Archive for the ‘BioPrinting in Regenerative Medicine’ Category

3D Print Shape-Shifting Smart Gel

Reporter: Irina Robu, PhD

Hydrogel scaffolds that mimic the native extracellular matrix (ECM) environment play a crucial role in tissue engineering and they are ubiquitously in our lives, including in contact lenses, diapers and the human body.

Researchers at Rutgers have invented a printing method for a smart gel that can be used to create materials for transporting small molecules like drugs to human organs. The approach includes printing a 3D object with a hydrogel that changes shape over time when temperature changes. The potential of the smart hydrogels could be to create a new are of soft robotics and enable new applications in flexible sensors and actuators, biomedical devices and platforms or scaffolds for cells to grow.

Rutgers engineers operated with a hydrogel that has been in use for decades in devices that generate motion and biomedical applications such as scaffolds for cells to grow on. The engineers learned how to precisely control hydrogel growth and shrinkage. In temperatures below 32 degrees Celsius, the hydrogel absorbs more water and swells in size. When temperatures exceed 32 degrees Celsius, the hydrogel begins to expel water and shrinks, the study showed.

According to the Rutgers engineers, the objects they can produce with the hydrogel range from the width of a human hair to several millimeters long. The engineers also showed that they can grow one area of a 3D-printed object by changing temperatures.



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New Liver Tissue Implants Showing Potential

Reporter: Irina Robu,PhD

To develop new tissues, researchers at the Medical Research Council Center for Regenerative Medicine at the University of Edinburgh have found that stem cells transformed into 3-D liver tissue can support liver function when implanted into the mice suffering with a liver disease.

The scientists stimulated human embryonic stem cells and induced pluripotent stem cells to mature pluripotent stem cells into liver cells, hepatocytes. Hepatocytes are the chief functional cells of the liver and perform an astonishing number of metabolic, endocrine and secretory functions. Hepatocytes are exceptionally active in synthesis of protein and lipids for export. The cells are grown in 3-D conditions as small spheres for over a year. However, keeping the stem cells as liver cells for a long time is very difficult, because the viability of hepatocytes decreases in-vitro conditions.

Succeeding the discovery, the team up with materials chemists and engineers to detect appropriate polymers that have already been approved for human use that can be developed into 3-D scaffolds. The best material to use a biodegradable polyester, called polycaprolactone (PCL).PCL is degraded by hydrolysis of its ester linkages in physiological conditions (such as in the human body) and it is especially interesting for the preparation of long term implantable devices, owing to its degradation which is even slower than that of polylactide. They spun the PCL into microscopic fibers that formed a scaffold one centimeter square and a few millimeters thick.

At the same time, hepatocytes derived from embryonic cells had been grown in culture for 20 days and were then loaded onto the scaffolds and implanted under the skin of mice.Blood vessels successfully grew on the scaffolds with the mice having human liver proteins in their blood, demonstrating that the tissue had successfully integrated with the circulatory system. The scaffolds were not rejected by the animals’ immune systems.

The scientists tested the liver tissue scaffolds in mice with tyrosinaemia,a potentially fatal genetic disorder where the enzymes in the liver that break down the amino acid tyrosine are defective, resulting in the accumulation of toxic metabolic products. The implanted liver tissue aided the mice with tyrosinaemia to break down tyrosine and the mice finally lost less weight, had less buildup of toxins in the blood and exhibited fewer signs of liver damage than the control group that received empty scaffolds.

According to Rob Buckle, PhD, Chief Science Officer at the MRC, “Showing that such stem cell-derived tissue is able to reproduce aspects of liver function in the lab also offers real potential to improve the testing of new drugs where more accurate models of human tissue are needed”. It is believed that the discovery could be the next step towards harnessing stem cell reprogramming technologies to provide renewable supplies of liver tissue products for transplantation.



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3-D Printed Ovaries Produce Healthy Offspring

Reporter: Irina Robu, PhD


Each year about 120,000 organs are transplanted from one human being to another and most of the time is a living volunteer. But lack of suitable donors, predominantly means the supply of such organs is inadequate. Countless people consequently die waiting for a transplant which has led researchers to study the question of how to build organs from scratch.

One promising approach is to print them, but “bioprinting” remains largely experimental. Nevertheless, bioprinted tissue is before now being sold for drug testing, and the first transplantable tissues are anticipated to be ready for use in a few years’ time. The first 3D printed organ includes bioprosthetic ovaries which are constructed of 3D printed scaffolds that have immature eggs and have been successful in boosting hormone production and restoring fertility was developed by Teresa K. Woodruff, a reproductive scientist and director of the Women’s Health Research Institute at Feinberg School of Medicine, at Northwestern University, in Illinois.

What sets apart these bioprosthetic ovaries is the architecture of the scaffold. The material is made of gelatin made from broken-down collagen that is safe to humans which is self-supporting and can lead to building multiple layers.

The 3-D printed “scaffold” or “skeleton” is implanted into a female and its pores can be used to optimize how follicles, or immature eggs, get wedged within the scaffold. The scaffold supports the survival of the mouse’s immature egg cells and the cells that produce hormones to boost production. The open construction permits room for the egg cells to mature and ovulate, blood vessels to form within the implant enabling the hormones to circulate and trigger lactation after giving birth. The purpose of this scaffold is to recapitulate how an ovary would function.
The scientists’ only objective for developing the bioprosthetic ovaries was to help reestablish fertility and hormone production in women who have suffered adult cancer treatments and now have bigger risks of infertility and hormone-based developmental issues.



Printed human body parts could soon be available for transplant


3D printed ovaries produce healthy offspring giving hope to infertile women


Brave new world: 3D-printed ovaries produce healthy offspring


3-D-printed scaffolds restore ovary function in infertile mice


Our Grandkids May Be Born From 3D-Printed Ovaries


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Nanostraws Developed at Stanford Sample a Cell’s Contents without Damage

Reporter: Irina Robu, PhD

Cells within our bodies change over time and divide, with thousands of chemical reactions happening within cell daily. Nicholas Melosh, Associate Professor of Materials Science and Engineering, developed a new, non-destructive system for sampling cells with nanoscale straws which could help uncover mysteries about how cells function.

Currently, cells are sampled via lysing which ruptures the cell membrane which means that it can’t ever be sampled again. The sample system that Dr. Melosh invented banks on, on tiny tubes 600 times smaller than a strand of hair that allow researchers to sample a single cell at a time. The nanostraws penetrate a cell’s outer membrane, without damaging it, and draw out proteins and genetic material from the cell’s salty interior.

The Nanostraw sampling technique, according to Melosh, will knowingly impact our understanding of cell development and could result to much safer and operational medical therapies because the technique allows for long term, non-destructive monitoring. The sampling technique could also inform cancer treatments and answer questions about why some cancer cells are resistant to chemotherapy while others are not. The sampling platform on which the nanostraws are grown is tiny, similar to the size of a gumball. It’s called the Nanostraw Extraction (NEX) sampling system, and it was designed to mimic biology itself.

The goal of developing this technology was to make an impact in medical biology by providing a platform that any lab could build.


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Topical Solution for Combination Oncology Drug Therapy: Patch that delivers Drug, Gene, and Light-based Therapy to Tumor

Reporter: Aviva Lev-Ari, PhD, RN


Self-assembled RNA-triple-helix hydrogel scaffold for microRNA modulation in the tumour microenvironment


  1. Massachusetts Institute of Technology, Institute for Medical Engineering and Science, Harvard-MIT Division for Health Sciences and Technology, Cambridge, Massachusetts 02139, USA
    • João Conde,
    • Nuria Oliva,
    • Mariana Atilano,
    • Hyun Seok Song &
    • Natalie Artzi
  2. School of Engineering and Materials Science, Queen Mary University of London, London E1 4NS, UK
    • João Conde
  3. Grup dEnginyeria de Materials, Institut Químic de Sarrià-Universitat Ramon Llull, Barcelona 08017, Spain
    • Mariana Atilano
  4. Division of Bioconvergence Analysis, Korea Basic Science Institute, Yuseong, Daejeon 169-148, Republic of Korea
    • Hyun Seok Song
  5. Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA
    • Natalie Artzi
  6. Department of Medicine, Biomedical Engineering Division, Brigham and Womens Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA
    • Natalie Artzi


J.C. and N.A. conceived the project and designed the experiments. J.C., N.O., H.S.S. and M.A. performed the experiments, collected and analysed the data. J.C. and N.A. co-wrote the manuscript. All authors discussed the results and reviewed the manuscript.

Nature Materials
22 April 2015
26 October 2015
Published online
07 December 2015

The therapeutic potential of miRNA (miR) in cancer is limited by the lack of efficient delivery vehicles. Here, we show that a self-assembled dual-colour RNA-triple-helix structure comprising two miRNAs—a miR mimic (tumour suppressor miRNA) and an antagomiR (oncomiR inhibitor)—provides outstanding capability to synergistically abrogate tumours. Conjugation of RNA triple helices to dendrimers allows the formation of stable triplex nanoparticles, which form an RNA-triple-helix adhesive scaffold upon interaction with dextran aldehyde, the latter able to chemically interact and adhere to natural tissue amines in the tumour. We also show that the self-assembled RNA-triple-helix conjugates remain functional in vitro and in vivo, and that they lead to nearly 90% levels of tumour shrinkage two weeks post-gel implantation in a triple-negative breast cancer mouse model. Our findings suggest that the RNA-triple-helix hydrogels can be used as an efficient anticancer platform to locally modulate the expression of endogenous miRs in cancer.




Patch that delivers drug, gene, and light-based therapy to tumor sites shows promising results

In mice, device destroyed colorectal tumors and prevented remission after surgery.

Helen Knight | MIT News Office
July 25, 2016

Approximately one in 20 people will develop colorectal cancer in their lifetime, making it the third-most prevalent form of the disease in the U.S. In Europe, it is the second-most common form of cancer.

The most widely used first line of treatment is surgery, but this can result in incomplete removal of the tumor. Cancer cells can be left behind, potentially leading to recurrence and increased risk of metastasis. Indeed, while many patients remain cancer-free for months or even years after surgery, tumors are known to recur in up to 50 percent of cases.

Conventional therapies used to prevent tumors recurring after surgery do not sufficiently differentiate between healthy and cancerous cells, leading to serious side effects.

In a paper published today in the journal Nature Materials, researchers at MIT describe an adhesive patch that can stick to the tumor site, either before or after surgery, to deliver a triple-combination of drug, gene, and photo (light-based) therapy.

Releasing this triple combination therapy locally, at the tumor site, may increase the efficacy of the treatment, according to Natalie Artzi, a principal research scientist at MIT’s Institute for Medical Engineering and Science (IMES) and an assistant professor of medicine at Brigham and Women’s Hospital, who led the research.

The general approach to cancer treatment today is the use of systemic, or whole-body, therapies such as chemotherapy drugs. But the lack of specificity of anticancer drugs means they produce undesired side effects when systemically administered.

What’s more, only a small portion of the drug reaches the tumor site itself, meaning the primary tumor is not treated as effectively as it should be.

Indeed, recent research in mice has found that only 0.7 percent of nanoparticles administered systemically actually found their way to the target tumor.

“This means that we are treating both the source of the cancer — the tumor — and the metastases resulting from that source, in a suboptimal manner,” Artzi says. “That is what prompted us to think a little bit differently, to look at how we can leverage advancements in materials science, and in particular nanotechnology, to treat the primary tumor in a local and sustained manner.”

The researchers have developed a triple-therapy hydrogel patch, which can be used to treat tumors locally. This is particularly effective as it can treat not only the tumor itself but any cells left at the site after surgery, preventing the cancer from recurring or metastasizing in the future.

Firstly, the patch contains gold nanorods, which heat up when near-infrared radiation is applied to the local area. This is used to thermally ablate, or destroy, the tumor.

These nanorods are also equipped with a chemotherapy drug, which is released when they are heated, to target the tumor and its surrounding cells.

Finally, gold nanospheres that do not heat up in response to the near-infrared radiation are used to deliver RNA, or gene therapy to the site, in order to silence an important oncogene in colorectal cancer. Oncogenes are genes that can cause healthy cells to transform into tumor cells.

The researchers envision that a clinician could remove the tumor, and then apply the patch to the inner surface of the colon, to ensure that no cells that are likely to cause cancer recurrence remain at the site. As the patch degrades, it will gradually release the various therapies.

The patch can also serve as a neoadjuvant, a therapy designed to shrink tumors prior to their resection, Artzi says.

When the researchers tested the treatment in mice, they found that in 40 percent of cases where the patch was not applied after tumor removal, the cancer returned.

But when the patch was applied after surgery, the treatment resulted in complete remission.

Indeed, even when the tumor was not removed, the triple-combination therapy alone was enough to destroy it.

The technology is an extraordinary and unprecedented synergy of three concurrent modalities of treatment, according to Mauro Ferrari, president and CEO of the Houston Methodist Research Institute, who was not involved in the research.

“What is particularly intriguing is that by delivering the treatment locally, multimodal therapy may be better than systemic therapy, at least in certain clinical situations,” Ferrari says.

Unlike existing colorectal cancer surgery, this treatment can also be applied in a minimally invasive manner. In the next phase of their work, the researchers hope to move to experiments in larger models, in order to use colonoscopy equipment not only for cancer diagnosis but also to inject the patch to the site of a tumor, when detected.

“This administration modality would enable, at least in early-stage cancer patients, the avoidance of open field surgery and colon resection,” Artzi says. “Local application of the triple therapy could thus improve patients’ quality of life and therapeutic outcome.”

Artzi is joined on the paper by João Conde, Nuria Oliva, and Yi Zhang, of IMES. Conde is also at Queen Mary University in London.


Other related articles published in thie Open Access Online Scientific Journal include the following:

The Development of siRNA-Based Therapies for Cancer

Author: Ziv Raviv, PhD


Targeted Liposome Based Delivery System to Present HLA Class I Antigens to Tumor Cells: Two papers

Reporter: Stephen J. Williams, Ph.D.


Blast Crisis in Myeloid Leukemia and the Activation of a microRNA-editing Enzyme called ADAR1

Curator: Larry H. Bernstein, MD, FCAP


First challenge to make use of the new NCI Cloud Pilots – Somatic Mutation Challenge – RNA: Best algorithms for detecting all of the abnormal RNA molecules in a cancer cell

Reporter: Aviva Lev-Ari, PhD, RN


miRNA Therapeutic Promise

Curator: Larry H. Bernstein, MD, FCAP

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BioPrinting Basics

Curator: Larry H. Bernstein, MD, FCAP



The ABCs of 3D Bioprinting of Living Tissues, Organs   5/06/2016 

(Credit: Ozbolat Lab/Penn State University)
(Credit: Ozbolat Lab/Penn State University)

Although first originated in 2003, the world of bioprinting is still very new and ambiguous. Nevertheless, as the need for organ donation continues to increase worldwide, and organ and tissue shortages prevail, a handful of scientists have started utilizing this cutting-edge science and technology for various areas of regenerative medicine to possibly fill that organ-shortage void.

Among these scientists is Ibrahim Tarik Ozbolat, an associate professor of Engineering Science and Mechanics Department and the Huck Institutes of the Life Sciences at Penn State University, who’s been studying bioprinting and tissue engineering for years.

While Ozbolat is not the first to originate 3D bioprinting research, he’s the first one at Penn State University to spearhead the studies at Ozbolat Lab, Leading Bioprinting Research.

“Tissue engineering is a big need. Regenerative medicine, biofabrication of tissues and organs that can replace the damage or diseases is important,” Ozbolat told R&D Magazine after his seminar presentation at Interphex last week in New York City, titled 3D Bioprinting of Living Tissues & Organs.”

3D bioprinting is the process of creating cell patterns in a confined space using 3D-printing technologies, where cell function and viability are preserved within the printed construct.

Recent progress has allowed 3D printing of biocompatible materials, cells and supporting components into complex 3D functional living tissues. The technology is being applied to regenerative medicine to address the need for tissues and organs suitable for transplantation. Compared with non-biological printing, 3D bioprinting involves additional complexities, such as the choice of materials, cell types, growth and differentiation factors, and technical challenges related to the sensitivities of living cells and the construction of tissues. Addressing these complexities requires the integration of technologies from the fields of engineering, biomaterials science, cell biology, physics and medicine, according to

“If we’re able to make organs on demand, that will be highly beneficial to society,” said Ozbolat. “We have the capability to pattern cells, locate them and then make the same thing that exists in the body.”

3D bioprinting of tissues and organs

Sean V Murphy & Anthony Atala
Nature Biotechnology 32,773–785(2014)       doi:10.1038/nbt.2958


Additive manufacturing, otherwise known as three-dimensional (3D) printing, is driving major innovations in many areas, such as engineering, manufacturing, art, education and medicine. Recent advances have enabled 3D printing of biocompatible materials, cells and supporting components into complex 3D functional living tissues. 3D bioprinting is being applied to regenerative medicine to address the need for tissues and organs suitable for transplantation. Compared with non-biological printing, 3D bioprinting involves additional complexities, such as the choice of materials, cell types, growth and differentiation factors, and technical challenges related to the sensitivities of living cells and the construction of tissues. Addressing these complexities requires the integration of technologies from the fields of engineering, biomaterials science, cell biology, physics and medicine. 3D bioprinting has already been used for the generation and transplantation of several tissues, including multilayered skin, bone, vascular grafts, tracheal splints, heart tissue and cartilaginous structures. Other applications include developing high-throughput 3D-bioprinted tissue models for research, drug discovery and toxicology.


Future Technologies : Bioprinting

3D printing is increasingly permitting the direct digital manufacture (DDM) of a wide variety of plastic and metal items. While this in itself may trigger a manufacturing revolution, far more startling is the recent development of bioprinters. These artificially construct living tissue by outputting layer-upon-layer of living cells. Currently all bioprinters are experimental. However, in the future, bioprinters could revolutionize medical practice as yet another element of the New Industrial Convergence.

Bioprinters may be constructed in various configurations. However, all bioprinters output cells from a bioprint head that moves left and right, back and forth, and up and down, in order to place the cells exactly where required. Over a period of several hours, this permits an organic object to be built up in a great many very thin layers.

In addition to outputting cells, most bioprinters also output a dissolvable gel to support and protect cells during printing. A possible design for a future bioprinter appears below and in the sidebar, here shown in the final stages of printing out a replacement human heart. Note that you can access larger bioprinter images on the Future Visions page. You may also like to watch my bioprinting video.



Bioprinting Pioneers

Several experimental bioprinters have already been built. For example, in 2002 Professor Makoto Nakamura realized that the droplets of ink in a standard inkjet printer are about the same size as human cells. He therefore decided to adapt the technology, and by 2008 had created a working bioprinter that can print out biotubing similar to a blood vessel. In time, Professor Nakamura hopes to be able to print entire replacement human organs ready for transplant. You can learn more about this groundbreaking work here or read this message from Professor Nakamura. The movie below shows in real-time the biofabrication of a section of biotubing using his modified inkjet technology.


Another bioprinting pioneer is Organovo. This company was set up by a research group lead by Professor Gabor Forgacs from the University of Missouri, and in March 2008 managed to bioprint functional blood vessels and cardiac tissue using cells obtained from a chicken. Their work relied on a prototype bioprinter with three print heads. The first two of these output cardiac and endothelial cells, while the third dispensed a collagen scaffold — now termed ‘bio-paper’ — to support the cells during printing.

Since 2008, Organovo has worked with a company called Invetech to create a commercial bioprinter called the NovoGen MMX. This is loaded with bioink spheroids that each contain an aggregate of tens of thousands of cells. To create its output, the NovoGen first lays down a single layer of a water-based bio-paper made from collagen, gelatin or other hydrogels. Bioink spheroids are then injected into this water-based material. As illustrated below, more layers are subsequently added to build up the final object. Amazingly, Nature then takes over and the bioink spheroids slowly fuse together. As this occurs, the biopaper dissolves away or is otherwise removed, thereby leaving a final bioprinted body part or tissue.


bioprinting stages

As Organovo have demonstrated, using their bioink printing process it is not necessary to print all of the details of an organ with a bioprinter, as once the relevant cells are placed in roughly the right place Nature completes the job. This point is powerfully illustrated by the fact that the cells contained in a bioink spheroid are capable of rearranging themselves after printing. For example, experimental blood vessels have been bioprinted using bioink spheroids comprised of an aggregate mix of endothelial, smooth muscle and fibroblast cells. Once placed in position by the bioprint head, and with no technological intervention, the endothelial cells migrate to the inside of the bioprinted blood vessel, the smooth muscle cells move to the middle, and the fibroblasts migrate to the outside.

In more complex bioprinted materials, intricate capillaries and other internal structures also naturally form after printing has taken place. The process may sound almost magical. However, as Professor Forgacs explains, it is no different to the cells in an embryo knowing how to configure into complicated organs. Nature has been evolving this amazing capability for millions of years. Once in the right places, appropriate cell types somehow just know what to do.

In December 2010, Organovo create the first blood vessels to be bioprinted using cells cultured from a single person. The company has also successfully implanted bioprinted nerve grafts into rats, and anticipates human trials of bioprinted tissues by 2015. However, it also expects that the first commercial application of its bioprinters will be to produce simple human tissue structures for toxicology tests. These will enable medical researchers to test drugs on bioprinted models of the liver and other organs, thereby reducing the need for animal tests.

In time, and once human trials are complete, Organovo hopes that its bioprinters will be used to produce blood vessel grafts for use in heart bypass surgery. The intention is then to develop a wider range of tissue-on-demand and organs-on-demand technologies. To this end, researchers are now working on tiny mechanical devices that can artificially exercise and hence strengthen bioprinted muscle tissue before it is implanted into a patient.

Organovo anticipates that its first artificial human organ will be a kidney. This is because, in functional terms, kidneys are one of the more straight-forward parts of the body. The first bioprinted kidney may in fact not even need to look just like its natural counterpart or duplicate all of its features. Rather, it will simply have to be capable of cleaning waste products from the blood. You can read more about the work of Organovoand Professor Forgac’s in this article from Nature.

Regenerative Scaffolds and Bones

A further research team with the long-term goal of producing human organs-on-demand has created the Envisiontec Bioplotter. Like Organovo’s NovoGen MMX, this outputs bio-ink ’tissue spheroids’ and supportive scaffold materials including fibrin and collagen hydrogels. But in addition, the Envisontech can also print a wider range of biomaterials. These include biodegradable polymers and ceramics that may be used to support and help form artificial organs, and which may even be used as bioprinting substitutes for bone.

Talking of bone, a team lead by Jeremy Mao at the Tissue Engineering and Regenerative Medicine Lab at Columbia University is working on the application of bioprinting in dental and bone repairs. Already, a bioprinted, mesh-like 3D scaffold in the shape of an incisor has been implanted into the jaw bone of a rat. This featured tiny, interconnecting microchannels that contained ‘stem cell-recruiting substances’. In just nine weeks after implantation, these triggered the growth of fresh periodontal ligaments and newly formed alveolar bone. In time, this research may enable people to be fitted with living, bioprinted teeth, or else scaffolds that will cause the body to grow new teeth all by itself. You can read more about this development in this article from The Engineer.

In another experient, Mao’s team implanted bioprinted scaffolds in the place of the hip bones of several rabbits. Again these were infused with growth factors. As reported inThe Lancet, over a four month period the rabbits all grew new and fully-functional joints around the mesh. Some even began to walk and otherwise place weight on their new joints only a few weeks after surgery. Sometime next decade, human patients may therefore be fitted with bioprinted scaffolds that will trigger the grown of replacement hip and other bones. In a similar development, a team from Washington State University have also recently reported on four years of work using 3D printers to create a bone-like material that may in the future be used to repair injuries to human bones.

In Situ Bioprinting

The aforementioned research progress will in time permit organs to be bioprinted in a lab from a culture of a patient’s own cells. Such developments could therefore spark a medical revolution. Nevertheless, others are already trying to go further by developing techniques that will enable cells to be printed directly onto or into the human body in situ. Sometime next decade, doctors may therefore be able to scan wounds and spray on layers of cells to very rapidly heal them.

Already a team of bioprinting researchers lead by Anthony Alata at the Wake Forrest School of Medicine have developed a skin printer. In initial experiments they have taken 3D scans of test injuries inflicted on some mice and have used the data to control a bioprint head that has sprayed skin cells, a coagulant and collagen onto the wounds. The results are also very promising, with the wounds healing in just two or three weeks compared to about five or six weeks in a control group. Funding for the skin-printing project is coming in part from the US military who are keen to develop in situ bioprinting to help heal wounds on the battlefield. At present the work is still in a pre-clinical phase with Alata progressing his research usig pigs. However, trials of with human burn victims could be a little as five years away.

The potential to use bioprinters to repair our bodies in situ is pretty mind blowing. In perhaps no more than a few decades it may be possible for robotic surgical arms tipped with bioprint heads to enter the body, repair damage at the cellular level, and then also repair their point of entry on their way out. Patients would still need to rest and recuperate for a few days as bioprinted materials fully fused into mature living tissue. However, most patients could potentially recover from very major surgery in less than a week.

Cosmetic Applications …

Bioprinting Implications …

More information on bioprinting can be found in my books 3D Printing: Second Editionand The Next Big Thing. There is also a bioprinting section in my 3D Printing Directory. Oh, and there is also a great infographic about bioprinting here. Enjoy!


How to print out a blood vessel

New work moves closer to the age of organs on demand.

Blood vessels can now be ‘printed out’ by machine. Could bigger structures be in the future?SUSUMU NISHINAGA / SCIENCE PHOTO LIBRARY

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New method for 3D imaging of brain tumors

Larry H. Bernstein, MD, FCAP, Curator




Third-Harmonic Generation Microscopy Provides In Situ Brain Tumor Imaging

AMSTERDAM, Netherlands, April 25, 2015 — A technique involving third-harmonic generation microscopy could allow neurosurgeons to image and assess brain tumor boundaries during surgery, providing optical biopsies in near-real time and increasing the accuracy of tissue removal.

Pathologists typically use staining methods, in which chemicals like hematoxylin and eosin turn different tissue components blue and red, revealing its structure and whether there are any tumor cells. A definitive diagnosis can take up to 24 hours, meaning surgeons may not realize some cancerous tissue has escaped from their attention until after surgery — requiring a second operation and more risk.

Tissue from a patient diagnosed with low-grade glioma.

Tissue from a patient diagnosed with low-grade glioma. The green image is taken with the new method, while the pink uses conventional hematoxylin and eosin staining. From the upper left to the lower right, both images show increasing cell density due to more tumor tissue. The insets reveal the high density of tumor cells. Courtesy of N.V. Kuzmin et al./VU University Amsterdam.

Brain tumors — specifically glial brain tumors — are often spread out and mixed in with the healthy tissue, presenting a particular challenge. Surgery, irradiation and chemotherapy often cause substantial collateral damage to the surrounding brain tissue.

Now researchers from VU University Amsterdam, led by professor Marloes Groot, have demonstrated a label-free optical method for imaging cancerous brain tissue. They were able to produce most images in under a minute; smaller ones took <1 s, while larger images of a few square millimeters took 5 min.

The study involved firing short, 200-fs, 1200-nm laser pulses into the tissue. When three photons converged at the same time and place, the photons interacted with the nonlinear optical properties of the tissue. Through the phenomena of third harmonic generation, the interactions produced a single 400- or 600-nm photon (in the case of third or second harmonic generation, respectively).

The shorter-wavelength photon scatters in the tissue, and when it reaches a detector — in this case a high-sensitivity GaAsP photomultiplier tube — it reveals what the tissue looks like inside. The resulting images enabled clear recognition of cellularity, nuclear pleomorphism and rarefaction of neuropil in the tissue.

While this technique has been used in other applications — to image insects and fish embryos, for example — the researchers said this is the first time it’s been used to analyze glial brain tumors.

Groot and her team are now developing a handheld device for tumor border detection during surgery. The incoming laser pulses can only reach a depth of about 100 μm into the tissue currently; to reach further, Groot envisions attaching a needle that can pierce the tissue and deliver photons deeper.

The research was published in Biomedical Optics Express, a publication of The Optical Society (OSA) (doi: 10.1364/boe.7.001889).


Third harmonic generation imaging for fast, label-free pathology of human brain tumors

Biomedical Optics Express 2016  7(5):1889-1904    doi: 10.1364/BOE.7.001889

In brain tumor surgery, recognition of tumor boundaries is key. However, intraoperative assessment of tumor boundaries by the neurosurgeon is difficult. Therefore, there is an urgent need for tools that provide the neurosurgeon with pathological information during the operation. We show that third harmonic generation (THG) microscopy provides label-free, real-time images of histopathological quality; increased cellularity, nuclear pleomorphism, and rarefaction of neuropil in fresh, unstained human brain tissue could be clearly recognized. We further demonstrate THG images taken with a GRIN objective, as a step toward in situ THG microendoscopy of tumor boundaries. THG imaging is thus a promising tool for optical biopsies.


Glial tumors (gliomas) account for almost 80% of the tumors originating from brain tissue. The vast majority of these tumors are so-called ‘diffuse gliomas’ as they show very extensive (‘diffuse’) growth into the surrounding brain parenchyma. With surgical resection, irradiation, and/or chemotherapy it is impossible to eliminate all glioma cells without serious damage to the brain tissue. As a consequence, until now, patients with a diffuse glioma have had a poor prognosis, a situation which strongly contributes to the fact that brain tumor patients experience more years of life lost than patients with any other type of cancer [1,2].

Meanwhile it has also been demonstrated that the prognosis of patients with a diffuse glioma correlates with the extent of resection [3–5]. During brain surgery, however, it is extremely difficult for the neurosurgeon to determine the boundary of the tumor, i.e. whether a brain area contains tumor cells or not. If the neurosurgeon could have histopathological information on the tumor boundaries during brain surgery, then recognition of these tumor boundaries and with that, the surgical resection, could be significantly improved.

Occasionally, intra-operative analysis using hematoxylin-and-eosin (H&E) stained sections of snap-frozen material or smear preparations is performed by the pathologist to help establish brain tumor boundaries, but this procedure only allows analysis of small, selected regions, can only be performed on tissue fragments that are already resected, and is rather time consuming (frozen section diagnosis) or does not allow analysis of tumor in the histological context (smear preparations). Fluorescence imaging techniques are increasingly used during surgery [6,7] but are associated with several drawbacks, such as heterogeneous delivery and nonspecific staining [8,9]. In particular, low-grade gliomas and normal brain tissue have an intact blood-brain barrier and take up little circulating dye [10–12]. Alternative techniques are therefore required, that can detect the presence of tumor cells in tissue without fluorescent labels and with a speed that enables ‘live’ feedback to the surgeon while he/she operates.

The past year has seen exciting new developments in which optical coherence tomography [13] and stimulated Raman microscopy [14,15] were reported to reliably detect tumor tissue in the brain of human glioma patients, and a handheld Raman spectroscopy device was even implemented intra-surgical to assess brain tissue prior to excision [16]. These techniques are especially sensitive in densely tumor-infiltrated areas, and for the Raman spectroscopy device study a sensitivity limit of 17 tumor cells in an area of 150 × 150 μm2 was reported. The discriminating power of the Raman techniques is based on subtle differences in the vibrational spectra of tumor tissue and healthy tissue, and they require extensive comparison of experimental spectra against libraries of reference spectra. A technique capable of directly visualizing the classical histopathological hallmark criteria currently used by pathologists for classification of tumor tissue could potentially be even more reliable and make the transition from the current practice—histopathological analysis of fixated tissue—to in situ optical biopsy easier. Diffuse gliomas are histopathologically characterized by variably increased cellularity, nuclear pleomorphism and—especially in higher-grade neoplasms—brisk mitotic activity, microvascular proliferation, and necrosis. To visualize these features in live tissue, a technique that elucidates the morphology of tissue is required. In this context, third harmonic generation (THG) microscopy is a promising tool because of its capacity to visualize almost the full morphology of tissue. THG is a nonlinear optical process that relies on spatial variations of the third-order non-linear susceptibility χ(3) intrinsic to the tissue and (in the case of brain tissue) mainly arises from interfaces with lipid-rich molecules [17–27]. SHG signals arise from an optical nonlinear process involving non-centrosymmetric molecules present in, for example, microtubules and collagen. THG has been successfully applied to image unstained samples such as insect embryos, plant seeds and intact mammalian tissue [28], epithelial tissues [29–31], zebra fish embryos [32], and the zebra fish nervous system [33]. In brain tissue of mice, augmented by co-recording of SHG signals, THG was shown to visualize cells, nuclei, the inner and outer contours of axons, blood cells, and vessels, resulting in the visualization of both gray and white matter (GM and WM) as well as vascularization, up to a depth of 350 μm [24,26]. Here, we explore the potential of THG and SHG imaging for real time analysis of ex-vivo human brain tissue in the challenging cases of diffuse tumor invasion in low-grade brain tumors as well as of high-grade gliomas and structurally normal brain tissues.


Multiphoton imaging

THG and SHG are nonlinear optical processes that may occur in tissue depending on the nonlinear susceptibility coefficients χ(3) and χ(2) of the tissue and upon satisfying phase matching conditions [17–19,21,23–27]. In the THG process, three incident photons are converted into one photon with triple energy and one third of the wavelength (Fig. 1(A)). In the SHG process, signals result from the conversion of an incident photon pair into one photon with twice the energy and half the wavelength. Two- and three photon excited fluorescence signals (2PF, 3PF) may simultaneously be generated by intrinsic proteins (Fig. 1(B)). As a result, a set of distinct (harmonic) and broadband (autofluorescence) spectral peaks is generated in the visible range. The imaging setup (Fig. 1(C)) to generate and collect these signals consisted of a commercial two-photon laser-scanning microscope (TriMScope I, LaVision BioTec GmbH) and a femtosecond laser source. The laser source was an optical parametric oscillator (Mira-OPO, APE) pumped at 810 nm by a Ti-sapphire oscillator (Coherent Chameleon Ultra II). The OPO generates 200 fs pulses at 1200 nm with a repetition rate of 80 MHz. We selected this wavelength as it falls in the tissue transparency window, providing deeper penetration and reduced photodamage compared to the 700–1000 nm range, as well as harmonic signals generated in the visible wavelength range, facilitating their collection and detection with conventional objectives and detectors. We focused the OPO beam on the sample using a 25 × /1.10 (Nikon APO LWD) water-dipping objective (MO). The 1200 nm beam focal spot size on the sample was dlateral ~0.7 μm and daxial ~4.1 μm. It was measured with 0.175 μm fluorescent microspheres (see Section 3.4) yielding two- and three-photon resolution values Δ2P,lateral ~0.5 μm, Δ2P,axial ~2.9 μm, Δ3P,lateral ~0.4 μm, and Δ3P,axial ~2.4 μm. Two high-sensitivity GaAsP photomultiplier tubes (PMT, Hamamatsu H7422-40) equipped with narrowband filters at 400 nm and 600 nm were used to collect the THG and SHG signals, respectively, as a function of position of the focus in the sample. The signals were filtered from the 1200 nm fundamental photons by a dichroic mirror (Chroma T800LPXRXT, DM1), split into SHG and THG channels by a dichroic mirror (Chroma T425LPXR, DM2), and passed through narrow-band interference filters (F) for SHG (Chroma D600/10X) and THG (Chroma Z400/10X) detection. The efficient back-scattering of the harmonic signals allowed for their detection in epi-direction. The laser beam was transversely scanned over the sample by a pair of galvo mirrors (GM). THG and SHG modalities are intrinsically confocal and therefore provide direct depth sectioning. We obtained a full 3D image of the tissue volume by scanning the microscope objective with a stepper motor in the vertical (z) direction. The mosaic imaging of the sample was performed by transverse (xy) scanning of the motorized translation stage. Imaging data was acquired with the TriMScope I software (“Imspector Pro”); image stacks were stored in 16-bit tiff-format and further processed and analyzed with “ImageJ” software (ver. 1.49m, NIH, USA). All images were processed with logarithmic contrast enhancement.

Fig. 1 THG/SHG microscopy for brain tissue imaging. (A) Energy level diagram of the second (SHG) and third (THG) harmonic generation process. (B) Energy level diagram of the two- (2PF) and three-photon (3PF) excited auto-fluorescence process. (C) Multiphoton microscope setup: Laser producing 200 fs pulses at 1200 nm; GM – X-Y galvo-scanner mirrors; SL – scan lens; TL – tube lens; MO – microscope objective; DM1 – dichroic mirror reflecting back-scattered THG/SHG photons to the PMT detectors; DM2 – dichroic mirror splitting SHG and THG channels; F – narrow-band SHG and THG interference filters; L – focusing lenses; PMT – photomultiplier tube detectors. (D) Infrared photons (white arrow) are focused deep in the brain tissue, converted to THG (green) and SHG (red) photons, scattered back (green/red arrows) and epi-detected. The nonlinear optical processes result in label-free contrast images with sub-cellular resolution and intrinsic depth sectioning. (E and F) Freshly-excised low-grade (E) and high-grade (F) glioma tissue samples in artificial cerebrospinal fluid (ACSF) in a Petri dish with a millimeter paper underneath for scale. (G) An agar-embedded tumor tissue sample under 0.17 mm glass cover slip with the microscope objective (MO) on top.   Download Full Size | PPT Slide

Endomicroscopy imaging

For endomicroscopic imaging we used a commercial high-numerical-aperture (NA) multi-element micro-objective lens (GT-MO-080-018-810, GRINTECH) composed of a plano-convex lens and two GRaded INdex (GRIN) lenses with aberration compensation, object NA = 0.80 and object working distance 200 µm (in water), image NA = 0.18 and image working distance 200 µm (in air), magnification × 4.8 and field-of-view diameter of 200 μm. The GRIN lenses and the plano-convex lens were mounted in a waterproof stainless steel housing with an outer diameter of 1.4 mm and a total length of 7.5 mm. Originally designed for a wavelength range of 800–900 nm [36–41], this micro-objective lens was used for focusing of 1200 nm femtosecond pulses and collection of back-scattered harmonic and fluorescence photons. A coupling lens with f = 40 mm (NA = 0.19, Qioptiq, ARB2 NIR, dia. 25 mm) focused the scanned laser beam in the image plane of the micro-objective lens and forwarded the epi-detected harmonic and fluorescence photons to the PMTs.

We characterized the lateral (x) and axial (z) resolution of the micro-objective lens by 3D imaging of fluorescence microspheres (PS-Speck Microscope Point Source Kit, P7220, Molecular Probes). We used “blue” and “deep red” microspheres, 0.175 ± 0.005 μm in diameter, with excitation/emission maxima at 360/440 nm and 630/660 nm to obtain three-photon (3P) and two-photon (2P) point spread function (PSF) profiles. The excitation wavelength was 1200 nm, and fluorescence signals were detected in the 400 ± 5 nm (3P) and 600 ± 5 nm (2P) spectral windows, just as in the brain tissue imaging experiments. 1 μL of “blue” and “deep red” sphere suspensions were applied to a propanol-cleaned 75 × 26 × 1 mm3 glass slide. The mixed microsphere suspension was left to dry for 20 min and was then imaged with the micro-objective lens via a water immersion layer. The assembly of the coupling lens and the micro-objective lens was vertically (z) scanned with a step of 0.5 μm, and stacks of two-/three-photon images were recorded. The line profiles were then taken over the lateral (xy) images of the fluorescent spheres with maximal intensity (in focus), and fluorescence counts were plotted as function of the lateral coordinate (x). The axial (z) scan values of the two- and three-photon fluorescence signals were acquired by averaging of the total fluorescence counts of the corresponding spheres and were plotted as function of the axial coordinate (z). Lateral (x) and axial (z) 2P/3P points were then fitted with Gaussian functions and full width at half-maximum (FWHM) values were measured.

……. Results….  Conclusions

The results shown here provide the first evidence that—by applying the same microscopic criteria that are used by the pathologist, i.e. increased cellularity, nuclear pleomorphism, and rarefaction of neuropil—THG/SHG ex-vivo microscopy can be used to recognize the presence of diffuse infiltrative glioma in fresh, unstained human brain tissue. Images and a first diagnosis can be provided in seconds, with the ‘inspection mode’, by moving the sample under the scanning microscope (see Visualization 4 and Visualization 5), or in about 5 minutes if an area has to be inspected with sub-cellular detail. The sensitivity of THG to interfaces provides images with excellent contrast in which cell-by-cell variations are visualized. The quality of the images and the speed with which they can be recorded make THG a promising tool for quick assessment of the nature of excised tissue. Importantly, because THG/SHG images are very close to those of histological slides, we expect that the surgeon (or pathologist) will need very little additional training for adequate interpretation of the images. We are planning to construct a THG/SHG ex-vivo tabletop device consisting of a compact laser source and a laser-scanning microscope requiring a physical footprint of only 1 m2, to be placed in an operating room, enabling immediate feedback to the surgeon on the nature of excised tissue, during the operation. With this device, we will perform a quantitative study of the added value of rapid THG/SHG pathological feedback during surgery for the final success of the neurosurgery. Finally, we note that THG/SHG imaging does not induce artifacts associated with fixation, freezing, and staining; therefore, tissue fragments examined ex-vivo can still be used for subsequent immunochemical and/or molecular analysis.

The microendoscopy THG/SHG imaging results represent an important step toward the development of a THG/SHG-based bioptic needle, and show that the use of such a needle for in situ optical sampling for optimal resection of gliomas is indeed a viable prospect, as has been demonstrated also before for multi-photon microscopies [38,49–54]. Although there are several issues associated with the operation of a needle-like optical device, such as the fact that blood in the surgical cavity may obscure the view, and the fact that only small areas can be biopsied with a needle, it may be a valuable tool in cases where sparing healthy tissue is of such vital importance as in brain surgery. Therefore, the reasonably good quality of the THG images taken with the GRIN micro-objective shown here, together with the developments in the field of microendoscopy, warrant further development of THG/SHG into a true handheld device. This next step, a true handheld bioptic needle, requires an optical fiber to transport the light from a small footprint laser to the GRIN micro-objective, and a small 2D scanner unit, to enable placing the laser at a sufficient distance from the patient. Patient-safe irradiation levels for THG imaging will have to be determined but are expected to lie in the 10–50 mW range [55–58]. This implies that only minor optimization of signal collection efficiency needs to be achieved, because the images of Fig. 10 were measured with 50 mW incident power.

THG/SHG imaging thus holds great promise for improving surgical procedures, thereby reducing the need for second surgeries and the loss of function by excising non-infiltrated brain tissue, as well as improving survival and quality of life of the patients. In addition, the success in the challenging case of diffuse gliomas promises great potential of THG/SHG-based histological analysis for a much wider spectrum of diagnostic applications.

References and links

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6. U. Pichlmeier, A. Bink, G. Schackert, and W. Stummer, “Resection and survival in glioblastoma multiforme: An RTOG recursive partitioning analysis of ALA study patients,” Neuro-oncol. 10(6), 1025–1034 (2008). [CrossRef]   [PubMed]  

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Tables (1)

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Table 1 Pre-operative diagnoses and cell densities observed in the studied brain tissue samples by THG imaging and corresponding H&E histopathology.

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