3-D Printing to Make Replacement Body Parts


Advances in three-dimensional (3D) printing have produced a swell of interest in artificial organs that are designed to replace, or even enhance, human tissues.

3-D printed organs

At the Inside 3D Printing conference in New York on April 15–17, 2015, researchers from academia and industry are gathering to discuss the growing interest in using three-dimensional (3D) printing to make replacement body parts. Although surgeons are already using 3D-printed metal and plastic implants to replace bones, researchers are looking ahead to printing organs using cells as “ink.”  All the structures shown here were all 3D printed at Wake Forest Baptist Medical Center in Winston-Salem, North Carolina, and include a rudimentary proto-kidney (top left), complete with living cells.

Printed organs, such as a prototype outer ear that was developed by researchers at Princeton University in New Jersey and Johns Hopkins University in Baltimore, Maryland, will be featured at the conference.  This ear is printed from a range of materials: a hydrogel to form an ear-shaped scaffold, cells that will grow to form cartilage, and silver nanoparticles to form an antenna (see M.S. Mannoor et al. Nano Lett. 13, 2634−2639; 2013. This is just one example of the increasing versatility of 3D printing.

This New York meeting, which is being advertised as the largest event in the industry, will provide exposure for a whole world of devices and novelties. But it will also feature serious discussions on the emerging market for printed body parts.

The dream of bioprinting is to print organs that can be used for transplant. For example, at the Wake Forest Baptist Medical Center in Winston-Salem, North Carolina, researchers are developing a 3D-printed kidney. The project is in its early stages and the kidney is far from functional and some doubt that researchers will ever be able to print such a complex organ. Perhaps a more achievable near-term goal might be to print sheets of kidney tissue that could be grafted onto existing kidneys.

Printed replacement for skull

Printed structures made of hard metal or polymers are already on the market for people in need of an artificial hip, finger bone or facial reconstruction. This skull implant (grey) was made by Oxford Performance Materials of South Windsor, Connecticut, and was approved by US regulators in 2013. It is made of a polymer meant to encourage bone growth, to aid integration of the implant into the surrounding skeleton. The company also sells implants for facial reconstruction and for replacing small bones in the feet and hands.

3-D printed lung tree

One of the key advantages of using 3D printing for surgical implants is the opportunity to model the implant to fit the patient. This airway splint (shown on the right branch of the model trachea) was designed by researchers at the University of Michigan in Ann Arbor to fit an infant with a damaged airway. The splint was made out of a material that is gradually absorbed by the body as the airway heals. The research team benefited from the concentration of 3D-printing expertise that has built up in Michigan because of the US automobile industry, which uses the technology for printing prototypes and design samples.

The business of 3-D printing also includes titanium replacement hip joints, which can be tailored to fit individual people, and made-to-order polymer bones to reconstruct damaged skulls and fingers. Printed body parts brought in US $537 million last year, up about 30% on the previous year, says Terry Wohlers, president of Wohlers Associates, a business consultancy firm in Fort Collins, Colorado, that specializes in 3D printing.

3-D printed prosthetics

3D printing can also be used to generate cheap — and creative — prostheses.  A prosthetic hand can cost thousands of dollars, which is a burdensome expense when fitting it to a growing child.  Jon Schull founded a company called e-NABLE that provides free printed prosthetics to those in need, harnessing the efforts of hundreds of volunteers who own consumer-grade 3D printers. “When people get tired of printing Star Wars figurines, they give us a call,” he says.  The cost of materials for a printed prosthesis is about US $35.

3-D animal prosthesis

Also, 3-D printed prostheses are not just for humans.  For example, a duck named Buttercup was born with its left foot turned backwards.  The Feathered Angels Waterfowl Sanctuary in Arlington, Tennessee, arranged for the fowl to receive a new foot, complete with a bendable ankle.  Also in the an eagle, a box turtle and a handful of dogs also have been fitted with 3-D printed prostheses.

Scientists are looking ahead to radical emerging technologies that use live cells as ‘ink’, assembling them layer-by-layer into rudimentary tissues, says Jennifer Lewis, a bioengineer at Harvard University in Cambridge, Massachusetts. Bioprinting firm Organovo of San Diego, California, already sells such tissues to researchers aiming to test experimental drugs for toxicity to liver cells. The company’s next step will be to provide printed tissue patches to repair damaged livers in humans, says Organovo’s chief executive, Keith Murphy.

Lewis hesitates to say that 3D printing will ever yield whole organs to relieve the shortage of kidneys and livers available for transplant. “I would love for that to be true,” she says. “But these are highly complicated architectures.”

Induced Pluripotent Stem Cells from Bone Cancer Patients Provide Crucial Insights into the Genesis of Bone Cancer


A team of Mount Sinai researchers have utilized induced pluripotent stem cells (iPSCs) to elucidate the genetic changes that seem to convert a well-known anti-cancer signaling gene into a driver bone cancers. When it comes to bone cancers, the survival rate has not improved in 40 years despite advances in treatment. Since this study might provide new targets and suggest new strategies for attacking such cancers. it represents a welcome addition to the cancer literature.

This study, which was published in the journal Cell, revolves around iPSCs, which were discovered in 2006 by Nobel laureate Shinya Yamanaka. iPSCs use genetic engineering and cell culture techniques to reprogram mature, adult cells to become like embryonic stem cells. These iPSCs are “pluripotent,” which means that they are able to differentiate into any adult cell type and can also divide in culture indefinitely.

For therapeutic purposes, iPSCs can be derived from a patient’s own cells, differentiated into the cells the patient needs to be replaced, and then implanted into the patient’s body to augment tissue healing or even organ reconstruction. Since iPSCs can be successfully differentiated into heart muscle, nerve cells, bone, and other cell types, they have the potential advance the field of regenerative medicine by leaps and bounds.

iPSCs have already made their presence known in the clinic by serving as model systems for research and diagnosis. The new Mount Sinai study used iPSCs to construct an accurate model of a genetic disease “in a dish.” The culture dishes contain self-renewing patient-specific iPSCs or a specific cell line that enable in-depth study diseases that are driven by each person’s genetic differences. When matched with patient records, iPSCs and iPSC-derived target cells have the ability to help physicians predict a patient’s prognosis and whether or not a given drug will be effective for him or her.

In this study, skin cells from healthy patients and patients with a genetic disease called Li-Fraumeni syndrome were isolated and reprogrammed into patient-specific iPSC lines. These iPSCs were then differentiated into bone-making cells (osteoblasts), which are the cells where particular rare and common bone cancers start. Li-Fraumeni syndrome greatly predisposes patients to a variety of cancers in several different types of tissues.

The patient-derived osteoblasts were then tested for their tendencies to become tumor cells and to make bone. This particular bone cancer model did a better job of recapitulating the characteristics of bone cancer than previously used mouse or cellular models.

LFS iPSCs for stem cell production

“Our study is among the first to use induced pluripotent stem cells as the foundation of a model for cancer,” said lead author Dung-Fang Lee, PhD, a postdoctoral fellow in the Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai. “This model, when combined with a rare genetic disease, revealed for the first time how a protein known to prevent tumor growth in most cases, p53, may instead drive bone cancer when genetic changes cause too much of it to be made in the wrong place.”

The Mount Sinai disease model research uses a simple fact of human life as its basis: human genes undergo mutations at a certain rate that tends to increase as we age, and the formation of new mutations in relentless and constant. Some mutations make no difference, a few some confer advantages, and others cause disease. Beyond inherited mutations that contribute to cancer risk, the combination of random, accumulated DNA changes in our cells as we age also increase our cancer risk.

The current study focused on those genetic pathways involved in Li-Fraumeni Syndrome or LFS, a rare genetic disease that causes high risk for many cancers in affected families. Osteosarcoma (bone cancer) is a common cancer observed in LFS patients and many of them are diagnosed before the age of 30. Additionally, osteosarcoma is the most common type of bone cancer in all children, and after leukemia, the second leading cause of cancer death for them.

 

Importantly, about 70 percent of LFS families have a mutation in their copy of a genes called TP53, which encodes the p53 protein. P53 is a “the tumor suppressor,” which means that it functions to preserve the integrity of the genome and keep the rate of cell division in check. Common forms of osteosarcoma, which are driven by somatic or inherited mutations, have also been closely linked by past studies to defects in p53 when mutations interfere with the ability of the protein to function properly.

p53

Crystal Structure of p53 protein bound to DNA

 

Rare genetic diseases like LFS provide excellent model systems because they tend to result from a change in a single gene, instead of the diverse and overlapping mutations observed in common diseases, and, in this case, more common, non-inherited bone cancers. The LFS-iPSC based modeling highlights the contribution of p53 alone to osteosarcoma.

By analyzing iPSC lines, and bone cancer driven by p53 mutations in LFS patients, the Munt Sinai research team showed, for the first time, that the LFS bone cancer results from an overactive p53 gene. Too much p53 in osteoblasts dampens the function of a gene, H19, and a related protein, decorin, that would otherwise help stem cells differentiate into normal osteoblasts.

The inability of cells to differentiate makes them vulnerable to genetic mistakes that drive cancer, since more “stemness” means a tendency toward rapid, abnormal growth, like that observed in tumors. One tragic feature of osteosarcoma is the rapid, error-prone production of weaker bone by cancerous bone-making cells, where a young person surprisingly breaks a bone to reveal undiagnosed, advanced cancer.

Dung-Fang Lee and his colleagues discovered that the H19 gene seems to control a network of interconnected genes that fine-tune the balance between cell growth and resistance to growth. Decorin is a protein that is part of connective tissue like bone, but that also plays a signaling role, interacting with growth factors to slow the rate that cells divide and multiply, unless turned off by too much p53.

“Our experiments showed that restoring H19 expression hindered by too much p53 restored “protective differentiation” of osteoblasts to counter events of tumor growth early on in bone cancer,” said co-author, Ihor Lemischka, PhD, Director of The Black Family Stem Cell Institute within the Icahn School of Medicine. “The work has implications for the future treatment or prevention of LFS-associated osteosarcoma, and possibly for all forms of bone cancer driven by p53 mutations, with H19 and p53 established now as potential targets for future drugs.”

A New Way to Prepare Fat-Based Stem Cells to Treat Wounds


An Italian laboratory headed by Dr. Raposio at the University of Parma has designed a simple and fast technique for preparing fat-based stem cells for use in the clinic.

Fat contains an alternative source of mesenchymal stem cells with characteristics similar to those found in bone marrow, but the fat-based stem cells are easier to isolate and have been shown to be effective enhancers of wound healing.

Raposio and his colleagues used fat contributed by liposuction patients. Each patient provided about 80 cubic centimeters of fat in liposuction procedures that were collected under anesthesia. Once the cells from this fat were isolated, they were mixed with platelet-rich plasma (PRP) that had been previously collected. Mixing PRP with stem cells enhances the capabilities of the fat-based stem cells and generates a concoction called “e-PRP.”  This simple procedure that consisted of fat collection, stem cell collection and mixing the cells with PRP to make e-PRP quickly made a produce that was ready for grafting onto wounds on the skin.

Detailed analyses of the cells isolated from the fat showed that they consisted of about 50,000 fat-based mesenchymal stem cells or ASCs. They represented about 5% of all cells in the sample. The remaining cells were blood-derived cell and blood vessel-making endothelial cells.

The significance of this procedure lies in the fact that most of the protocols used to isolate stem cells from fat take about two hours and require animal-derived reagents. However, the number of ASCs isolated with this new procedure is sufficient for application to wounds without the need of expanding the cells in culture. Also, this new procedure does not require serum or animal-derived reagents, and it takes only 15 minutes.

Thus this method of ASC isolation is innovative, feasible, and represents an advance in the stem cell-based treatment of chronic wounds.

Amniotic Fluid Stem Cells Make Robust Blood Vessel Networks


The growth of new blood vessels in culture received in new boost from researchers at Rice University and Texas Children’s Hospital who used stem cells from amniotic fluid to promote the growth of robust, functional blood vessels in healing hydrogels.

These results were published in the Journal of Biomedical Materials Research Part A.

Engineer Jeffrey Jacot thinks that amniotic fluid stem cells are valuable for regenerative medicine because of their ability to differentiate into many other types of cells, including endothelial cells that form blood vessels. Amniotic fluid stem cells are taken from the discarded membranes in which babies are encased in before birth. Jacot and others combined these cells with an injectable hydrogel that acted as a scaffold.

In previous experiments, Jacot and his colleagues used amniotic fluid cells from pregnant women to help heal infants born with congenital heart defects. Amniotic fluids, drawn during standard tests, are generally discarded but show promise for implants made from a baby’s own genetically matched material.

“The main thing we’ve figured out is how to get a vascularized device: laboratory-grown tissue that is made entirely from amniotic fluid cells,” Jacot said. “We showed it’s possible to use only cells derived from amniotic fluid.”

Researchers from Rice, Texas Children’s Hospital and Baylor College of Medicine combined amniotic fluid stem cells with a hydrogel made from polyethylene glycol and fibrin. Fibrin is the proteins formed during blood clots, but it is also used for cellular-matrix interactions, wound healing and angiogenesis (the process by which new vessels are made). Fibrin is widely used as a bioscaffold but it suffers from low mechanical stiffness and is degraded rapidly in the body. When fibrin was combined with polyethylene glycol, the hydrogel became much more robust, according to Jacot.

Additionally, these groups used a growth factor called vascular endothelial growth factor to induce the stem cells to differentiate into endothelial cells. Furthermore, when induced in the presence of fibrin, these cells infiltrated the native vasculature from neighboring tissue to make additional blood vessels.

When mice were injected with fibrin-only hydrogels, thin fibril structures formed. However if those same hydrogels were infused with amniotic fluid stem cells that had been induced with vascular endothelial growth factor, the cell/fibrin hydrogel concoctions showed far more robust vasculature.

In similar experiments with hydrogels seeded with bone marrow-derived mesenchymal cells, once again, vascular growth was observed, but these vessels did not have the guarantee of a tissue match. Interestingly, seeding with endothelial cells didn’t work as well as the researchers expected, he said.

Jacot and others will continue to study the use of amniotic stem cells to build biocompatible patches for the hearts of infants born with birth defects and for other procedures.

Scientists Use Stem Cells to Grow Three-Dimensional Mini Lungs.


In research done in several laboratories, lung tissue was derived from flat cell culture systems or by growing cells on scaffolds made from donated organs.

Now in a new study published in the online journal eLife, a multi-institution team has defined a culture system for generating self-organizing human lung organoids, which are three-dimensional structures that mimic the structure and complexity of human lungs.

“These mini lungs can mimic the responses of real tissues and will be a good model to study how organs form, change with disease, and how they might respond to new drugs,” said study senior author Jason R. Spence, Ph.D., an assistant professor of internal medicine and cell and developmental biology at the University of Michigan Medical School.

Spence and his colleagues successfully grew structures that resembled both the large airways or bronchi and small lung sacs, known as alveoli.

These mini lung structures were developed in a cell culture system. Therefore, they lack several components of the human lung, including blood vessels, which are a critical component of gas exchange during breathing.

Despite that, these cultured organoids can serve as a unique research model system for researchers as they grind out basic science ideas that are turned into clinical innovations. These three-dimensional mini-lungs should be an excellent complement to research in liver laboratory animals.

Traditionally, the behavior of cells has been investigated in the laboratory in two-dimensional culture systems where cells are grown in thin layers on cell-culture dishes. Most cells in the body, however, exist in a three-dimensional environment as part of complex tissues and organs. Tissue engineered have been trying to re-create these environments in the laboratory by successfully generating small version of particular organs known as organoids, which serve as models of the stomach, brain, liver and human intestine. The advantage of growing three-dimensional structures of lung tissue, according to Dr. Spence, is that the organization of organoids bears greater similarity to the human lung.

To make these lung organoids, researchers at the U-M’s Spence Lab and colleagues from the University of California, San Francisco; Cincinnati Children’s Hospital Medical Center; Seattle Children’s Hospital and University of Washington, Seattle manipulated several of the cell signaling pathways that control the formation of organs.

First, stem cells were induced to form a type of tissue called endoderm, which is found in early embryos and gives rise to the lung, liver and several other internal organs. Second, the group activated two important development pathways (FGF and WNT signaling ) that are stimulate endoderm to form three-dimensional tissue. By inhibiting two other key development pathways at the same time (BMP and TGFβ signaling), the endoderm became tissue that resembles the early lung found in embryos.

In the laboratory, this early culture-derived lung-like tissue spontaneously formed three-dimensional spherical structures as it developed. Afterwards, they had to expand these structures and develop them into lung tissue. In order to do this, Spence and his colleagues and collaborators exposed the cells to additional proteins involved in lung development (FGF and Hedgehog).

After all this manipulation, the resulting lung organoids survived in the laboratory for over 100 days.

“We expected different cells types to form, but their organization into structures resembling human airways was a very exciting result,” said author Briana Dye, a graduate student in the U-M Department of Cell and Developmental Biology.

While this type of experiment is remarkable, this is only the beginning of lung tissue engineering.  These mini-lungs  will hopefully serve and new model systems for drug testing and researching genetic diseases that affect the lungs, such as cystic fibrosis, sarcoidosis, or inherited forms of emphysema.  It will be a while before scientists can make replacement lungs for human patients, but these experiments by Spence and others are a remarkable start.

A Patient’s Own Stem Cells Treats Their Crohn’s Disease


Stem cells isolated from the fat of patients with Crohn’s disease, an inflammatory disease of the bowel, relieved them from fistulas, which are a common, and potentially dangerous side effect of the disease. This is according to the results of a phase 2 clinical trial published in the latest issue of STEM CELLS Translational Medicine (SCTM).

Patients with Crohn’s disease suffer from a painful, chronic disease in which the body’s immune system attacks its own gastrointestinal tract. In Crohn’s patients, inflammation within the bowel can sometimes extend completely through the intestinal wall and create a what is known as a “fistula.”. Fistulas are abnormal connections between the intestine and another organ or even the skin. If left untreated, a fistula can become infected and form an abscess that can be life threatening.

Chang Sik Yu, M.D., Ph.D., of the Asan Medical Center in Seoul, Korea, who is a senior author of the SCTM paper, describes the results of a clinical trial that was conducted in collaboration with four other hospitals in South Korea. According to Dr. Yu: “Crohn’s fistula is one of the most distressing diseases as it decreases patient’s quality of life and frequently recurs. It has been reported to occur in up to 38 percent of Crohn’s patients and over the course of the disease, 10 to 18 percent of them must undergo a proctectomy, which is a surgical procedure to remove the rectum.”

Overall, the treatments currently available for Crohn’s fistula remain unsatisfactory because they fail to achieve complete closure, lower recurrence of the fistulas and do not limit adverse effects, Dr. Yu said. Given the challenges and unmet medical needs in Crohn’s fistula, attention has turned to stem cell therapy as a possible treatment.

Several studies, including those undertaken by Dr. Yu’s team, have shown that mesenchymal stem cells (MSCs) do indeed improve Crohn’s disease and Crohn’s fistula. Their phase II trial enrolled 43 patients for a term of one year, over the period from January 2010 to August 2012. These patients received injections of their own fat-based MSCs, and 82 percent of them experienced complete closure of fistula eight weeks after the final ASC injection. 75 percent of the trial participants remained fistula-free two years later.

“It strongly demonstrated MSCs derived from ASCs are a safe and useful therapeutic tool for the treatment of Crohn’s fistula,” Dr. Yu said.

The latest study was intended to evaluate the long-term outcome by following 41 of the original 43 patients for yet another year. Dr. Yu reported, “Our long-term follow-up found that one or two doses of autologous ASC therapy achieved complete closure of the fistulas in 75 percent of the patients at 24 months, and sustainable safety and efficacy of initial response in 83 percent. No adverse events related to ASC administration were observed. Furthermore, complete closure after initial treatment was well sustained.”

“These results strongly suggest that autologous ASCs may be a novel treatment option for Crohn’s fistulae,” he said.

“Stem cells derived from fat tissue are known to regulate the immune response, which may explain these successful long-term results treating Crohn’s fistulae with a high risk of recurrence,” said Anthony Atala, M.D., Editor-in-Chief of STEM CELLS Translational Medicine and director of the Wake Forest Institute for Regenerative Medicine.

Dead Heart Muscle Regrown in Rodents


If you cut a piece of tissue from the heart of a salamander or zebrafish, they wild simply grow new heart tissue. Unfortunately, humans are unable to easily regenerate heart cells, and this males it difficult to recover from the permanent damage caused by heart attacks.

Fortunately, life scientists from the Weizmann Institute of Science in Israel and the Victor Chang Institute in Sydney have discovered a way to stimulate heart muscle cells in mammals to grow. This finding could have major implications for future heart attack sufferers.

Even though human blood, hair and skin cells renew themselves throughout life, cell division in the heart comes to a virtual standstill shortly after birth, according to Prof. Richard Harvey, from the Victor Chang cardiac research institute, and one of the authors of this research. Harvey said, “So there’s always been an intense interest in the mechanism salamanders and fish use which makes them capable of heart regeneration, and one thing they do is send their cardiomyocytes, or muscle cells, into a dormant state, which they then come out of to go into a proliferative state, which means they start dividing rapidly and replacing lost cardiomyocytes.”

Harvey continued: “There are various theories why the human heart can not do that, one being that our more sophisticated immune system has come at a cost, and because human cardiomyocytes are in a deeper state of quiescence, that has made it very difficult to stimulate them to divide.”

Today, for the first time in history, more people in developing countries die from strokes and heart attacks than infectious diseases. Fortunately there are cost-effective ways to save lives

By studying mice, Harvey and his colleagues found a way to overcome that regenerative barrier – at least in the rodents.

Harvey and others found that by stimulating a cell signaling pathway in the heart that is driven by a hormone called neuregulin, heart muscle cells divided in a spectacular way in both adolescent and adult mice. In humans, neuregulin expression is usually muted about one week after birth, and by about 20 weeks after birth in mice.

By triggering of the neuregulin pathway following a heart attack in mice, Harvey and others induced the replacement of lost muscle, which repaired the heart to a level close to that prior to the heart attack. Harvey said that he and other scientists should be able to determine with in the next five years if it is possible to replicate these results in humans.

“This is such a significant finding that it will harness research activities in many labs around the world, and there will be much more attention now on how this neuregulin-response could be maximised,” Harvey said.

“We will now examine what else we can use, other than genes, to activate that pathway, and it could be that there are already drugs out there, used for other conditions and regarded as safe, that can trigger this response in humans.”

When one of the blood vessels that provide blood to the heart muscle becomes blocked, the patient suffers a heart attack. Heart attacks or “myocardial infractions” cause billions of cardiomyocytes to die. Even if you survive a heart attack, you usually experience diminished quality of life because of it.

“The dream is that one day we will be able to regenerate damaged heart tissue, much like a salamander can regrow a new limb if it is bitten off by a predator,” Harvey said.

Molecular biologist Gabriele D’Uva lead this research, which was published in the scientific journal Nature Cell Biology.