Building a better recipe for 3D printed bones

A Johns Hopkins biomedical engineer at the School of Medicine has found that a blend of natural and man-made materials works best to create a better bone replacement with 3D printing technology. Warren Grayson, an affiliated member of Johns Hopkins Institute for NanoBioTechnology, reports his findings in the journal ACS Biomaterials Science and Engineering.

Read more below.


A sample 3-D printed scaffold that matches the lower jaw of a female patient. (Credit: Johns Hopkins Medicine)

To make a good framework for filling in missing bone, mix at least 30 percent pulverized natural bone with some special man-made plastic and create the needed shape with a 3-D printer. That’s the recipe for success reported by researchers at The Johns Hopkins University in a paper published April 18 online in ACS Biomaterials Science & Engineering.

Each year, the Johns Hopkins scientists say, birth defects, trauma or surgery leave an estimated 200,000 people in need of replacement bones in the head or face. Historically, the best treatment required surgeons to remove part of a patient’s fibula (a leg bone that doesn’t bear much weight), cut it into the general shape needed and implant it in the right location. But, according toWarren Grayson, Ph.D., associate professor of biomedical engineering at the Johns Hopkins University School of Medicine and the report’s senior author, the procedure not only creates leg trauma but also falls short because the relatively straight fibula can’t be shaped to fit the subtle curves of the face very well.

That has led investigators to 3-D printing, or so-called additive manufacturing, which creates three-dimensional objects from a digital computer file by piling on successive, ultrathin layers of materials. The process excels at making extremely precise structures — including anatomically accurate ones — from plastic, but “cells placed on plastic scaffolds need some instructional cues to become bone cells,” says Grayson. “The ideal scaffold is another piece of bone, but natural bones can’t usually be reshaped very precisely.”

In their experiments, Grayson and his team set out to make a composite material that would combine the strength and printability of plastic with the biological “information” contained in natural bone.

They began with polycaprolactone, or PCL, a biodegradable polyester used in making polyurethane that has been approved by the FDA for other clinical uses. “PCL melts at 80 to 100 degrees Celsius (176 to 212 Fahrenheit) — a lot lower than most plastics — so it’s a good one to mix with biological materials that can be damaged at higher temperatures,” says Ethan Nyberg, a graduate student on Grayson’s team.

PCL is also quite strong, but the team knew from previous studies that it doesn’t support the formation of new bone well. So they mixed it with increasing amounts of “bone powder,” made by pulverizing the porous bone inside cow knees after stripping it of cells.

“Bone powder contains structural proteins native to the body plus pro-bone growth factors that help immature stem cells mature into bone cells,” Grayson says. “It also adds roughness to the PCL, which helps the cells grip and reinforces the message of the growth factors.”

The first test for the composite materials was printability, Grayson says. Five, 30 and 70 percent bone powder blends performed well, but 85 percent bone powder had too little PCL “glue” to maintain clear lattice shapes and was dropped from future experiments. “It was like a chocolate chip cookie with too many chocolate chips,” Nyberg says.

To find out whether the scaffolds encourage bone formation, the researchers added human fat-derived stem cells taken during a liposuction procedure to scaffolds immersed in a nutritional broth lacking pro-bone ingredients.

After three weeks, cells grown on 70 percent bone powder scaffolds showed gene activity hundreds of times higher in three genes indicative of bone formation, compared to cells grown on pure PCL scaffolds. Cells on 30 percent bone powder scaffolds showed large but less impressive increases in the same genes.

After the scientists added the key ingredient beta-glycerophosphate to the cells’ broth to enable their enzymes to deposit calcium, the primary mineral in bone, the cells on 30 percent scaffolds produced about 30 percent more calcium per cell, while those on 70 percent scaffolds produced more than twice as much calcium per cell, compared to those on pure PCL scaffolds.

Finally, the team tested their scaffolds in mice with relatively large holes in their skull bones made experimentally. Without intervention, the bone wounds were too large to heal. Mice that got scaffold implants laden with stem cells had new bone growth within the hole over the 12 weeks of the experiment. And CT scans showed that at least 50 percent more bone grew in scaffolds containing 30 or 70 percent bone powder, compared to those with pure PCL.

“In the broth experiments, the 70 percent scaffold encouraged bone formation much better than the 30 percent scaffold,” says Grayson, “but the 30 percent scaffold is stronger. Since there wasn’t a difference between the two scaffolds in healing the mouse skulls, we are investigating further to figure out which blend is best overall.”

Although the use of “decellularized” cow bone has been FDA-approved for clinical use, in future studies, the researchers say, they hope to test bone powder made from human bone since it is more widely used clinically. They also want to experiment with the designs of the scaffolds’ interior to make it less geometric and more natural. And they plan to test additives that encourage new blood vessels to infiltrate the scaffolds, which will be necessary for thicker bone implants to survive.

Other authors of the report include Ben Hung, Bilal Naved, Miguel Dias, Christina Holmes, Jennifer Elisseeff and Amir Dorafshar of the Johns Hopkins University School of Medicine.

This work was supported by the National Institute of Dental and Craniofacial Research (F31 DE024922), the Russell Military Scholar Award, the Department of Defense, the Maryland Stem Cell Research Fund and the American Maxillofacial Surgery Society Research Grant Award.

Press release by Catherine Gara; 443-287-2251; and Shawna Williams; 410-955-8236;

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Researchers honored with Presidential career awards

Two Johns Hopkins researchers were honored by the White House for their research achievements, including one biomedical engineer affiliated with Johns Hopkins Institute for NanoBioTechnology (INBT).

Namandje Bumpus, Ph.D., and Jordan Green, Ph.D., of the Johns Hopkins University School of Medicine are among 105 winners of Presidential Early Career Awards for Scientists and Engineers, which were announced by the White House on Feb. 18. The awards recognize young researchers who are employed or funded by federal agencies “whose early accomplishments show the greatest promise for assuring America’s pre-eminence in science and engineering and contributing to the awarding agencies’ missions,” according to a White House statement.

“These early-career scientists are leading the way in our efforts to confront and understand challenges from climate change to our health and wellness,” President Barack Obama said in the statement. “We congratulate these accomplished individuals and encourage them to continue to serve as an example of the incredible promise and ingenuity of the American people.”

Namandje Bumpus, left, and Jordan Green. CREDIT Keith Weller, Johns Hopkins Medicine

Namandje Bumpus, left, and Jordan Green.
Keith Weller, Johns Hopkins Medicine

Bumpus, an associate professor of medicine and of pharmacology and molecular sciences, also serves as the school of medicine’s associate dean for institutional and student equity. Her research focuses on how the body processes HIV medications, converting them into different molecules, and the actions of those molecules. In recent studies, she has found genetic differences in how people process popular HIV drugs, suggesting genetic testing should have a greater role to play in combating the virus. “Since joining Johns Hopkins in 2010, Namandje has made tremendous progress toward ultimately making HIV treatment more personalized and effective,” says Mark Anderson, M.D., Ph.D., director of the Department of Medicine. “This is a well-deserved recognition of her work, and I look forward to seeing how she will continue to advance the field.”

Green, an associate professor of biomedical engineering, neurosurgery, oncology and ophthalmology, and a member of INBT, was named one of Popular Science’s Brilliant Ten in 2014. He develops nanoparticles that could potentially deliver therapeutics to the precise place in the body where they’re needed — to make tumor cells self-destruct, for example, while leaving healthy cells intact. “Jordan’s innovations and productivity are exceptional, and his findings have very exciting implications for patients,” says Leslie Tung, Ph.D., interim director of the Department of Biomedical Engineering. “He is truly an extraordinary and exemplary early-career scientist, and a wonderful colleague as well.”

The 105 award winners will be recognized at a White House ceremony this spring.

Source: Johns Hopkins Medicine

Symposium speakers 2015: Jordan Green

Neuro X is the title and theme for the May 1 symposium hosted by Johns Hopkins Institute for NanoBioTechnology. The event kicks off with a continental breakfast at 8 a.m. in the Owens Auditorium, between CRB I and CRB II on the Johns Hopkins University medical campus. Talks begin at 9 a.m. Posters featuring multidisciplinary research from across many Hopkins divisions and departments will be on display from 1 p.m. to 4 p.m.

One of this year’s speakers is Jordan J. Green, PhD.

Jordan Green, PhD

Jordan Green, PhD

Jordan Green is an associate professor in the Department of Biomedical Engineering at Johns Hopkins University. He graduated from Carnegie Mellon University with a bachelor’s degree in Biomedical Engineering, Chemical Engineering and then attended Massachusetts Institute of Technology to earn his doctorate in Biological Engineering. Green joined the Johns Hopkins faculty in 2008 His research focuses on cellular engineering and nanobiotechnology, with special interests in biomaterials, controlled drug delivery, and gene therapy. The potential of gene therapy and genetic medicine to benefit human health is tremendous as almost all human diseases have a genetic component, from cancer to cardiovascular disease. Methods for drug and gene delivery that are both safe and effective have remained elusive. New insights into understanding and controlling the mechanisms of delivery are required to further advance the field. To accomplish this, Green’s research team is developing a framework where biomaterials and nanoparticles can be rationally designed and computationally modeled. These same biomedical insights can also be used more broadly in the fields of regenerative medicine and nanomedicine.

Dr. Green is working at the chemistry/biology/engineering interface to answer fundamental scientific questions and create innovative technologies and therapeutics that can directly benefit human health. In 2014, Dr. Green was named one of Popular Science magazine’s “Brilliant Ten” list, highlighting young scientists who are revolutionizing their fields. He is also a member of the USA Science and Engineering Festival’s Nifty Fifty, which includes 200 of the most dynamic scientists and engineers in the United States who were selected for their unique ability to inspire the next generation of students to pursue careers in the STEM fields. He and Dr. Alfredo Quiñones-Hinojosa recently won a BioMaryland Center Biotechnology Development Award to advance their work on a biodegradable nanoparticle therapy enabling effective transfection of a patient’s stem cells derived from adipose tissue that are applied directly to the post-operative site of brain cancer.

Additional speakers will be profiled in the next few weeks. To register your poster and for more details visit

For all press inquiries regarding INBT, its faculty and programs, contact Mary Spiro, or 410-516-4802.




Podcast: Shaping polymers for biomedical use

In this edition of the Johns Hopkins Institute for NanoBioTechnology Nanobyte podcast, Hai-Quan Mao, professor of materials science and engineering at Johns Hopkins University, discusses his work with polymers and their potential applications for medicine.

Slide1 In the Mao lab, researchers are using multi-molecule structures called polymers and forming them into different shapes for biomedical applications such as tissue engineering, nerve regeneration, and drug delivery. Mao uses natural models, such virus, as shape templates for designing nanoparticles with specific capabilities.

Listen to the podcast on Mixcloud here.

Visit the Mao Research Group here.

For all press inquiries regarding INBT, its faculty and programs, contact Mary Spiro, or 410-516-4802.

Engineering bacteria for medical uses

According to the National Institutes of Health and Centers for Disease Control, drug resistant pathogens are responsible for 2 million illnesses, 23,000 premature deaths, and an estimated $20 billion dollars in health care costs per year (1,2). The rapid emergence of drug resistant pathogens threatens to undo nearly a century’s worth of biomedical advances, and the situation has become so dire that President Obama has recently made fighting antibiotic resistant pathogens a top national priority.

Engineering Bacteria

Figure 1. Azido modified KDO was used to metabolically glycoengineer the LPS core of E. Coli

Newly emerging molecular engineering techniques may lead the way for next generation therapies designed to attack resistant microbes. One such strategy is metabolic glycoengineering, which is using unnatural monosaccharides to intercept the metabolic machinery of a cell to artificially install chemical “handles” on the surface. These chemical handles can then be exploited by performing reactions known as “click chemistry” to connect almost anything a researcher can think of to the surface of any cell.

Some of the most important structures of bacteria such as the peptidoglycan layer, lipopolysaccharides (LPS), teichoic acids, and capsule are comprised of extensive amounts of carbohydrates. Using glycoengineering, a physician may one day be able engineer those structures with unnatural monosaccharides to disrupt the adhesive properties, directly image, or target drugs to bacteria in a species specific manner–an unprecedented level of selectivity currently unachievable with our current regimen of antibiotics (Fig. 1).

For further reading:

About the Author: Christopher Saeui is a fourth year Biomedical Engineering PhD student in the Kevin J. Yarema Laboratory for Cell and Carbohydrate Engineering studying the epigenetic and metabolic mechanisms that alter glycosylation in cancer.


REU student profile: Christopher Glover

Christopher Glover is a rising senior in bioengineering at the University of Missouri. He worked this summer as an REU intern in the laboratory of professor Jeff Tza-Huei Wang, who has joint appointments in mechanical engineering, biomedical engineering and oncology. The Research Experience for Undergraduates, hosted by Johns Hopkins Institute for NanoBioTechnology, attracts nearly 800 undergraduate applicants for just 10 research positions.

Christopher Glover

Christopher Glover

Christopher’s project involved a proof-of-concept experiment to test a device used to digitally sort and amplify DNA samples.

The device consists of a silicone chip imprinted with 3,000 tiny wells to contain DNA. A thermoplastic lid covers the top of the chip to keep the DNA in place in the wells. After a segment of DNA is added to the chip, the number of copies of that DNA segment is amplified using a device called a thermal cycler. “The goal is to either get zero or one copy of the DNA segment in each well, which makes the device “digital,” he said.

“We aren’t concerned about the type of DNA we are amplifying but just to see if it will work,” Christopher said. “This could be used for medical screening where a specific allele could be detected within a gene to see if someone is more susceptible to getting a disease,” he said.

Christopher said that working in the Wang lab has helped him learn much more about nanotechnology than he had previously known. His future plans include earning a PhD in biomedical engineering.

For all press inquiries regarding INBT, its faculty and programs, contact Mary Spiro, or 410-516-4802.

REU student profile: Rebecca Majewski

DNA, the genetic sequence that tells cells what proteins to manufacture, typically resides inside the nucleus of a cell, but not always. Rebecca Majewski is studying the uptake of DNA into cell nuclei using a different polymer chains. Rebecca is a rising senior in BioMolecular Engineering from the Milwaukee School of Engineering and is working as a summer intern in the Johns Hopkins Institute for Nanobiotechnology’s REU program.

“We are interested in how much of the DNA with the polyplex can get into the nucleus,” she said, but explains that DNA associated outside of the nucleus can cause false higher measurements.

Rebecca Majewski. Photo by Mary Spiro

Rebecca Majewski. Photo by Mary Spiro

Rebecca is washing the cells with the nuclei to get rid of DNA outside the nucleus and then comparing the measurement of uptake of the DNA by the cell versus the measurement of the uptake of DNA by the nucleus.

“We are interested in what DNA gets inserted into the nucleus because that is what is ultimately expressed. It is important to find out how much makes it to the final destination and then is expressed. The goal of this work is to test different polymer chains to see which one actually does the better job of getting the DNA into the nucleus,” she said.

Rebecca works alongside PhD students and postdoctoral fellows in the biomedical engineering lab of Jordan Green lab at the Johns Hopkins School of Medicine. She says she highly values the opportunity for a research experience through INBT’s REU because her undergraduate institution does not train graduate students.

For all press inquiries regarding INBT, its faculty and programs, contact Mary Spiro, or 410-516-4802.

REU student profile: Ian Reucroft

Sitting at what looks like a pottery wheeled turned on its side, Ian Reucroft is using a method called electrospinning to create a nano-scale polymer fiber embedded with a drug that encourages nerve growth. The strand is barely visible to the eye, but the resulting fibers resemble spider web.

Ian Reucroft, a rising junior in Biomedical Engineering at Rutgers University, is working in the medical school campus laboratory of Hai-Quan Mao, professor of materials sciences and engineering at Johns Hopkins University. He is part of Johns Hopkins Institute for NanoBioTechnology’s summer REU, or research experience for undergraduates program.

Ian Reucroft in the Mao lab. Photo by Mary Spiro.

Ian Reucroft in the Mao lab. Photo by Mary Spiro.

“We are developing a material to help regrow nerves, either in central or peripheral nervous systems,” said Ian. One method of doing that he explained is to make nanofibers and incorporating a drug into those fibers, drugs that promote neuronic growth or cell survival or various other beneficial qualities. The Mao lab is looking into a relatively new and not well-studied drug called Sunitinib that promotes neuronal survival.

“We make a solution of the component to make the fiber, which is this case is polylactic acid (PLA), and the drug, which I have to dissolve into the solution,” Ian said. Although the drug seems to remain stable in solution, one of the challenges Ian has faced has been improving the distribution of the drug along the fiber.

This is Ian’s first experience with electrospinning but not his first time conducting research. He plans to pursue a PhD in biomedical engineering and remain in academia.

For all press inquiries regarding INBT, its faculty and programs, contact Mary Spiro, or 410-516-4802.

Konstantopoulos to present distinguished lecture on tumor cell migration

Biomedical Engineering 8 5 x 11 4-7Biomedical Engineering 8 5 x 11 4-7Biomedical Engineering 8 5 x 11 4-7Professor and Chair of the Department of Chemical and Biomolecular Engineering, Konstantinos Konstantopoulos will present a distinguished lecture for the Department of Biomedical Engineering on Monday, April 7 at 4 p.m. in the Mason Hall Auditorium on the Homewood campus of Johns Hopkins University.  His talk. “Joining Forces with Biology: A Bioengineering Perspective on Tumor Cell Migration,” will reveal some of his laboratory’s current findings on metastasis. The talk is free and open the Johns Hopkins University community. Refreshments follow the lecture.

Here’s the abstract of his talk:

“Understanding the mechanisms of cell migration is a fundamental question in cell, developmental and cancer biology. Unraveling key, physiologically relevant motility mechanisms is also crucial for developing technologies that can control, manipulate, promote or stop cell migration in vivo. Much of what we know about the mechanisms of cell migration stems from in vitro studies using two-dimensional (2D) surfaces. Cell locomotion in 2D is driven by cycles of actin protrusion, integrin-mediated adhesion and myosin-dependent contraction. A major pitfall of 2D assays is that they fail to account for the physical confinement that cells  encounter within the physiological tissue environment. The seminar will challenge the conventional wisdom regarding cell motility mechanisms, and show that migration through physically constricted spaces does not require beta1 integrin dependent adhesion or myosin contractility. Importantly, confined migration persists even when filamentous actin is disrupted. This seminar will also discuss a novel mechanism of confined cell migration based on an osmotic en

Nanotechnology for gene therapy

Editor’s Note: The following is a summary of one of the talks from the 2013 Nano-bio Symposium hosted by Johns Hopkins Institute for NanoBioTechnology held May 17. This summary was written by Randall Meyer, a doctoral candidate in the biomedical engineering and a member of the Cancer Nanotechnology Training Center. Look for other symposium summaries on the INBT blog.

One of the key features of nanotechnology is its wide range of applicability across multiple biological scenarios ranging from gene therapy to immune system modulation. Jordan Green, an assistant professor of Biomedical Engineering at Johns Hopkins University, summarized some of the fascinating applications of nanotechnology on which his laboratory has been working. Green is an INBT affiliated faculty member.

One of the Green lab projects involves the design and implementation of nanoparticle based vectors for delivery of genetic material to the cell. Green demonstrated how these particles could be used to deliver DNA and induce expression of a desired gene, or small interfering RNA (siRNA) to silence the expression of a target gene. These genetic therapeutics are being developed to target a wide variety of retinal diseases and cancers.

Jordan Green (Photo by Marty Katz)

Jordan Green (Photo by Marty Katz)


As opposed to viral based vectors for gene therapy, nonviral vectors such as nanoparticles are safer, more flexible in their range of cellular targets, and can carry larger cargoes than viruses, Green explained.


Another project in the Green lab involves the development of micro and nano dimensional artificial antigen presenting cells (aAPCs) for cancer immunotherapy. These aAPCs mimic the natural signals that killer T-cells receive when there is an invader (bacteria, virus, cancer cell, etc.) in the body. The Green lab is currently working with these particles to stimulate the immune system to fight melanoma.


Green Group