cells differentiated endothelial cells and smooth muscle cells attached to and migrated into the decellularized porcine vascular scaffolding in a bioreactor. The histology of the conduits revealed viable and layered tissue. Scanning electron microscopy showed confluent, homogeneous tissue surfaces. The mechanical strength of the pulsed constructs was similar to that of the human artery. In vivo, the A20 gene-transfected tissue-engineered blood vessels were transplanted into the carotid artery of a rat for 6 months. Blood vessel xenotransplantation caused hyperacute rejection; all transplanted control blood vessels were completely rejected, but A20-transfected tissue-engineered blood vessels demonstrated good flow on implantation, and remained open for 6 months postoperatively, as assessed by Doppler. The HE stain demonstrated that the vessels were patent, without evidence of stenosis or dilatation after 6 months. These results demonstrate that transgenic tissue-engineered blood vessels have long-term patency and unique anti-stenotic properties.
Keywords: Vascular graft; Restenosis; Stem cell; Gene transfer; Arterial tissue engineering;
Bioadhesive hydrogel microenvironments to modulate epithelial morphogenesis by I.-Ming Chung; Nduka O. Enemchukwu; Sirajud D. Khaja; Niren Murthy; Athanasios Mantalaris; Andrés J. García (2637-2645).
Epithelial cells polarize and differentiate into organotypic cell aggregates in response to cell–cell and cell–matrix interactions. For example, Madin–Darby Canine Kidney (MDCK) cells form spherical cell aggregates (cysts) with distinct apical and basolateral polarity when cultured three dimensionally (embedded) in type I collagen gels. To investigate the effects of individual extracellular factors on epithelial morphogenesis, we engineered fast degrading protease-responsive polyethylene glycol (PEG) hydrogels functionalized with controlled densities of various bioligands (RGD peptide, laminin-1 (LN)) to allow 3D culturing of MDCK cells, cyst expansion, and morphogenesis/polarization. Cysts formed after 15 days of culture in these hydrogels were analyzed with multiphoton fluorescence microscopy for markers of apical and basolateral membrane domains. Epithelial cysts formed in bioadhesive ligand-functionalized PEG gels exhibited a higher frequency of central lumen and interior apical pole formation as well as basolateral polarization compared to those of unmodified PEG hydrogels. These results demonstrate that incorporation of specific bioadhesive motifs into synthetic hydrogels provides 3D culture environments that support epithelial morphogenesis. These microenvironments provide a flexible and controlled system for systematic investigations into normal and pathologic morphogenic behaviours as well as synthetic environments for promoting tissue morphogenesis for regenerative medicine applications.
Keywords: Cell adhesion; Extracellular matrix; Differentiation; Laminin; RGD;
Individually programmable cell stretching microwell arrays actuated by a Braille display by Yoko Kamotani; Tommaso Bersano-Begey; Nobuhiro Kato; Yi-Chung Tung; Dongeun Huh; Jonathan W. Song; Shuichi Takayama (2646-2655).
Cell culture systems are often static and are therefore nonphysiological. In vivo, many cells are exposed to dynamic surroundings that stimulate cellular responses in a process known as mechanotransduction. To recreate this environment, stretchable cell culture substrate systems have been developed, however, these systems are limited by being macroscopic and low throughput. We have developed a device consisting of 24 miniature cell stretching chambers with flexible bottom membranes that are deformed using the computer-controlled, piezoelectrically actuated pins of a Braille display. We have also developed efficient image capture and analysis protocols to quantify morphological responses of the cells to applied strain. Human dermal microvascular endothelial cells (HDMECs) were found to show increasing degrees of alignment and elongation perpendicular to the radial strain in response to cyclic stretch at increasing frequencies of 0.2, 1, and 5 Hz, after 2, 4, and 12 h. Mouse myogenic C2C12 cells were also found to align in response to the stretch, while A549 human lung adenocarcinoma epithelial cells did not respond to stretch.
Keywords: Cell stretching; Polydimethylsiloxane; Cell morphology; Endothelial cell; Epithelial cell; Lung;
Atorvastatin and uptake of ultrasmall superparamagnetic iron oxide nanoparticles (Ferumoxtran-10) in human monocyte–macrophages: Implications for magnetic resonance imaging by Karin Müller; Jeremy N. Skepper; Tjun Y. Tang; Martin J. Graves; Andrew J. Patterson; Claire Corot; Eric Lancelot; Paul W. Thompson; Andrew P. Brown; Jonathan H. Gillard (2656-2662).
Ferumoxtran-10 is an ultrasmall superparamagnetic iron oxide nanoparticle potentially useful as a contrast material in magnetic resonance imaging for the diagnosis of inflammatory and degenerative disorders associated with high macrophage activity. In clinical trials, it is currently applied to monitor the effect of atorvastatin therapy on macrophage activity in human carotid plaques. A recent study reported the inhibition of iron oxide nanoparticle uptake in macrophages by lovastatin, an effect which could compromise the suitability of Ferumoxtran-10 as an MRI contrast material in patients on statin therapy. Therefore, we examined the effect of atorvastatin on human monocyte–macrophage uptake of Ferumoxtran-10 in vitro using biochemical assays, magnetic resonance imaging and transmission electron microscopy. Our study showed that non-toxic concentrations of atorvastatin did not affect the amount of Ferumoxtran-10 taken up by HMMs. Furthermore, the intracellular distribution of iron oxide nanoparticles and the resulting MRI signal intensities remained unchanged by statin treatment. These results were obtained using atorvastatin concentrations probably vastly exceeding those reached in patient plasma in vivo. Atorvastatin therapy itself is therefore unlikely to affect Ferumoxtran-10 based macrophage detection by MRI, a prerequisite for the use of this contrast material to monitor lesion macrophage burden during lipid-lowering therapy.
Keywords: Nanoparticle; Macrophage; Uptake; Atorvastatin; Magnetic resonance imaging; Electron microscopy;
Targeted delivery of paclitaxel using folate-decorated poly(lactide)–vitamin E TPGS nanoparticles by Jie Pan; Si-Shen Feng (2663-2672).
We synthesized nanoparticles (NPs) of the blend of two-component copolymers for targeted chemotherapy with paclitaxel used as model drug. One component is poly(lactide)–d-α-tocopheryl polyethylene glycol succinate (PLA–TPGS), which is of desired hydrophobic–lipophilic balance, and another is TPGS–COOH, which facilitates the folate conjugation for targeting. The nanoparticles of the two-copolymer blend at various component ratio were prepared by the solvent extraction/evaporation single emulsion method and then decorated by folate, which were characterized by laser light scattering (LLS) for particles' size and size distribution, zeta potential analyzer for surface charge, and X-ray photoelectron spectroscopy (XPS) for surface chemistry. The drug encapsulation efficiency (EE) and in vitro drug release were measured by high performance liquid chromatography (HPLC). The targeting effect was investigated in vitro by cancer cell uptake of coumarin-6-loaded NPs and further confirmed by cytotoxicity of cancer cells treated with the drug formulated in the NPs. We showed that the NP formulation has great advantages vs the pristine drug in achieving better therapeutic effect, which increased 8.68% for MCF-7 breast cancer cells, and that the folate-decoration can significantly promote targeted delivery of the drug into the corresponding cancer cells and thus enhance its therapeutic effect, which increased 24.4% for the NP formulation of 16.7% TPGS–COOH component and 31.1% for the NP formulation of 33.3% TPGS–COOH component after 24 h treatment at the same 25 μg/ml paclitaxel concentration. The experiments on C6 glioma cells further confirmed these advantages.
Keywords: Cancer nanotechnology; Biodegradable polymers; Breast cancer; Nanobiotechnology; Nanomedicine;
Using thermal energy produced by irradiation of Mn–Zn ferrite magnetic nanoparticles (MZF-NPs) for heat-inducible gene expression by Qiu-sha Tang; Dong-sheng Zhang; Xiao-ming Cong; Mei-ling Wan; Li-qiang Jin (2673-2679).
One of the main advantages of gene therapy over traditional therapy is the potential to target the expression of therapeutic genes in desired cells or tissues. To achieve targeted gene expression, we developed a novel heat-inducible gene expression system in which thermal energy generated by Mn–Zn ferrite magnetic nanoparticles (MZF-NPs) under an alternating magnetic field (AMF) was used to activate gene expression. MZF-NPs, obtained by co-precipitation method, were firstly surface modified with cation poly(ethylenimine) (PEI). Then thermodynamic test of various doses of MZF-NPs was preformed in vivo and in vitro. PEI–MZF-NPs showed good DNA binding ability and high transfection efficiency. In AMF, they could rise to a steady temperature. To analyze the heat-induced gene expression under an AMF, we combined P1730OR vector transfection with hyperthermia produced by irradiation of MZF-NPs. By using LacZ gene as a reporter gene and Hsp70 as a promoter, it was demonstrated that expression of a heterogonous gene could be elevated to 10 to 500-fold over background by moderate hyperthermia (added 12.24 or 25.81 mg MZF-NPs to growth medium) in tissue cultured cells. When injected with 2.6 or 4.6 mg MZF-NPs, the temperature of tumor-bearing nude mice could rise to 39.5 or 42.8 °C, respectively, and the β-gal concentration could increase up to 3.8 or 8.1 mU/mg proteins accordingly 1 day after hyperthermia treatment. Our results therefore supported hyperthermia produced by irradiation of MZF-NPs under an AMF as a feasible approach for targeted heat-induced gene expression. This novel system made use of the relative low Curie point of MZF-NPs to control the in vivo hyperthermia temperature and therefore acquired safe and effective heat-inducible transgene expression.
Keywords: Magnetic nanoparticles (MNPs); Mn–Zn ferrite; Gene expression; Hyperthermia; Induced;
Overexpression of Bcl-2 as a proxy redox stimulus to enhance activity of non-viral redox-responsive delivery vectors by Devika S. Manickam; Aiko Hirata; David A. Putt; Lawrence H. Lash; Fusao Hirata; David Oupický (2680-2688).
Redox-sensitive non-viral delivery systems exploit intracellular reducing environment to improve the efficacy of the delivery of nucleic acids by selectively releasing the cargo in the subcellular space. Bcl-2 overexpression is frequently observed in human cancers and is closely associated with increased resistance to chemotherapy and radiotherapy. One of the biochemical alterations accompanying Bcl-2 overexpression is the increase in cellular glutathione (GSH) levels. In this study, we hypothesize that such increase of GSH concentration will selectively enhance the transfection activity of redox-sensitive delivery systems in cells overexpressing Bcl-2. Transfection studies were conducted in MCF-7 mammary carcinoma cells and MCF-7 clones overexpressing Bcl-2. It was confirmed that Bcl-2 overexpression resulted in the expected increase in GSH concentration. Redox-sensitive complexes containing plasmid DNA, mRNA, antisense oligodeoxynucleotides, and siRNA exhibited selectively increased activity in cells overexpressing Bcl-2 compared to non-redox complexes. The effect of Bcl-2 overexpression on the selective enhancement of transfection was highly dependent on the type of the delivered nucleic acid, and was most pronounced for mRNA. This study shows that Bcl-2 overexpression can serve as a proxy redox stimulus to enhance the activity of all major classes of potential nucleic acid therapeutics, when delivered using redox-sensitive vectors.
Keywords: Non-viral gene delivery; Polyplexes; Transfection; Bcl-2 overexpression; Glutathione;
Reversible electronic nanoswitch based on DNA G-quadruplex conformation: A platform for single-step, reagentless potassium detection by Zai-Sheng Wu; Chen-Rui Chen; Guo-Li Shen; Ru-Qin Yu (2689-2696).
A novel on/off electronic nanoswitch is for the first time described based on the conformational change of DNA sequence possessing a single guanine (G)-rich stretch. Here, a thiolated, amine-containing G-rich DNA sequence is immobilized on the surface of gold electrode by means of facile sulfur–gold chemistry, followed by being labeled with redox-active ferrocene molecules serving as the signaling species. The surface-confined DNA sequence is able to change its configuration between rigid tetramolecular G-quadruplex and flexible single-stranded structures. The large conformational change enables the probes to perform an inchworm like extending–shrinking motion, which is reflected by the fluctuation in current intensity that depends on the electron-transfer distance between the electrode surface and the redox labels. Since potassium ion can specifically bind to G-quadruplex, using this reagentless reusable electrochemical sensing platform, the simple, rapid and selective detection of potassium ion can be accomplished without the use of exogenous reagents. Success in the present electronic nanoswitch is expected to promote the exploitation of functional DNA-based nanosystems.
Keywords: G-quadruplex structure; Conformational change; Electronic nanoswitch;
On the critical parameters that regulate the deformation behaviour of tooth enamel by Zonghan Xie; Michael Swain; Paul Munroe; Mark Hoffman (2697-2703).
Tooth enamel is the hardest tissue in the human body with a complex hierarchical structure. Enamel hypomineralisation – a developmental defect – has been reported to cause a marked reduction in the mechanical properties of enamel and loss of dental function. We discover a distinctive difference in the inelastic deformation mechanism between sound and hypomineralised enamels that is apparently controlled by microstructural variation. For sound enamel, when subjected to mechanical forces the controlling deformation mechanism was distributed shearing within nanometre thick protein layer between its constituent mineral crystals; whereas for hypomineralised enamel microcracking and subsequent crack growth were more evident in its less densely packed microstructure. We develop a mechanical model that not only identifies the critical parameters, i.e., the thickness and shear properties of enamels, that regulate the mechanical behaviour of enamel, but also explains the degradation of hypomineralised enamel as manifested by its lower resistance to deformation and propensity for catastrophic failure. With support of experimental data, we conclude that for sound enamel an optimal microstructure has been developed that endows enamel with remarkable structural integrity for durable mechanical function.
Keywords: Tooth enamel; Microstructure; Nanoindentation; Protein; Modelling; Electron microscopy;