Body by Science

Ned Shaw Margaret Atwood's novel Oryx and Crake describes a gruesome future for organ transplantation: Pigoons, genetically altered pigs that grow surplus human organs. Though this scenario may never come to pass, it is easy to see why the science of human replacement parts ignites the dystopian imagination: It was not too long ago that Charles Vacanti of the University of Massachusetts and coworkers injected a polymer scaffold seeded with cartilage cells into the back of the mouse and created

By | October 6, 2003

Ned Shaw

Margaret Atwood's novel Oryx and Crake describes a gruesome future for organ transplantation: Pigoons, genetically altered pigs that grow surplus human organs. Though this scenario may never come to pass, it is easy to see why the science of human replacement parts ignites the dystopian imagination: It was not too long ago that Charles Vacanti of the University of Massachusetts and coworkers injected a polymer scaffold seeded with cartilage cells into the back of the mouse and created an eerie-looking, but otherwise intact, mouse with a human ear growing out of its back.1

If today's tissue engineers have their way, transplantable human organs, or organ substitutes, may instead be grown in laboratory bioreactors in the next 20 to 30 years. Engineered skin and cartilage substitutes already are available to patients, and a wide range of engineered human body parts, including teeth, bladders, and blood vessels, are in the works. "For almost every tissue of the body you can engineer at least a rudimentary component of such organic tissues that look and behave like the tissues you are trying to create," says Peter Johnson, coeditor of the journal Tissue Engineering and CEO of Pittsburgh-based TissueInformatics.

The tools of today's tissue engineers seem deceptively simple: modified polymers, stem cells, and inkjet printers. Yet significant technical hurdles remain. And, if engineered parts are ever to find their way into patients in large numbers, researchers must find a way to make them cost-effective.

"I don't see anything else that is going to be able to redefine tissue repair and transplantation the way tissue engineering can," says Gail Naughton, president and chief operating office for the now-defunct tissue-engineering firm Advanced Tissue Sciences, La Jolla, Calif. "We just have to have the time to figure out how to get the product specifications right and how to go and manufacture the tissues in a cost-effective way."

DESKTOP ORGAN PRINTING Far-fetched as it may seem, several researchers are finding some success building organs from the ground up using desktop printing technology. One multidisciplinary team from the Medical University of South Carolina, Clemson University, and the University of Missouri, Columbia recently described the use of a modified inkjet printer to print a "bio-ink" composed of cell aggregates into a three-dimensional, biodegradable polymer gel.2

The work is a variation of three-dimensional printing, a rapid prototyping technology in which a computer model of a structure guides the formation or "printing" of an object, such as a tissue scaffold, layer by layer. Cells and growth factors can then be seeded on the scaffold and grown to produce functional tissue. The problem, says Vladimir Mironov, Medical University of South Carolina, is that this approach has not been shown to work with large tissues that require a complex vascular network. To solve this problem, the scientists came up with a workaround: Rather than printing a scaffold for cells, they printed living cells and relied on cell biology to guide formation of the desired structures.

Embryonic tissues exhibit a fundamental property called viscoelasticity, which allows cell aggregates in close proximity to flow together and fuse. Gabor Forgacs, one of the papers' authors, has studied tissue liquidity extensively and reasoned that if the cell aggregates were able to fuse under the conditions of printing, they would be able to form three-dimensional organ structures.

Courtesy of Karoly Jakab
 BUT CAN IT PRINT IN COLOR? (A and B) Two views of a tissue printer cartridge, a glass micro-pipette with a 500-micron inner diameter. A computer-controlled piston expels one aggregate at a time into the collagen gel. (C) Initial printed configuration of 10 Chinese hamster ovary cell aggregates. (D) Fused configuration after 72 hours.

"That's exactly what happened," says Mironov. "If you placed aggregates close to each other in [a] geometrical structure like ring or tube, they fused," creating ringed homocellular aggregates (see figure at right). This research represents the first stage in the process of actually printing an entire organ, Mironov says; the next stage will involve the use of heterocellular aggregates, or "multicolor" bio-ink.

Tissue engineers still must ultimately find a way to print vascular networks, as newly fashioned organs will neither survive nor function without adequate oxygenation and nutrients. Though functional human blood vessels have been developed--Laura Niklason and Chris Counter of Duke University recently grew arteries in vitro from human vascular cells3--such vessels are unable to vascularize bioengineered organs. A recent collaboration between researchers at Massachusetts Institute of Technology (MIT), Harvard Medical School, and the Charles Stark Draper Laboratory in Cambridge, Mass., though, represents a step in this direction.

The team combined computer modeling of vascular networks with microfabrication of biodegradable polymer films to engineer microfluidic channels akin to blood vessels. Mohammad Kaazempur-Mofrad of MIT, who presented the work at the American Society for Microbiology's Conference on Bio-, Micro-, and Nanosystems last July, says his team has seen promising results. The devices have lasted as long as one week when implanted in rats, and two weeks in vitro.

BUILDING BETTER PRINTERS Some re-searchers are working to develop faster, gentler, and more precise printing technologies. Forgacs, for example, is collaborating with Stillwater, Okla.-based Sciperio to develop a new printer that can fabricate heterogeneous constructs in three dimensions. Bill Warren, president of Sciperio, says this new technology, which uses multiple dispensing nozzles, can work with a range of viscosities and print on any type of surface. The printer, called the BioAssembly Tool, can even print on moving objects such as a beating heart or breathing lung. "We can actually print inside of a person if we want to," says Warren.

Mironov's team has made progress in adding 3-D capabilities to its inkjet-based printing technology, too. "We have printed many structures, including tubes with lumens, and we have used many cell types, including endothelial and smooth muscle cells," says team member Thomas Boland, who adds that, like Sciperio's, his printer can use multiple nozzles and handle a range of viscosities and surfaces. It is also portable, and, because it is based on off-the-shelf technology, relatively inexpensive.

Using another technique, Valerie Liu, working in Sangeeta Bhatia's laboratory at the University of California, San Diego, developed complex, 3-D tissue-like structures that contained three different cell types in different layers.4 The team used photomasks to selectively solidify certain portions of a photosensitive, cell-impregnated hydrogel. By repeating the process over successive layers, they generated a solid, 3-D construct containing living cells.

It will be years before researchers will be able to create functional organs using this technique, says Bhatia, who argues that a more realistic short-term goal is the construction of pieces of functional tissue for drug screening. Nevertheless, if developments proceed as researchers hope, functional complex organ printing could soon become a reality. "I think within five years we'll be able to print realistic tubular organ structures," Forgacs says.

BANKING ON STEM CELLS The next big advances in tissue engineering will likely come from the use of stem cells, predicts David Mooney of the University of Michigan. In the last few years, he says, "the biggest thing that has changed the [tissue-engineering] landscape [is] the very significant shift to the emphasis on stem cells." Reasons include the cells' proliferative capacity and pluripotency, as well as their theoretically lower immunogenicity, Mooney says.

Tissue-engineering researchers using stem cells have made significant progress recently. Smadar Cohen and colleagues at Ben-Gurion University, Israel, used scaffolds composed of alginate, a biodegradable seaweed derivative, and fetal cardiac cells, to create a patch to prevent left ventricle remodeling after myocardial infarction. These biografts were able to integrate with the infarct area and ultimately generate new vascularization.5

Surprisingly, subsequent work showed similar results could be obtained using the scaffold alone, but others have had success with stem cells, too. Victor Dzau and colleagues at Brigham and Women's Hospital and Harvard Medical School, for instance, demonstrated that transplantation of mesenchymal stem cells engineered to overexpress the serine/threonine kinase Akt could repair infarcted myocardium and prevent left ventricle remodeling in rats.6

Scientists in Teruo Okano's laboratory at Tokyo Women's Medical University designed cardiac grafts composed solely of sheets of chicken embryonic or neonatal rat cardiomyocyte cells, without the use of scaffolds.7 The team developed a cell culture method using a temperature-responsive polymer surface to allow the release of cells by changing the temperature alone. The process avoids the disruption of cell junctions that normally occurs when cells are harvested enzymatically, and the resulting sheets maintain cell-to-cell junctions and adhesiveness. Okano and colleagues demonstrated that the cells exhibited intralayer electrical communication and beat simultaneously.

But stem cells are not the answer to every tissue-engineering problem. Duke's Niklason says her laboratory has experimented with stem cells for the last three years and has one stem cell-based publication in press, but has not focused on this area to a large extent. "We're interested in using cells as ... building blocks to build pretty large structures," Niklason explains. "We need millions and millions of cells that all act the same and work together in concert." So far, though, she has not been able to coax stem cells to convert in large numbers to the appropriate cell types needed to create those building blocks.

Nevertheless, developments in stem cell biology have spurred the growth of tissue engineering companies. Baltimore-based Osiris Therapeutics, for example, focuses on the use of adult mesenchymal stem cells for use in applications such as bone, neural, and cardiac regeneration and cartilage repair. Likewise, Paul Sharpe of King's College, London, has formed a startup company called Odontis to develop and ultimately market dental implants grown from stem cells harvested from the teeth of patients.

Sharpe's group studies how teeth develop in the embryo and has successfully coaxed stem cells from mice to form tooth precursors. He hopes to one day apply this knowledge to replace traditional dental implants in humans. "If somebody loses a tooth or teeth, we would go in and make a small incision in the gum, implant our tissue engineered explant, and that would then grow in the adult as it would if it was part of the embryo," he says.

Courtesy of Thomas Boland, Clemson University
 FIRST GENERATION TISSUE ENGINEERING: Tubes of endothelial cells embedded in an agarose hydrogel matrix. The tubes are constructed through layer-by-layer printing of circular cell patterns onto agarose "biopaper" using printers that have been modified for 3-D printing by attaching a chamber with a vertical-translation stage. Tubes reaching one inch in length have been fabricated with the modified inkjet printers.

THE BUSINESS OF TISSUE ENGINEERING Tissue engineers agree that what drives growth in the field is a steady demand for organ/tissue replacement therapies coupled with a chronically low supply of transplantable organs. As a result, organ replacement technology is a huge industry: Michael Lysaght of Brown University estimates that, worldwide, the total cost of providing organ replacement therapy is $350 billion (US) and growing at a rate of 10% per year. Cumulative private spending on tissue engineering research over the past decade, despite declines in recent years owing to the overall lackluster economy, has reached $4 billion to $5 billion, Lysaght notes. "It's a high-vision, high-concept field," he says. "It's a field that when the successful product hits, there are already markets, patterns for reimbursement, and huge opportunities for financial gains."

But it is also an industry hampered by financial failure. Two of the first companies to launch tissue engineered products filed for bankruptcy within weeks of each other at the end of 2002. Both of these companies, Organogenesis of Canton, Mass., and Advanced Tissue Sciences (ATS), marketed functional skin-replacement products engineered for treatment of burns and diabetic skin ulcers. But lower-than-anticipated sales coupled with high manufacturing costs led to profit margins that were too low to offset the high cost of FDA approval for these products.8

"We saw an industry that was really fraught with many problems, one in which the investors basically gave up, and one in which it was impossible to ... become profitable in the short term," explains ATS's Naughton, currently dean of the College of Business Administration, San Diego State University.

Yet the demise of ATS and Organogenesis does not necessarily presage the demise of the tissue engineering industry as a whole; instead, some argue, such setbacks are to be expected. "More often than not, a groundbreaking technology is not immediately accepted or widely adopted," says Lysaght. "Eventually a few blockbusters emerge and pave the way for investment acceptance of others. I believe this is exactly what you're going to see in tissue engineering, though it is not clear how long this will take."

Jason Rushton, an associate at UK-based venture capital firm Merlin Biosciences, says his company has seen many opportunities to invest in tissue engineering-related companies within the last three years and that two factors will influence continued growth in the field: overcoming the challenges of putting tissue engineered products into late-stage clinical trials, and bringing manufacturing costs into line with competing, standard therapies. These challenges are not insurmountable, and Rushton believes some of the current players in the market will ultimately succeed.

Indeed, Naughton says ATS's experiences can be viewed as a lesson for the industry. She notes that a number of the products developed by ATS have been acquired by other companies and are currently doing well on the market.

Courtesy of Anthony Atala
 BUILDING A BETTER BLADDER: An engineered bladder in a beaker.

For example, the company had developed a bioengineered human collagen for cosmetic uses to replace existing bovine collagen therapies--a patient-paid product that is now sold under the trade name CosmoDerm™ by Santa Barbara, Calif.-based Inamed. Had ATS adopted a different business model, it may have been more likely to stay afloat. "If I were going to redo ... what we did, I would have focused more on some of the marketplace that is less sensitive to reimbursement and cost," Naughton says. She adds that she also would have focused on markets in which new technologies are readily adopted by physicians, such as the cardiovascular market.

While adopting a different business strategy may help offset regulatory costs, some companies and researchers are choosing to avoid altogether these costs and other hurdles associated with meeting FDA approval. "The tissue engineering field has been dominated by the US, but more and more activity is developing overseas," says Christine Kelley of the National Institute of Biomedical Imaging and Bioengineering.

Companies that sell tissue-engineered products overseas often have a shorter time-to-market than their US-based counterparts, owing to a more flexible regulatory environment. Naughton notes, for example, that Swiss biosurgery company IsoTis, which supplies autologous skin grafts for treatment of burn wounds, was not required to conduct a pivotal clinical trial before going to market. "They've saved hundreds of millions of dollars over what traditional tissue-engineering companies had to spend here in the US to get FDA approval," says Naughton.

Overseas researchers also have an advantage in the level of government funding available to tissue engineering-related research. TissueInformatics' Johnson points out that tissue engineering in the United States began with significant financial support from private industry and less from the federal government, which limited intellectual property development to specific companies. "Other countries--England, Japan, Germany--have done quite the opposite," says Johnson. "They've made very substantial outlays to support tissue engineering, where the support for the academic side has been stronger, with relatively less support for the private financing of companies." Further, development of stem cell technologies for tissue engineering applications, particularly those based on the use of embryonic stem cells, is occurring at a more rapid pace in countries that do not limit federal funding of such research. "In stem cell development, we have the potential to undergo a brain drain here," says Johnson.

European scientists point out that US government funding for applied tissue engineering research is still high enough to give US researchers an edge. King's College's Sharpe notes that while the UK government is pouring money into stem cell biology, it still perceives applied tissue engineering research as something that should be industry-funded. "In America, it's very clear that people have latched onto tissue engineering and have created ring-fence monies to specifically fund that," he says. "In England, it's very different."

Tissue engineering research and development around the world continues, and with advances in technology and stem cell biology, the future looks bright. Even Naughton, her company burned by an unresponsive market, sees promise: "I think that you're going to really see a huge revolution in medicine because of it."

Aileen Constans can be contacted at aconstans@the-scientist.com.

References
1. Y. Cao et al., "Transplantation of chondrocytes utilizing a polymer-cell construct to produce tissue-engineered cartilage in the shape of a human ear," Plast Reconstr Surg, 100:297-302; discussion 303-4, 1997.

2. V. Mironov et al., "Organ printing: Computer-aided jet-based 3D tissue engineering," Trends Biotechnol, 21:157-61, April 2003.

3. J.A. McKee et al., "Human arteries engineered in vitro," EMBO Reports, 4:633-8, June 2003.

4. V. Liu, S.N. Bhatia, "Three-dimensional photopatterning of hydrogels containing living cells," Biomedical Microdevices, 4:257-66, 2002.

5. J. Leor et al., "Bioengineered cardiac grafts: A new approach to repair the infarcted myocardium," Circulation, 102:56-61, 2000.

6. A.A. Mangi et al., "Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts," Nat Med, E-pub ahead of print, doi:10.1038/nm912, Aug. 10, 2003.

7. T. Shimizu et al., "Cell sheet engineering for myocardial tissue reconstruction," Biomaterials, 24:2309-16, June 2003.

8. A. Stone, "FDA issues felled Advanced Tissue Sciences," Gen Eng News, 23:25, 28, June 1, 2003.

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