Artificial Skin

Background

Skin, the human body's largest organ, protects the body from disease and physical damage, and helps to regulate body temperature. It is composed of two major layers, the epidermis and the dermis. The epidermis, or outer, layer is composed primarily of cells: keratinocytes, melanocytes, and langerhans. The dermis, composed primarily of connective tissue fibers such as collagen, supplies nourishment to the epidermis.

When the skin has been seriously damaged through disease or burns, the body cannot act fast enough to manufacture the necessary replacement cells. Wounds, such as skin ulcers suffered by diabetics, may not heal and limbs must be amputated. Burn victims may die from infection and the loss of plasma. Skin grafts were developed as a way to prevent such consequences as well as to correct deformities. As early as the sixth century b.c., Hindu surgeons were involved in nose reconstruction, grafting skin flaps from the patient's nose. Gaspare Tagliacozzo, an Italian physician, brought the technique to Western medicine in the sixteenth century.

Until the late twentieth century, skin grafts were constructed from the patient's own skin (autografts) or cadaver skin (allografts). Infection or, in the case of cadaver skin, rejection were primary concerns. While skin grafted from one part of a patient's body to another is immune to rejection, skin grafts from a donor to a recipient are rejected more aggressively than any other tissue graft or transplant. Although cadaver skin can provide protection from infection and loss of fluids during a burn victim's initial healing period, a subsequent graft of the patient's own skin is often required. The physician is restricted to what skin the patient has available, a decided disadvantage in the case of severe burn victims.

In the mid-1980s, medical researchers and chemical engineers, working in the fields of cell biology and plastics manufacturing, joined forces to develop tissue engineering to reduce the incidences of infection and rejection. One of the catalysts for tissue engineering was the growing shortage of organs available for transplantation. In 1984, a Harvard Medical School surgeon, Joseph Vacanti, shared his frustration over the lack of available livers with his colleague Robert Langer, a chemical engineer at the Massachusetts Institute of Technology. Together, they pondered whether new organs could be grown in the laboratory. The first step was to duplicate the body's production of tissue. Langer came up with the idea of constructing a biodegradable scaffolding on which skin cells could be grown using fibroblasts, cells extracted from donated neonatal foreskins removed during circumcision.

In a variation of this technique developed by other researchers, the extracted fibroblasts are added to collagen, a fibrous protein found in connective tissue. When the compound is heated, the collagen gels and traps the fibroblasts, which in turn arrange themselves around the collagen, becoming compact, dense, and fibrous. After several weeks, keratinocytes, also extracted from the donated foreskins, are seeded onto the new dermal tissue, where they create an epidermal layer.

An artificial skin graft offers several advantages over those derived from the patient and cadavers. It eliminates the need for tissue typing. Artificial skin can be made in large quantities and frozen for storage and shipping, making it available as needed. Each culture is screened for pathogens, severely curtailing the chance of infection. Because artificial skin does not contain immunogenic cells such as dendritic cells and capillary endothelial cells, it is not rejected by the body. Finally, rehabilitation time is significantly reduced.

Raw Materials

The raw materials needed for the production of artificial skin fall into two categories, the biological components and the necessary laboratory equipment. Most of the donated skin tissue comes from neonatal foreskins removed during circumcision. One foreskin can yield enough cells to make four acres of grafting material. Fibroblasts are separated from the dermal layer of the donated tissue. The fibroblasts are quarantined while they are tested for viruses and other infectious pathogens such as IIV, hepatitis B and C, and mycoplasma. The mother's medical history is recorded. The fibroblasts are stored in glass vials and frozen in liquid nitrogen at -94°F (-70°C). Vials are kept frozen until the fibroblasts are needed to grow cultures. In the collagen method, keratinocytes are also extracted from the foreskin, tested, and frozen.

If the fibroblasts are to be grown on mesh scaffolding, a polymer is created by combining molecules of lactic acid and glycolic acid, the same elements used to make dissolving sutures. The compound undergoes a chemical reaction resulting in a larger molecule that consists of repeating structural units.

In the collagen method, a small amount of bovine collagen is extracted from the extensor tendon of young calves. The collagen is mixed with an acidic nutrient, and stored in a refrigerator at 39.2°F (4°C).

Laboratory equipment includes glass vials, tubing, roller bottles, grafting cartridges, molds, and freezers.

The Manufacturing
Process

The manufacturing process is deceptively simple. Its main function is to trick the extracted fibroblasts into believing that they are in the human body so that they communicate with each other in the natural way to create new skin.

Mesh scaffolding method

• 1 Fibroblasts are thawed and expanded. The fibroblasts are transferred from the vials into roller bottles, which resemble liter soda bottles. The bottles are rotated on their sides for three to four weeks. The rolling action allows the circulation of oxygen, essential to the growth process.
• 2 Cells are transferred to a culture system. The cells are removed from the roller bottles, combined with a nutrient-rich media, flowed through tubes into thin, cassette-like bioreactors housing the biodegradable mesh scaffolding, and sterilized with e-beam radiation. As the cells flow into the cassettes, they adhere to the mesh and begin to grow. The cells are flowed back and forth for three to four weeks. Each day, leftover cell suspension is removed and fresh nutrient is added. Oxygen, pH, nutrient flow, and temperature are controlled by the culture system. As the new cells create a layer of dermal skin, the polymer disintegrates.
• 3 Growth cycle completed. When cell growth on the mesh is completed, the tissue is rinsed with more nutrient-rich media. A cryoprotectant is added. Cassettes are stored individually, labeled, and frozen.

Collagen method

• 4 Cells are transferred to a culture system. A small amount of the cold collagen and nutrient media, approximately 12% of the combined solution, is added to the fibroblasts. The mixture is dispensed into molds and allowed to come to room temperature. As the collagen warms, it gels, trapping the fibroblasts and generating the growth of new skin cells.
• 5 Keratinocytes added. Two weeks after the collagen is added to the fibroblasts, the extracted keratinocytes are thawed and seeded onto the new dermal skin. They are allowed to grow for several days and then exposed to air, inducing the keratinocytes to form epidermal layers.
• 6 Growth cycle completed. The new skin is stored in sterile containers until needed.

The Future

The medical profession is using artificial skin technology to pioneer organ reconstruction. It is hoped that this so-called engineered structural tissue will, for example, someday replace plastic and metal prostheses currently used to replace damaged joints and bones. Ears and noses will be reconstructed by seeding cartilage cells on polymer mesh. The regeneration of breast and urethral tissues is currently under study in the laboratory. Through this technology, it is possible that one day, livers, kidneys, and even hearts, will be grown from human tissues.

Where to Learn More

Periodicals

Langer, Robert and Joseph P. Vacanti. "Artificial Organs," Scientific American, September 1995, pp. 130-133.

Langer, Robert and Joseph P. Vacanti. "Tissue Engineering," Science, May 14, 1993, pp. 920-921.

McCarthy, Michael. "Bio-engineered tissues move towards the clinic," The Lancet, August 17, 1996, p. 466.

Rundle, Rhonda L. "Cells 'Tricked' To Make Skin For Burn Cases," The Wall Street Journal, March 17, 1994.

—MaryF.McNulty

Artificial skin

Artificial skin is a synthetic (laboratory produced) substitute for human skin that can dramatically save the lives of severely burned patients. Skin, composed of two layers called epidermis (the outer layer) and dermis (the inner layer), is the largest human organ. It covers the entire body, keeping harmful bacteria out and vital fluids in. The epidermis is the outer layer; the dermis is the inner layer that contains the blood vessels, nerves, and hair, oil, and sweat glands.

A severe burn leaves the body dangerously vulnerable to infection and dehydration (drying out). Keeping burn patients in sterile (germ free) rooms can protect against infection, and covering burned areas with grafts (a piece of skin or bone transplanted from one area of the body to another) from the patient's own skin or temporary grafts from other humans or pigs can help save some patients. Still, many burn patients die because their bodies cannot produce large quantities of new skin quickly enough, or because their bodies reject the skin grafts.

Burke and Yannas Create Synthetic Skin

The medical community has long been looking for a more dependable alternative. The first synthetic skin was invented by John F. Burke, chief of Trauma Services at Massachusetts General Hospital, and Ioannis V. Yannas, chemistry professor at the Massachusetts Institute of Technology (MIT) in Cambridge, Massachusetts. Burke had treated many burn victims and realized the need for a human skin replacement. Yannas had been studying collagen, a protein found in human skin. Teaming up during the 1970s, the two made a polymer (a chemical compound made of multiple repeating units). Using collagen fibers and a long sugar molecule, they formed a porous (full of small holes) material resembling skin. When placed on the wounds of lab animals, this material seemed to encourage the growth of new skin cells around it.

Burke and Yannas then created a kind of artificial skin using polymers from shark cartilage and collagen from cowhide. This mixture was dried and sterilized to make a thin membrane (a covering through which things can pass) similar to the human dermis layer. Added to the membrane was a protective top layer of silicone that acted like the human epidermis.

Burke and Yannas's experiments with their synthetic skin, called Silastic, showed that it acted like a framework onto which new skin tissue and blood vessels could grow (although these new cells never produced hair follicles or sweat glands, which normally form in the dermis). As the new skin grew, the cowhide and shark substances from the artificial skin broke down and were absorbed by the body. In 1979 Burke and Yannas used their artificial skin on their first patient, a woman whose burns covered over half her body. After peeling away the burned tissue, Burke applied a layer of artificial skin and, where possible, grafted on some of her own unburned skin. Three weeks later, the woman's new skin—the same color as her unburned skin—was growing at an amazingly healthy rate.

Graftskin

At nearby Harvard University, Howard Green was culturing human skin cells under sterile conditions and growing a sheet of human epider-mis cells from just a tiny piece of a person's skin. When the cultured skin was placed on a wound area, however, it was rejected by the body's immune system (an internal mechanism for fighting off disease). Green later collaborated with Eugene Bell of MIT, who founded a research group called Organogenesis. The research goal at Organogenesis was to make artificial skin that would both include an epidermis layer and resist rejection by the patient's immune system. Organogenesis teams eventually created an extraordinary product called Graftskin, a living skin equivalent made of purified bovine (ox or cow) collagen into which some of the patient's own dermal cells are "seeded" (placed for growth). On top of this layer is an epidermal layer of cultured human skin cells. The Graftskin is formed into four-by-eight inch sheets that can be sutured (sewn) or stapled onto a patient during surgery.

In clinical trials Graftskin grafts have not been rejected by patients' immune systems. Hospital trials have studied burn victims as well as patients needing skin grafts after cancer surgery, and those with chronic (nonhealing) wounds. After further testing, synthetic skin may become a more common treatment for burns and other serious skin disorders. A welcome side effect of this research is that synthetic skin is a source of human tissue that can also be used to test dermatological (skin) products without lab animals.