This article discusses biomedical engineers who are producing materials on which human skin cells can thrive to be used as grafts to treat wounds. Advanced Tissue Sciences of La Jolla, Calif., and Molecular Geodesics of Cambridge, MA, are employing not just awe-inspiring molecular and cell biology and biochemical knowledge, but the most advanced skills in computer-aided design, exacting refrigeration systems, micron-level stereolithography, and an understanding of FDA regulations. Advanced Tissue Sciences’ (ATS) ability to create the tissue involves many disciplines-not just cell biology, but also biochemistry and biomedical engineering. ATS takes stromal cells, like fibroblasts from neonatal tissue (which reproduces the fastest and is the most likely to be healthy), and expands and seeds them onto biocompatible scaffolds. Molecular Geodesics Inc. (MGI) uses laser-based rapid-prototyping technologies to render its biomimetic material designs in solid form. The geodesic scaffolds may incorporate polymeric hydrogels with solid-phase catalytic or chemosensing capabilities, optical fibers for delivery of sterilizing radiation, or any other desired chemical or structural features required by the user.
Biomedical engineers on both V.S. coasts are moving ever closer to a medical treatment once thought to be purely within the realm of Star Trek. We now have the capability to produce materials on which human skin cells can thrive to be used as grafts to treat wounds. One might be hard-pressed to find a more interdisciplinary engineering exercise.
Two companies, and soon more, are advancing the field. Advanced Tissue Sciences of La Jolla, Calif., and Molecular Geodesics of Cambridge, Mass., are employing not just awe-inspiring molecular and cell biology and biochemical knowledge, but the most advanced skills in computer-aided design, exacting refrigeration systems, micron-level stereolithography, and-not to be underestimated-an understanding of FDA regulations.
Skin is the largest single organ in the body, with a surface area ranging from 17 to 20 square feet in adults. Skin is the body's first defense against disease-causing organisms: It prevents dehydration, holds the extensive capillary networks and sweat glands , and maintains body temperature. Skin houses the nerves that receive stimuli of touch, pressure, heat, cold, and pain, and relay them to central nervous system. Skin accommodates vitamin D synthesis, which is essential for normal bone and tooth structure.
Skin cells, in all their forms, know very well how to regenerate and repair themselves in many instances. But with deep second-degree (partial thickness) or third-degree (full thickness) burns, the capacity for regeneration of tissue is very limited and can be destroyed altogether. About 5,000 people a year die from burn wounds, according to the American Burn Association.
A common health threat for diabetics is ulcers on the soles of their feet that do not heal, a condition that can become severe enough to require amputation. A replacement that could offer an alternative treatment must mimic a thick complex of skin called stratum lucidum, found only on the palms and soles. Advanced Tissue Sciences reports that some 800,000 diabetics per year develop these ulcers.
Human cadaver skin is currently the most commonly used biologic temporary covering for surgically excised thermal and chemical burns, but there are drawbacks.
Foremost is insufficient supply: The American Red Cross estimates that only one-sixth of the skin supply needed for burn victims is available from the nation's tissue banks. Other drawbacks include epidermal sloughing (requiring painful and costly removal and reapplication) and disease transmission.
Biomedical engineers have been attacking the problem for a couple of decades. They do not get easy breaks, but they have developed a means of generating replacement tissue free from most of the complications of using borrowed human skin.
Straight From the Human Cell
Gail K. Naughton, president and chief operating officer of Advanced Tissue Sciences, spoke to an ASME-sponsored gathering of congressional members and staffers last fall. She explained the work the company does.
ATS makes artificial skin, which it markets under the name Dermagraft, for treating severe wounds. The product is grown under laboratory conditions from human cells on a chemical base called a scaffold.
ATS's ability to create this tissue involves many disciplines-not just cell biology, but also biochemistry and biomedical engineering. ATS takes stromal cells, like fibroblasts from neonatal tissue (which reproduces the fastest and is the most likely to be healthy), and expands and seeds them onto biocompatible scaffolds.
The scaffolds are made of polyglycolic acids, the basis of many "resorbable" medical materials, such as surgical sutures and new surgical glues. According to Gary Gentzkow of ATS, Dermagraft is grown on Vicryl, a Johnson & Johnson trademarked blend of polylactic and polyglycolic acids. When Dermagraft is applied to the body, the scaffold breaks down into glycolic acid and lactic acid, which are carried away by the bloodstream and metabolized to carbon dioxide, oxygen, and water.
As they develop over a period of a few weeks, the cells on the scaffold are kept under optimal conditions in bioreactors. The bioreactors are challenging to build because they have to create perfect conditions to grow the implants. In them, one neonatal sample of a square inch or so yields cells sufficient for manufacturing 250,000 square feet of final product; each 3x4-inch lot produces 1,000 units of Dermagraft.
The tissue must be cryopreserved at a sufficiently low temperature to maintain sterility. Dermagraft is stored at -70DC and shipped in dry ice to the clinical site.
Tough demands are put on ATS for quality control. The tissue must be free of all pathogens. The rinse for sterility requires that the scaffold be laser z-welded to the inside of the transport bioreactor for stability. The reactor must also be translucent to allow tracing of the wound and precise trimming of the implant with sterile scissors.
Then, after cryopreservation and just before implantation, the material is tested to ensure proper metabolic activity-that is, that the cells can generate enough energy on their own for growth and reproduction. ATS found out the hard way in a long clinical trial that if tissue metabolic activity was not within a certain range, healing suffered.
ATS also makes and markets TransCyte, which is a temporary skin for partial thickness burns, to help new cells grow. Dermagraft is a total skin replacement for full thickness burns and chronic wounds like diabetic foot ulcers.
The evidence ATS provides is inspiring. Patients on whom Dermagraft is used have shown reduction of pain within 30 minutes, and can be released from the hospital within one or two days rather than the 10 to 12 typically needed for healing. Pictures show cosmetic improvement in days.
ATS has a British partner, Smith and Nephew. According to ATS, applications of skin scaffold tissue are more widely applied in Britain than in the United States because of differing medical regulations.
Getting Under the Skin
Naughton told the gathering in Washington that wound healing is just the beginning for tissue engineering. ATS has developed bioreactors and methods of producing implants for human cartilage, blood vessels', bone, ligaments, even liver and pancreatic tissue.
The cartilage produced in bioreactors can be used for orthopedic repair and facial reconstruction. Another ATS bioreactor system has allowed for building vascular grafts, stents, heart valves, and heart muscle repair patches.
The promise of tissue-engineered liver implants is great, since today fewer than 3,000 liver donors arise each year as compared with the 30,000 patients who die from liver failure. The cell biology involved in the liver is impressive. The tissue must have a fully formed structure with liver-specific proteins and particular enzyme activity to perform the liver's complicated function. ATS has accomplished this in lab apparatus and in living, cellular conditions for several months, Naughton said last fill.
Companies in the field have passed a dark period for them, when liability threats became so huge that " the best suppliers just left the field ," Naughton said. The federal Biomaterials Act of 1998 plugged this gap, she said.
Microstructures in the Lab
Molecular Geodesics Inc. is making scaffolds to further the tissue engineering field of study and, for now, is leaving wound care to companies like Advanced Tissue Sciences.
MGr is developing a "core" biomimetic, or biologymimicking, technology for tissue replacement scaffolds that can immediately bear physiological loads. In other words, MGI's scaffolds mimic the geodesic microstructure and mechanics of living tissues as well as key molecular determinants.
The initial focus for MGI is the development of scaffolds for orthopedic applications, including the weight-bearing alternative to current bone graft substitutes.
MGI's plan for a scaffold has a defined structure on the micron scale to provide desired mechanical behavior. The scaffold will incorporate bioactive molecular components to induce new tissue growth and regeneration, and be fully biocompatible and bioresorbable.
Other applications under exploration include cartilage repair, synthetic ligaments and tendons, and artificial vertebral disks.
According to A.J. Meuse, MGr's chief operating officer: "You can make scaffolds that are flexible and dynamic, so that the scaffold transmits mechanical loads to the adherent cells. If you engineer your scaffolds right, the cells will experience differential loading conditions, and those conditions can stimulate tissue remodeling and regeneration. That's how the cells do it in the body."
It is MGr's aim to apply new insights into the mechanical basis of cellular regulation, including recognition of the importance of microarchitecture and mechanics, to advance the field of tissue engineering.
Although its current focus is research and development, MGI also has to be forward-looking, so marketing wound care products is not ruled out. Meuse said, when asked if the company was targeting wound care applications," Not in the next 12 months." And as for corporate partners for organ replacement applications, he said he couldn't talk about that yet.
MGI produces its biomimetic materials through rapid pro to typing technologies, such as stereolithography, as well as novel proprietary manufacturing methods, including self-assembly. The company said it can design, pretest, and manufacture biomimetic materials with desired material properties, including increased strength, surface area, and porosity, that are dictated by the microstructure.
This approach offers the possibility of fabricating scaffolds and foams that are lighter, stronger, and more flexible than conventional fabrics for industrial as well as biomedical applications. Strength, compliance, surface area, and any other structural features can be varied on demand.
Using proprietary software, MGI is able to search through libraries encompassing millions of computergenerated biomimetic structures to identify the designs possessing the material properties (for example, porosity, flexibility, strength, surface, and volume) desired for a particular application.
MGI uses its own "combinatorial geodesics" software to generate biomimetic structures that possess unique material properties, to test the designs, and to select optimal configurations prior to manufacturing. This approach to scaffold design is analogous to computer-based design methods currently used in the pharmaceutical industry.
According to MGI, the software also could be potentially useful for applications that involve design and analysis of other types of discrete mechanical systems, including robotics, surgical instruments, and molecular modeling.
MGI is currently using this, its own CAD/ CAE approach, to create improved medical devices for minimally invasive surgery and interventional cardiology that offer advantages over existing devices, including greater expansion and finer control.
Skin Like Armor
Mgi's Manufacturing process can be described as micron-scale rapid prototyping, engineering expertise that has been evolving for about 15 years.
The company is developing manufacturing approaches to create porous scaffolds that mimic the microstructure of living materials, and lately has turned its skill to a research project on biofiltration funded by the Defense Advanced Research Projects Agency.
DARPA went through a metamorphosis during the 1990s as the Cold War ended and defense spending was reevaluated. Founded in 1958 when the technology of Sputnik put the fear of Russian technological prowess into America, the agency seeded all manner of defense and aerospace technologies over the past four decades.
In the 1970s and 1980s, DARPA millions could be won by corporations developing products relatively close to market (this included ARPAnet, ancestor of the Internet, and many computer chip innovations). By the middle of this decade, DARPA had to return to its roots-ensuring the most advanced defense technologies, such as high-end missile avoidance components, and keeping the United States up on technological change so the country would not fall behind some future adversary. A project like Molecular Geodesics' falls into this league.
In April 1997, MGI was awarded a two-year $6.4 million contract from DARPA in the area of "biomimetic materials for pathogen neutralization."
Under the contract, MGI will develop a biological detoxification capability in the form of synthetic biomimetic materials that will capture and neutralize biological agents before they enter the body.
These materials will be applied to the development of protective masks, cooling vents for conventional battle dress overgarments, and artificial bioskins (the battle dress of the future) in order to provide generic countermeasures to biological warfare agents.
The major advantage of the MGI approach over other forms of defensive shielding is that these new porous materials will incorporate chemical, enzymatic, and physical features to destroy toxic agents.
The protective fabrics and filtration devices will be lightweight, easily worn, and interfere only minimally with the wearer's mobility, yet effectively resist inadvertent tearing or puncture.
The porosity and hydrogel-like features of the biomimetic material will allow it to soak up pathogens and toxins like a hydrated sponge. Due to the incorporation of biologically active chemicals and enzymes within the hydrogel-like layer, toxic agents will be neutralized and inactivated once they enter the interstices of the material.
These materials also will incorporate optical fibers within their weave to deliver local ultraviolet radiation for generic sterilization of pathogens and for decontamination of garments, treatments that likely will be necessary for extended use in the field.
MGI is coating these porous scaffolds with a synthetic "protoplasm" composed of bioactive hydrogels, which exhibit a range of biochemical processes at levels of efficiency similar to that in living cells and tissues.
Polymer synthesis and hydrogel fabrication capabilities have been developed through a subcontract with Tony Mikos, a professor in the Department of Bioengineering at Rice University in Houston.
The company needed to bring in Mikos at this point, according to A.J. Meuse, MGI's chief operating officer. "Hydrogel development and formulation is known to us, but he does it better than anyone else," Meuse said. "We share researchers and technology."
Additional programs in development at MGI include heat dissipators for the computer industry, and novel replacements for foams, honeycombs, and impact-resistant building materials.
In addition, MGI has a proprietary self-assembly-based manufacturing technique, which can be used to create cylindrical metal microdevices, such as stents, for the biomedical market.
For its work in rapid prototyping, the company has a corporate alliance with Laser Fare Inc. of Warwick, R.1. Laser Fare's technology is overseen by two veterans of stereolithography, Terry Feeley and Paul Jacobs.
MGI uses laser-based rapid-prototyping technologies to render its biomimetic material designs in solid form.
As in all stereolithography, a liquid polymer resin is selectively solidified by a laser beam under the control of the computer. The system creates a solid replica of the computer design that is precise in its structural features down to the micron scale.
MGI's in-house capabilities in this area permit direct CAD/ CAM-based fabrication of porous materials with individual elements that are about 100 to 125 microns wide using a 75-micron beam spot. For comparison, collagen bundles in human skin are on the order of 35 microns.
One of MGI's core strengths is the ability to controllably microposition these materials. "We will use our stereolithography, very fine beam-spot SLA machine for the development of tissue scaffolds and to create substrates for cell culture," Meuse said.
According to Me use, few stereolithography systems are as precise. MGI's system "allows us to make very, very fine scaffolds," he said. "By combining the ability to control the scaffold microarchitecture with novel surface chemistry, we can develop controlled 3-D microenvironments that will provide critical mechanical features as well as enhanced tissue growth and repair."
A Care Delivery Platform
Molecular Geodesics and Advanced Tissue Sciences see the future in care delivery systems that are developed on these scaffolds.
Naughton of ATS predicted at last fall's congressional briefing that tissue engineering and genetic engineering could be combined to build a tissue that would deliver a physiological drug-a missing hormone, for instance.
MGI says geodesic scaffolds may incorporate polymeric hydrogels with solid-phase catalytic or chemosensing capabilities, optical fibers for delivery of sterilizing radiation, or any other desired chemical or structural features required by the user. These biomimetic materials may be manufactured using polymers, metals, ceramic, glass, or natural biomaterials, including bone and collagen.
The intellectual property and liability perils of this field cannot be understated. Millions of dollars and thousands of man-hours in research could be lost if competitors were to copy a company's work.
The exact nature of the biocompatible materials that are employed is kept very close to the vest, both at Advanced Tissue Sciences and at Molecular Geodesics. Polyglycolic acid can be procured easily from commercial sources, but how ATS configures it to be utterly biocompatible is one of many proprietary skills.
Regardless of its chemistry, the scaffold materials will yield a richly textured "cell-friendly environment," Meuse said. "Cells will see friends on the roof and on the walls."