Posts Tagged ‘Materials science’

3D DNA Images  Nanoscale design of printed vascular tissue

Curator: Larry H. Bernstein, MD, FCAP


New Detailed 3D DNA Images Could Aid Nanoscale Designs

This video shows techniques that scientists used to produce 3-D reconstructions of shape fluctuations in double-helix DNA segments attached to gold nanoparticles [Lei Zhang, Dongsheng Lei, Jessica M. Smith, Meng Zhang, Huimin Tong, Xing Zhang, Zhuoyang Lu, Jiankang Liu, A. Paul Alivisatos, and Gang “Gary” Ren]


Flexible double-helix DNA segments connected to gold nanoparticles are revealed from the 3-D density maps (purple and yellow) reconstructed from individual samples using a Berkeley Lab-developed technique called individual-particle electron tomography or IPET. Projections of the structures are shown in the background grid.[Berkeley Lab]


The general chemical structure of the DNA helix was described by James Watson and Francis Crick in 1953. Over the years that followed, scientists intensely studied the molecular structure of DNA to understand its behaviorin vivo and to exploit its unique properties for nanotechnology purposes.

Now, an international team of scientists working at the Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab) has captured the first high-resolution 3D images from individual double-helix DNA segments attached at either end to gold nanoparticles. The images detail the flexible structure of the DNA segments, which appear as nanoscale jump ropes.

Using a cutting-edge electron microscopy (EM) technique, called individual-particle electron tomography (IPET), the researchers were able to visualize the shapes of the coiled DNA strands, which were sandwiched between polygon-shaped gold nanoparticles, and reconstruct high-resolution 3D images. The EM technique was coupled with a protein-staining process and sophisticated software that provided structural details to the scale of approximately 2 nanometers (two billionths of a meter).

“We had no idea about what the double-strand DNA would look like between the nanogold particles,” noted senior study author Gang Ren, Ph.D., staff scientist in the Molecular Foundry at Berkeley Lab. “This is the first time for directly visualizing an individual double-strand DNA segment in 3-D.”

The findings from this study were published recently in Nature Communications in an article entitled “Three-Dimensional Structural Dynamics and Fluctuations of DNA-Nanogold Conjugates by Individual-Particle Electron Tomography.”

Dr. Ren and his colleagues hope their unique imaging technique will aid in the use of DNA segments as building blocks for molecular devices that function as nanoscale drug-delivery systems, markers for biological research, and components for computer memory and electronic devices. Additionally, the research team speculates that the new method could also lead to images of important disease-relevant proteins that have proven elusive for other imaging techniques and of the assembly process that forms DNA from separate, individual strands.

The Berkeley Lab scientists flash froze samples to preserve their structure for study with cryo-EM imaging. The distance between the two gold particles in individual samples varied from 20 to 30 nanometers based on different shapes observed in the DNA segments. They then collected a series of tilted images of the stained objects and reconstructed 14 electron-density maps that detailed the structure of individual samples using the IPET technique. They gathered a dozen confirmations for the samples and found the DNA shape variations were consistent with those measured in the flash-frozen cryo-EM samples.

While the 3D reconstructions show the basic nanoscale structure of the samples, the investigators are looking at the next steps, which will be to work on improving the resolution to the subnanometer scale.

“Even in this current state we begin to see 3D structures at 1- to 2-nanometer resolution,” Dr. Ren explained. “Through better instrumentation and improved computational algorithms, it would be promising to push the resolution to that visualizing a single DNA helix within an individual protein.”

In future studies, Dr. Ren noted that researchers could attempt to improve the imaging resolution for complex structures that incorporate more DNA segments as a sort of “DNA origami”—with the hope of building and better characterizing nanoscale molecular devices using DNA segments that can, for example, store and deliver drugs to targeted areas of the body.

“DNA is easy to program, synthesize, and replicate, so it can be used as a special material to quickly self-assemble into nanostructures and to guide the operation of molecular-scale devices,” Dr. Ren stated. “Our current study is just a proof of concept for imaging these kinds of molecular devices’ structures.”


Three-dimensional structural dynamics and fluctuations of DNA-nanogold conjugates by individual-particle electron tomography

Lei ZhangDongsheng LeiJessica M. Smith, …., Jiankang LiuA. Paul Alivisatos & Gang Ren
Nature Communications7,Article number:11083

DNA base pairing has been used for many years to direct the arrangement of inorganic nanocrystals into small groupings and arrays with tailored optical and electrical properties. The control of DNA-mediated assembly depends crucially on a better understanding of three-dimensional structure of DNA-nanocrystal-hybridized building blocks. Existing techniques do not allow for structural determination of these flexible and heterogeneous samples. Here we report cryo-electron microscopy and negative-staining electron tomography approaches to image, and three-dimensionally reconstruct a single DNA-nanogold conjugate, an 84-bp double-stranded DNA with two 5-nm nanogold particles for potential substrates in plasmon-coupling experiments. By individual-particle electron tomography reconstruction, we obtain 14 density maps at ~2-nm resolution. Using these maps as constraints, we derive 14 conformations of dsDNA by molecular dynamics simulations. The conformational variation is consistent with that from liquid solution, suggesting that individual-particle electron tomography could be an expected approach to study DNA-assembling and flexible protein structure and dynamics.

Organic–inorganic-hybridized nanocrystals are a valuable class of new materials that are suitable for addressing many emerging challenges in biological and material sciences1, 2. Nanogold and quantum dot conjugates have been used extensively as biomolecular markers3, 4, whereas DNA base pairing has directed the self-assembly of discrete groupings and arrays of organic and inorganic nanocrystals in the formation of a network solid for electronic devices and memory components5. Discretely hybridized gold nanoparticles conjugated to DNA were developed as a molecular ruler to detect sub-nanometre distance changes via plasmon-coupling-mediated variations in dark-field light scattering3, 6. For many of these applications, it is desirable to obtain nanocrystals functionalized with discrete numbers of DNA strands7, 8. In all of these circumstances, the soft components can fluctuate, and the range of these structural deviations have not previously been determined with a degree of rigour that could help influence the future design and use of these assemblies.

Conformational flexibility and dynamics of the DNA-nanogold conjugates limit the structural determination by X-ray crystallography, nuclear magnetic resonance spectroscopy and single-particle cryo-electron microscopic (cryo-EM) reconstruction because they do not crystallize, are not sufficiently small for nuclear magnetic resonance studies and cannot be classified into a limited number of classes for single-particle EM reconstruction. In addition, three-dimensional (3D) structure averaged from tens of thousands of different macromolecular particles obtained without prior knowledge of the macromolecular structural flexibility could result in an absence of flexible domains upon using the single-particle reconstruction method, for example, two ankyrin repeated regions of TRPV1 were absent in its atomic resolution 3D density map9.

A fundamental experimental solution to reveal the structure of a flexible macromolecule should be based on the determination of each individual macromolecule’s structure10. Electron tomography (ET) provides high-resolution images of a single object from a series of tilted viewing angles11. ET has been applied to reveal the 3D structure of a cell section and an individual bacterium at nanometre-scale resolution12. However, reconstruction from an individual macromolecule at an intermediate resolution (1–3nm) remains challenging due to small molecular weight and low image contrast. Although, the first 3D map of an individual macromolecule, a fatty acid synthetase molecule, was reconstructed from negative-staining (NS) ET by Hoppe et al.13, serious doubts have been raised regarding the validity of this structure14, as this molecule received a radiation dose hundreds of times greater than the reported damage threshold15. Recently, we investigated the possibility based on simulated and real experimental NS and cryo-ET images10. We showed that a single-protein 3D structure at an intermediate resolution (1–3nm) is potentially achieved using our proposed individual-particle ET (IPET) method10, 16, 17, 18. IPET, an iterative refinement process using automatically generated dynamic filters and soft masks, requires no pre-given initial model, class averaging or lattice, but can tolerate small tilt errors and large-scale image distortion via decreasing the reconstruction image size to reduce the negative effects on 3D reconstruction. IPET allows us to obtain a ‘snapshot’ single-molecule 3D structure of flexible proteins at an intermediate resolution, and can be even used to reveal the macromolecular dynamics and fluctuation17.

Here we use IPET, cryo-EM and our previously reported optimized NS (OpNS)19, 20 techniques to investigate the morphology and 3D structure of hybridized DNA-nanogold conjugates. These conjugates were self-assembled from a mixture of two monoconjugates, each consisting of 84-bp single-stranded DNA and a 5-nm nanogold particle. The dimers were separated by anion-exchange high-performance liquid chromatography (HPLC) and agarose gel electrophoresis as potential substrates in plasmon-coupling experiments. By OpNS-ET imaging and IPET 3D reconstruction, we reconstruct a total of 14 density maps at a resolution of ~2nm from 14 individual double-stranded DNA (dsDNA)-nanogold conjugates. Using these maps as constraints, we derive 14 conformations of dsDNA by projecting a standard flexible dsDNA model onto the observed maps using molecular dynamics (MD) simulations. The variation of the conformations was largely consistent with that from liquid solution, and suggests that the IPET approach provides a most complete experimental determination of flexibility and fluctuation range of these directed nanocrystal assemblies to date. The general features revealed by this experiment can be expected to occur in a broad range of DNA-assembled nanostructures and flexible proteins.


Although the direct imaging of dsDNA has been previously reported using heavy metal shadowing32, 33 and NS methods34, 35, 36, to the best of our knowledge, the 3D structure of an individual dsDNA strand has not previously been achieved. It has been thought that individual dsDNA would be destroyed under the high energy of the electron beam before a 3D reconstruction, or even a 2D image, is able to be achieved. Our NS tilt images showing fibre-shaped dsDNA bridging two conjugated nanogold particles demonstrated that the dsDNA can in fact be directly visualized using EM, which is consistent with the recently reported single-molecule DNA sequencing technique via TEM36. The resolutions of our density maps ranged from ~14 to ~23Å, demonstrating that an intermediate-resolution 3D structure can be obtained for each individual macromolecule. This capability is consistent with our earlier report of a ~20-Å resolution 3D reconstruction of an individual IgG1 antibody using the same approach16, 17.

Notably, a total dose of ~2,000eÅ−2 used in our ET data acquisition is significantly above the limitation conventionally used in cryo-EM (~80–100eÅ−2), which can be suspected to have certain artefact from radiation damage. In cryo-EM, the radiation damage could cause sample bubbling, deformation and knockout effects; in NS, only the knockout phenomena is often observed, in which the protein is surrounded by heavy atoms that were kicked out by electron beam. Since the sample was coated with heavy metal atoms and were dried in air, the bubbling and deformation phenomena were not usually observed. The heavy metal atoms that coat the surface of the biomolecule can provide a much higher electron scattering than from a biomolecule only inside lighter atoms. The scattering is sufficiently high to provide enough image contrast at our 120-kV high tension; thus, a further increase to the scattering ability by reducing the high tension to 80kV may not be necessary for this NS sample. In addition, the heavy atoms can provide more radiation resistance and allow the sample to be imaged under a higher dose condition. The exact dose limitation for NS is still unknown. The radiation damage related artefact in NS samples is knockout, which could reduce the image contrast and lower the tilt image alignment accuracy and 3D reconstruction resolution. In our study, a total dose of 2,000eÅ−2 did not cause any obvious knockout phenomena, but provides a sufficiently high contrast for the otherwise barely visible DNA conformations in each tilt series. The direct confirmation of visible DNA in each tilt image is essentially important to us to validate each 3D reconstruction, especially considering this relatively new approach.

Our 3D reconstruction algorithm used an ab initio real-space reference-projection match iterative algorithm to correct the centres of each tilt images, in which the equal tilt angle step for 3D reconstruction of a low contrast and asymmetric macromolecule was used. This method is different from recently reported Fourier-based iterative algorithm, termed equally sloped tomography, in which the pseudo-polar fast Fourier transform, the oversampling method and internal lattice of a targeted nanoparticle are used to achieve 3D reconstruction at atomic resolution37.

It is generally challenging to achieve visualization and 3D reconstruction on an individual, small and asymmetric macromolecule by other conventional methods; our method demonstrated its capability for 3D reconstruction of 52kDa 84-bp dsDNA through these studies: IgG1 antibody 3D structural fluctuation17, peptide-induced conformational changes on flexible IgG1 antibody5, floppy liposome surface binding with 53-kDa proteins38, all of which suggest that this method could be used to serve the community as a novel tool for studying flexible macromolecular structures, dynamics and fluctuations of proteins, and for catching the intermediate 3D structure of protein assembling.

DNA-based self-assembling materials have been developed for use in materials science and biomedical research, such as DNA origami designed for targeted drug delivery. The structure, design and control require feedback from the 3D structure, which could validate the design hypothesis, optimize the synthesis protocol and improve the reproducible capability, while even providing insight into the mechanism of DNA-mediated assembly.


Printing Vascular Tissue

from Wyss Institute for Biologically Inspired Engineering at Harvard University


Printing vessel vasculature is essential for sustaining functional living tissues. Until now, bioengineers have had difficulty building thick tissues, lacking a method to embed vascular networks. A 3D bioprinting method invented at the Wyss Institute and Harvard SEAS embeds a grid of vasculature into thick tissue laden with human stem cells and connective matrix. Printed within a custom-made housing, this method can be used to create tissue of any shape. Once printed, an inlet and outlet own opposite ends are perfused with fluids, nutrients, and cell growth factors, which control stem cell differentiation and sustain cell functions. By flowing growth factors through the vasculature, stem cells can be differentiated into a variety of tissue cell types. This vascularized 3D printing process could open new doors to tissue replacement and engineering. Footage credit: David KA.S. Gladman, E. Matsumoto, L.K. Sanders, and J.A. Lewis / Wyss Institute at Harvard University For more information, please visit:

Scaling up tissue engineering


In this video, the Wyss Institute and Harvard SEAS team uses a customizable 3D bioprinting method to build a thick vascularized tissue structure comprising human stem cells, collective matrix, and blood vessel endothelial cells. Their work sets the stage for advancement of tissue replacement and tissue engineering techniques. Credit: Lewis Lab, Wyss Institute at Harvard University


Bioprinting technique creates thick 3D tissues composed of human stem cells and embedded vasculature, with potential applications in drug testing and regenerative medicine

(CAMBRIDGE, Massachusetts)  — A team at the Wyss Institute for Biologically Inspired Engineering at Harvard University and the Harvard John A. Paulson School for Engineering and Applied Sciences (SEAS) has invented a method for 3D bioprinting thick vascularized tissue constructs composed of human stem cells, extracellular matrix, and circulatory channels lined with endothelial blood vessel cells. The resulting network of vasculature contained within these deep tissues enables fluids, nutrients and cell growth factors to be controllably perfused uniformly throughout the tissue. The advance is reported March 7 in the journal Proceedings of the National Academy of Sciences.

“This latest work extends the capabilities of our multi-material bioprinting platform to thick human tissues, bringing us one step closer to creating architectures for tissue repair and regeneration,” says Wyss Core Faculty member Jennifer A. Lewis, Sc.D., senior author on the study, who is also the Hansörg Wyss Professor of Biologically Inspired Engineering at SEAS.

Three-dimensional bioprinting of thick vascularized tissues

David B. Koleskya,1Kimberly A. Homana,1Mark A. Skylar-Scotta,1, and Jennifer A. Lewisa,2     aSchool of Engineering and Applied Sciences, Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, MA 02138
PNAS  2016; 113(12): 3179–3184   http://dx.doi.org:/10.1073/pnas.1521342113


Current tissue manufacturing methods fail to recapitulate the geometry, complexity, and longevity of human tissues. We report a multimaterial 3D bioprinting method that enables the creation of thick human tissues (>1 cm) replete with an engineered extracellular matrix, embedded vasculature, and multiple cell types. These 3D vascularized tissues can be actively perfused with growth factors for long durations (>6 wk) to promote differentiation of human mesenchymal stem cells toward an osteogenic lineage in situ. The ability to construct and perfuse 3D tissues that integrate parenchyma, stroma, and endothelium is a foundational step toward creating human tissues for ex vivo and in vivo applications.

The advancement of tissue and, ultimately, organ engineering requires the ability to pattern human tissues composed of cells, extracellular matrix, and vasculature with controlled microenvironments that can be sustained over prolonged time periods. To date, bioprinting methods have yielded thin tissues that only survive for short durations. To improve their physiological relevance, we report a method for bioprinting 3D cell-laden, vascularized tissues that exceed 1 cm in thickness and can be perfused on chip for long time periods (>6 wk). Specifically, we integrate parenchyma, stroma, and endothelium into a single thick tissue by coprinting multiple inks composed of human mesenchymal stem cells (hMSCs) and human neonatal dermal fibroblasts (hNDFs) within a customized extracellular matrix alongside embedded vasculature, which is subsequently lined with human umbilical vein endothelial cells (HUVECs). These thick vascularized tissues are actively perfused with growth factors to differentiate hMSCs toward an osteogenic lineage in situ. This longitudinal study of emergent biological phenomena in complex microenvironments represents a foundational step in human tissue generation.

The ability to manufacture human tissues that replicate the essential spatial (1), mechanochemical (2, 3), and temporal aspects of biological tissues (4) would enable myriad applications, including 3D cell culture (5), drug screening (6, 7), disease modeling (8), and tissue repair and regeneration (9, 10). Three-dimensional bioprinting is an emerging approach for creating complex tissue architectures (10, 11), including those with embedded vasculature (1215), that may address the unmet needs of tissue manufacturing. Recently, Miller et al. (15) reported an elegant method for creating vascularized tissues, in which a sacrificial carbohydrate glass is printed at elevated temperature (>100 °C), protectively coated, and then removed, before introducing a homogeneous cell-laden matrix. Kolesky et al. (14) developed an alternate approach, in which multiple cell-laden, fugitive (vasculature), and extracellular matrix (ECM) inks are coprinted under ambient conditions. However, in both cases, the inability to directly perfuse these vascularized tissues limited their thickness (1–2 mm) and culture times (<14 d). Here, we report a route for creating thick vascularized tissues (≥1 cm) within 3D perfusion chips that provides unprecedented control over tissue composition, architecture, and microenvironment over several weeks (>6 wk). This longitudinal study of emergent biological phenomena in complex microenvironments represents a foundational step in human tissue generation.

Central to the fabrication of thick vascularized tissues is the design of biological, fugitive, and elastomeric inks for multimaterial 3D bioprinting. To satisfy the concomitant requirements of processability, heterogeneous integration, biocompatibility, and long-term stability, we first developed printable cell-laden inks and castable ECM based on a gelatin and fibrinogen blend (16). Specifically, these materials form a gelatin–fibrin matrix cross-linked by a dual-enzymatic, thrombin and transglutaminase (TG), strategy (Fig. 1and SI Appendix, Fig. S1). The cell-laden inks must facilitate printing of self-supporting filamentary features under ambient conditions as well as subsequent infilling of the printed tissue architectures by casting without dissolving or distorting the patterned construct (Fig. 1A). The thermally reversible gelation of the gelatin–fibrinogen network enables its use in both printing and casting, where gel and fluid states are required, respectively (SI Appendix, Fig. S2). Thrombin is used to rapidly polymerize fibrinogen (17), whereas TG is a slow-acting Ca2+-dependent enzymatic cross-linker that imparts the mechanical and thermal stability (18) needed for long-term perfusion. Notably, the cell-laden ink does not contain either enzyme to prevent polymerization during printing. However, the castable matrix contains both thrombin and TG, which diffuse into adjacent printed filaments, forming a continuous, interpenetrating polymer network, in which the native fibrillar structure of fibrin is preserved (SI Appendix, Fig. S3). Importantly, our approach allows arbitrarily thick tissues to be fabricated, because the matrix does not require UV curing (19), which has a low penetration depth in tissue (20) and can be readily expanded to other biomaterials, including fibrin and hyaluronic acid (SI Appendix, Fig. S4).


Fig. 1.

Fig. 1.

Three-dimensional vascularized tissue fabrication. (A) Schematic illustration of the tissue manufacturing process. (i) Fugitive (vascular) ink, which contains pluronic and thrombin, and cell-laden inks, which contain gelatin, fibrinogen, and cells, are printed within a 3D perfusion chip. (ii) ECM material, which contains gelatin, fibrinogen, cells, thrombin, and TG, is then cast over the printed inks. After casting, thrombin induces fibrinogen cleavage and rapid polymerization into fibrin in both the cast matrix, and through diffusion, in the printed cell ink. Similarly, TG diffuses from the molten casting matrix and slowly cross-links the gelatin and fibrin. (iii) Upon cooling, the fugitive ink liquefies and is evacuated, leaving behind a pervasive vascular network, which is (iv) endothelialized and perfused via an external pump. (B) HUVECs growing on top of the matrix in 2D, (C) HNDFs growing inside the matrix in 3D, and (D) hMSCs growing on top of the matrix in 2D. (Scale bar: 50 µm.) (E and F) Images of printed hMSC-laden ink prepared using gelatin preprocessed at 95 °C before ink formation (E) as printed and (F) after 3 d in the 3D printed filament where actin (green) and nuclei (blue) are stained. (G) Gelatin preprocessing temperature affects the plateau modulus and cell viability after printing. Higher temperatures lead to lower modulus and higher HNDF viability postprinting. (H) Photographs of interpenetrated sacrificial (red) and cell inks (green) as printed on chip. (Scale bar: 2 mm.) (I) Top-down bright-field image of sacrificial and cell inks. (Scale bar: 50 µm.). (J–L) Photograph of a printed tissue construct housed within a perfusion chamber (J) and corresponding cross-sections (K and L). (Scale bars: 5 mm.)

To construct thick, vascularized tissues within 3D perfusion chips, we coprinted cell-laden, fugitive, and silicone inks (Fig. 1 H and I). First, the silicone ink is printed on a glass substrate and cured to create customized perfusion chips (Movie S1 and SI Appendix, Fig. S1). Next, the cell-laden and fugitive inks are printed on chip, and then encapsulated with the castable ECM (Fig. 1 J–L and Movie S2). The fugitive ink, which defines the embedded vascular network, is composed of a triblock copolymer [i.e., polyethylene oxide (PEO)–polypropylene oxide (PPO)–PEO]. This ink can be removed from the fabricated tissue upon cooling to roughly 4 °C, where it undergoes a gel-to-fluid transition (14, 23). This process yields a pervasive network of interconnected channels, which are then lined with HUVECs. The resulting vascularized tissues are perfused via their embedded vasculature on chip over long time periods using an external pump (Movie S3) that generates smooth flow over a wide range of flow rates (24).


Movie S3.

Fluorescent microscopy video of different perfusion rates through the embedded vasculature within the printed 3D tissue microenvironments.


Fig. 2.

Three-dimensional vascularized tissues remain stable during long-term perfusion. (A) Schematic depicting a single HUVEC-lined vascular channel supporting a fibroblast cell-laden matrix and housed within a 3D perfusion chip. (B and C) Confocal microscopy image of the vascular network after 42 d, CD-31 (red), vWF (blue), and VE-Cadherin (magenta). (Scale bars: 100 µm.) (D) Long-term perfusion of HUVEC-lined (red) vascular network supporting HNDF-laden (green) matrix shown by top-down (Left) and cross-sectional confocal microscopy at 45 d (Right). (Scale bar: 100 µm.) (E) Quantification of barrier properties imparted by endothelial lining of channels, demonstrated by reduced diffusional permeability of FITC-dextran. (F) GFP-HNDF distribution within the 3D matrix shown by fluorescent intensity as a function of distance from vasculature.


Movie S4.

Confocal microscopy video of cross-section through vascularized tissue after 45 d of perfusion.

To explore emergent phenomena in complex microenvironments, we created a heterogeneous tissue architecture (>1 cm thick and 10 cm3 in volume) by printing a hMSC-laden ink into a 3D lattice geometry along with intervening in- and out-of-plane (vertical) features composed of fugitive ink, which ultimately transform into a branched vascular network lined with HUVECs. After printing, the remaining interstitial space is infilled with an HNDF-laden ECM (Fig. 3A) to form a connective tissue that both supports and binds to the printed stem cell-laden and vascular features. In this example, fibroblasts serve as model cells that surround the heterogeneously patterned stem cells and vascular network. These model cells could be replaced with either support cells (e.g., immune cells or pericytes) or tissue-specific cells (e.g., hepatocytes, neurons, or islets) in future embodiments. The embedded vascular network is designed with a single inlet and outlet that provides an interface between the printed tissue and the perfusion chip. This network is symmetrically branched to ensure uniform perfusion throughout the tissue, including deep within its core. In addition to providing transport of nutrients, oxygen, and waste materials, the perfused vasculature is used to deliver specific differentiation factors to the tissue in a more uniform manner than bulk delivery methods, in which cells at the core of the tissue are starved of factors (25). This versatile platform (Fig. 3A) is used to precisely control growth and differentiation of the printed hMSCs. Moreover, both the printed cellular architecture and embedded vascular network are visible macroscopically with this thick tissue (Fig. 3B).

Fig. 3.

Fig. 3.

Osteogenic differentiation of thick vascularized tissue. (A) Schematic depicting the geometry of the printed heterogeneous tissue within the customized perfusion chip, wherein the branched vascular architecture pervades hMSCs that are printed into a 3D lattice architecture, and HNDFs are cast within an ECM that fills the interstitial space. (B) Photographs of a printed tissue construct within and removed from the customized perfusion chip. (C) Comparative cross-sections of avascular tissue (Left) and vascularized tissue (Right) after 30 d of osteogenic media perfusion with alizarin red stain showing location of calcium phosphate. (Scale bar: 5 mm.) (D) Confocal microscopy image through a cross-section of 1-cm-thick vascularized osteogenic tissue construct after 30 d of active perfusion and in situ differentiation. (Scale bar: 1.5 mm.) (E) Osteocalcin intensity across the thick tissue sample inside the red lines shown in C. (F) High-resolution image showing osteocalcin (purple) localized within hMSCs, and they appear to take on symmetric osteoblast-like morphologies. (Scale bar: 100 µm.) After 30 d (Gand H), thick tissue constructs are stained for collagen-I (yellow), which appears to be localized near hMSCs. (Scale bars: 200 µm.) (I) Alizarin red is used to stain calcium phosphate deposition, and fast blue is used to stain AP, indicating tissue maturation and differentiation over time. (Scale bar: 200 µm.)

In summary, thick, vascularized human tissues with programmable cellular heterogeneity that are capable of long-term (>6-wk) perfusion on chip have been fabricated by multimaterial 3D bioprinting. The ability to recapitulate physiologically relevant, 3D tissue microenvironments enables the exploration of emergent biological phenomena, as demonstrated by observations of in situ development of hMSCs within tissues containing a pervasive, perfusable, endothelialized vascular network. Our 3D tissue manufacturing platform opens new avenues for fabricating and investigating human tissues for both ex vivo and in vivo applications.


Steve Dufourny Hello Mr Bernstein, it is relevant.I have a model with quantum sphères and spherical volumes correlated with my theory of spherisation in theoretical physics(in a very simple resume quant sphères…..spherisation encodings…….Cosmol sphères…….Universal sphères with a central BH)more two équations about matter and energy and sphères.The quantizations and properties can be computed.The adn, amino acids ,protiens INE ASE are the keys.I search the main gravitationalcodes.It is more far than our standard model in logic.Regards


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Nanotechnology and MRI imaging

Author: Tilda Barliya PhD

The recent advances of “molecular and medical imaging” as an integrated discipline in academic medical centers has set the stage for an evolutionary leap in diagnostic imaging and therapy. Molecular imaging is not a substitute for the traditional process of image formation and interpretation, but is intended to improve diagnostic accuracy and sensitivity.

Medical imaging technologies allow for the rapid diagnosis and evaluation of a wide range of pathologies. In order to increase their sensitivity and utility, many imaging technologies such as CT and MRI rely on intravenously administered contrast agents. While the current generation of contrast agents has enabled rapid diagnosis, they still suffer from many undesirable drawbacks including a lack of tissue specificity and systemic toxicity issues. Through advances made in nanotechnology and materials science, researchers are now creating a new generation of contrast agents that overcome many of these challenges, and are capable of providing more sensitive and specific information (1)

Magnetic resonance imaging (MRI) contrast enhancement for molecular imaging takes advantage of superb and tunable magnetic properties of engineered magnetic nanoparticles, while a range of surface chemistry offered by nanoparticles provides multifunctional capabilities for image-directed drug delivery. In parallel with the fast growing research in nanotechnology and nanomedicine, the continuous advance of MRI technology and the rapid expansion of MRI applications in the clinical environment further promote the research in this area.

It is well known that magnetic nanoparticles, distributed in a magnetic field, create extremely large microscopic field gradients. These microscopic field gradients cause substantial diphase and shortening of longitudinal relaxation time (T1) and transverse relaxation time (T2 and T2*) of nearby nuclei, e.g., proton in the case of most MRI applications. The magnitudes of MRI contrast enhancement over clinically approved conventional gadolinium chelate contrast agents combined with functionalities of biomarker specific targeting enable the early detection of diseases at the molecular and cellular levels with engineered magnetic nanoparticles. While the effort in developing new engineered magnetic nanoparticles and constructs with new chemistry, synthesis, and functionalization approaches continues to grow, the importance of specific material designs and proper selection of imaging methods have been increasingly recognized (2)

Earlier investigations have shown that the MRI contrast enhancement by magnetic nanoparticles is highly related to their composition, size, surface properties, and the degree of aggregation in the biological environment.

Therefore, understanding the relationships between these intrinsic parameters and relaxivities of nuclei under influence of magnetic nanoparticles can provide critical information for predicting the properties of engineered magnetic nanoparticles and enhancing their performance in the MRI based theranostic applications. On the other hand, new contrast mechanisms and imaging strategies can be applied based on the novel properties of engineered magnetic nanoparticles. The most common MRI sequences, such as the spin echo (SE) or fast spin echo (FSE) imaging and gradient echo (GRE), have been widely used for imaging of magnetic nanoparticles due to their common availabilities on commercial MRI scanners. In order to minimize the artificial effect of contrast agents and provide a promising tool to quantify the amount of imaging probe and drug delivery vehicles in specific sites, some special MRI methods, such as  have been developed recently to take maximum advantage of engineered magnetic NPs

  • off-resonance saturation (ORS) imaging
  • ultrashort echo time (UTE) imaging

Because one of the major limitations of MRI is its relative low sensitivity, the strategies of combining MRI with other highly sensitive, but less anatomically informative imaging modalities such as positron emission tomography (PET) and NIRF imaging, are extensively investigated. The complementary strengths from different imaging methods can be realized by using engineered magnetic nanoparticles via surface modifications and functionalizations. In order to combine optical or nuclear with MR for multimodal imaging, optical dyes and radio-isotope labeled tracer molecules are conjugated onto the moiety of magnetic nanoparticles

Since most functionalities assembled by magnetic nanoparticles are accomplished by the surface modifications, the chemical and physical properties of nanoparticle surface as well as surface coating materials have considerable effects on the function and ability of MRI contrast enhancement of the nanoparticle core.

The longitudinal and transverse relaxivities, Ri (i=1, 2), defined as the relaxation rate per unit concentration (e.g., millimole per liter) of magnetic ions, reflects the efficiency of contrast enhancement by the magnetic nanoparticles as MRI contrast agents. In general, the relaxivities are determined, but not limited, by three key aspects of the magnetic nanoparticles:

  1. Chemical composition,
  2. Size of the particle or construct and the degree of their aggregation
  3. Surface properties that can be manipulated by the modification and functionalization.

(It is also recognized that the shape of the nanoparticles can affect the relaxivities and contrast enhancement. However these shaped particles typically have increased sizes, which may limit their in vivo applications. Nevertheless, these novel magnetic nanomaterials are increasingly attractive and currently under investigation for their applications in MRI and image-directed drug delivery).

Composition Effect: The composition of magnetic nanoparticles can significantly affect the contrast enhancing capability of nanoparticles because it dominates the magnetic moment at the atomic level. For instance, the magnetic moments of the iron oxide nanoparticles, mostly used nanoparticulate T2 weighted MRI contrast agents, can be changed by incorporating other metal ions into the iron oxide.  The composition of magnetic nanoparticles can significantly affect the contrast enhancing capability of nanoparticles because it dominates the magnetic moment at the atomic level. For instance, the magnetic moments of the iron oxide nanoparticles, mostly used nanoparticulate T2 weighted MRI contrast agents, can be changed by incorporating other metal ions into the iron oxide.

Size Effect: The dependence of relaxation rates on the particle size has been widely studied both theoretically and experimentally. Generally the accelerated diphase, often described by the R2* in magnetically inhomogeneous environment induced by magnetic nanoparticles, is predicted into two different regimes. For the relatively small nanoparticles, proton diffusion between particles is much faster than the resonance frequency shift. This resulted in the relative independence of T2 on echo time. The values for R2 and R2*are predicted to be identical. This process is called “motional averaging regime” (MAR). It has been well demonstrated that the saturation magnetization Ms increases with the particle size. A linear relationship is predicted between Ms1/3 and d-1. Therefore, the capability of MRI signal enhancement by nanoparticles correlates directly with the particle size. 

Surface Effect: MRI contrast comes from the signal difference between water molecules residing in different environments that are under the effect of magnetic nanoparticles. Because the interactions between water and the magnetic nanoparticles occur primarily on the surface of the nanoparticles, surface properties of magnetic nanoparticles play important roles in their magnetic properties and the efficiency of MRI contrast enhancement. As most biocompatible magnetic nanoparticles developed for in vivo applications need to be stabilized and functionalized with coating materials, the coating moieties can affect the relaxation of water molecules in various forms, such as diffusion, hydration and hydrogen binding.

The early investigation carried at by Duan et al suggested that hydrophilic surface coating contributes greatly to the resulted MRI contrast effect. Their study examined the proton relaxivities of iron oxide nanocrystals coated by copolymers with different levels of hydrophilicity including: poly(maleic acid) and octadecene (PMO), poly(ethylene glycol) grated polyethylenimine (PEG-g-PEI), and hyperbranched polyethylenimine (PEI). It was found that proton relaxivities of those IONPs depend on the surface hydrophilicity and coating thickness in addition to the coordination chemistry of inner capping ligands and the particle size.

The thickness of surface coating materials also contributed to the relaxivity and contrast effect of the magnetic nanoparticles. Generally, the measured T2 relaxation time increases as molecular weight of PEG increases.

In Summary

Much progress has taken place in the theranostic applications of engineered magnetic nanoparticles, especially in MR imaging technologies and nanomaterials development. As the feasibilities of magnetic nanoparticles for molecular imaging and drug delivery have been demonstrated by a great number of studies in the past decade, MRI guiding and monitoring techniques are desired to improve the disease specific diagnosis and efficacy of therapeutics. Continuous effort and development are expected to be focused on further improvement of the sensitivity and quantifications of magnetic nanoparticles in vivo for theranostics in future.

The new design and preparation of magnetic nanoparticles need to carefully consider the parameters determining the relaxivities of the nanoconstructs. Sensitive and reliable MRI methods have to be established for the quantitative detection of magnetic nanoparticles. The new generations of magnetic nanoparticles will be made not only based on the new chemistry and biological applications, but also with combined knowledge of contrast mechanisms and MRI technologies and capabilities. As new magnetic nanoparticles are available for theranostic applications, it is anticipated that new contrast mechanism and MR imaging strategies can be developed based on the novel properties of engineered magnetic nanoparticles.











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