Posts Tagged ‘nanotechnology’

New ways to Heal Damage after a Heart Attack

Reporter: Irina Robu, PhD

More than a million Americans have heart attacks each year. Researchers at Northwestern University and University of California, San Diego have designed a minimally invasive platform to deliver nanomaterial that turns body’s inflammatory response into a signal rather than means of scarring following a heart attack. The researchers from Northwestern-UC San Diego established a novel way to deliver a bioactivated, biodegradable, regenerative substance through a noninvasive catheter without clogging in-vivo in a rat model.

When a person has a heart attack, the extracellular matrix is stripped away and scar tissue forms in its place, decreasing the heart’s functionality. The team injects a self-assembling peptide that seeks out a target, the heart’s damaged extracellular matrix and the solution is then activated by the inflammatory environment itself and gels.

The team’s preclinical research was led in rats and segmented into two proof-of-concept tests. The first test recognized that the material could be fed through a catheter without clogging and without interacting with human blood. The second determined whether the self-assembling peptides could find their way to the damaged tissue, bypassing healthy heart tissue. The scientists attached a fluorescent tag to the self-assembling peptides and imaged the heart to see where the peptides eventually settled.

Researchers now know that when they remove the fluorescent tag and replace it with a therapeutic, the self-assembling peptides will locate to the affected area of the heart. One hurdle is that catheter delivery in a rodent model is far more complicated than the same procedure in a human.

A major innovation occurred when sterically constrained cyclic peptides, which flow freely during delivery and rapidly assemble into hydrogels when they come in contact with disease associated enzymes. The process creates conditions for the peptides to better self-assemble on top one another and form the scaffold that resembles the native extracellular matrix.





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Carbon Nano-tube Design Halts Dust Harmful Protective Gear by Deterring Particles

Reporter: Irina Robu, PhD

A self-cleaning spacesuit was developed by engineers using carbon nanotube technology to purge itself from hyper-abrasive space dust. The sharp and sticky particles can cause noteworthy wear and tear on protective gear as well as causing them to overheat.
Kavya Manyapu, a flight crew operations and test engineer for Starliner Spacecraft at Boeing, has now created a way to magnetize flexible carbon nanotube fibers which make the fabric immune to the problematic dust particles. A magnetic field induces a process known as electrophoresis, which carries and moves charged particles away from an area to stop it building up in certain areas.
However, particles on our moon and other planets are sharper and abrasive because of the atmosphere which erodes bulging edges here on Earth. It is also often electrically charged due to the relentless and unfiltered UV rays from space which experts say make the dust particles ‘sticky’. Static electricity aids the dust cling to a spacesuit and then wears out the fabric – often in crevices and folds such as elbows and knees.

Carbon nano-tubes are already in use to stop dust settling on solar panels and other sensors in space but they are brittle and ill-suited for use in clothing. However, scientists have found a way to make technology flexible with the use of small magnetic field created a fabric that can repel the dust.
Boeing engineers created a fully functioning knee joint section to prove their concept was operative. The segment was fully pressurized, as it would be on future lunar and Martian missions. It can even be adapted to improved suit the circumstances and requirements of other planets.


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Unlocking the Secrets of 3D Printing

Reporter: Irina Robu, PhD

Researchers at Lawrence Livermore National Laboratory discovered interesting ways to advance the capabilities of two-photon lithography, a high-resolution 3D printing technique capable of producing nanoscale features which unleashes the potential for X-ray computed tomography to analyze stress or defects noninvasively in embedded in 3D printed medical devices or implants. Two-photon lithography stereotypically requires a thin glass slide, a lens and an immersion oil to help the laser light focus to a fine point where curing and printing occurs. The findings were published in the journal of ACS Applied Material and Interfaces.

In the paper, researchers describe cracking the code on resist materials improved for two-photon lithography and forming 3-D microstructures with features less than 150 nanometer which is better in comparison to previous techniques which build structures from ground up, limiting the height of the objects.

According to LLNL researcher James Oakdale, “In this paper, we have unlocked the secrets to making custom materials on two-photon lithography systems without losing resolution”, because the laser light refracts as it passes through the photoresist material, the cornerstone is discovering how to match the refractive index of the resist material to the immersion medium of the lens so the laser could pass through unimpeded.

Investigators can now use X-ray computed tomography as an analytical tool to copy the inside parts without cutting them open and to investigate 3D printed objects by fine-tuning the material’s x-ray absorption. The only limiting factor is the time it takes to build, so the researchers are investigating how to speed up the process.

These techniques could be used to harvest and probe the internal structure of targets for the National Ignition Facility, as well as optical and mechanical metamaterials and 3D-printed electrochemical batteries.



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Ferritin Cage Enzyme Encapsulation as a New Platform for Nanotechnology

 Reporter: Irina Robu, PhD

In bionanotechnology, biological systems such as viruses, protein complexes, lipid vesicles and artificial cells, are being developed for applications in medicine and materials science.  However, the paper published by Stephan Tetter and Donald Hilvert in Angewandte Chemie International Edition show that it is possible to encapsulate proteins such as ferritin by manipulating electrostatic interactions with the negatively charged interior of the cage.The primary role of ferritin is to protect cells from the damage caused by the Fenton reaction; where, in oxidizing conditions, free Fe(II) produces harmful reactive oxygen species that can damage the cellular machinery.

The ferritin family proteins are protein nanocages that evolved to safely store iron in an oxidizing world. Since ferritin family proteins are able to mineralize and store metal ions, they have been the focus of much research for the production of metal nanoparticles and as prototypes for semiconductor production. The ferritin cage itself is highly symmetrical, and is made up of 24 subunits arranged in an octahedral symmetry. Ferritins are smaller than other protein used for protein   encapsulation.   Their  outer  diameter is only 12 nm, whereas engineered lumazine synthase variants form cages with diameters ranging from about 20 to 60 nm.The ferritin cage displays remarkable thermal and chemical stability it is likely to modify the surface of the ferritin cage through the addition of peptide and protein tags. These characteristics have made ferritins attractive vectors for the delivery of drug molecules and as scaffolds for vaccine design.

In summary, the paper published in Angewandte Chemie International Edition is the first example of protein incorporation by a ferritin.  Dr. Donald Hilvert and colleagues have shown that AfFtn not only complexes positively charged guest proteins within its naturally negatively charged luminal cavity, but that the in vitro mixing technique can be extended to the encapsulation and protection of other functional  fusion proteins.

Hence, the recent discovery of encapsulated ferritins has identified an exciting new platform for use in bio nanotechnology. The use of synthetic biology tools will allow their rapid implementation in materials science, bio-nanotechnology and medical applications.


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Novel Blood Substitute – ErythroMer

Reporter: Irina Robu, PhD

For years, scientists have tried ineffectively to create an artificial molecule that emulates the oxygen-carrying function of human red blood cell but the efforts failed because of oxygen delivery and safety issues. Now, a group of researchers led by Dr. Alan Doctor at Washington University in Saint Louis, are trying to resuscitate blood substitutes with a new nanotechnology-based, artificial red blood cell may overcome the problems that killed products designed by a team of companies such as BiopureAlliance PharmaceuticalsNorthfield Labs and even Baxter. Dr. Alan Doctor’s new company, Kalocyte is advancing the development of the

The donut-shaped artificial cells, ErythroMer are one-fiftieth the size of human red blood cells. ErythroMer is a novel blood substitute composed of a patented nanobialys nanoparticle. A special lining and control system tied to changes in blood Ph allows Erythromer to grab onto oxygen in the lungs and then dispense the oxygen in tissues where it is needed. The new artificial cells are intended to sidestep problems with vasoconstriction or narrowing of blood vessels.

Erythromer is stored freeze dried and reconstituted with water when needed but it can also be stored at room temperature which makes it for military and civilian trauma.

Trials have been successful in rats, mice, and rabbits, and human trials are planned. However, moving Erythromer into human clinical trials is still 8-10 years away.


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Walking DNA Nanorobot

Reporter: Irina Robu, PhD

New research from California Institute of Technology headed by Anupama Thubagere and Lulu Qian built robots from DNA and programmed them to sort and deliver molecules to a specified location. These robots can potentially transform the drug delivery field to how body fights infections to how microscopic measurements are made. The dominant premise of DNA robots is that rather than creating molecular devices from scratch, we can use the power of molecular machinery by building microscopic-size robots and send them to places that are then impossible to reach, such as a cell or a hard-to-reach cancerous tumor. These robots demonstrated the ability to perform simple tasks, however this latest effort ramped up a path by programming DNA robots to perform a cargo‐sorting task and possibly many other tasks.

Each robot was built from a single-stranded DNA molecule of just 53 nucleotides equipped with “legs” for walking and “arms” for picking up objects. The robot are 20 nanometers tall and their walking strides measures six nanometers long, where one nanometer is a billionth of a meter. For the cargo, the researchers used two types of molecules, each being a distinct single-stranded piece of DNA. For the tests, the researchers placed the cargo onto a random location along the surface of a two-dimensional origami DNA test platform. The walking DNA robots moved in parallel along this surface, hunting for their cargo.

To see if a robot successfully picked up and dropped off the right cargo at the right location, the researchers used two fluorescent dyes to differentiate the molecules.

The researchers guess that each DNA robot took around 300 steps to complete its task, or roughly ten times more than in previous efforts. Though, more work is needed to figure out how these DNA robots perform under different environmental conditions. This new study suggests a worthwhile methodology for scientists to continue pursuing.



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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


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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