The AFIRM Burn Repair Program

Today, the U.S. Army is experiencing unprecedented burn wounds, in terms of both number and severity. Improvements in the resuscitation of burned patients, in patient transport, and in training of medical personnel have resulted in larger numbers of severely burned soldiers surviving to reach the upper echelons of care. These patients have a high total body surface area (TBSA) burned, experience longer delays in reaching a burn center, and have a higher injury severity score than their civilian counterparts. They are at high risk of death due to secondary complications, primarily infection, particularly if concomitant lung injury is present. If they do survive, the majority do not return to active military duty.

Survival of what were once lethal burn injuries presents a new challenge to physicians and nurses. Because they have lost the barrier function the skin normally provides, these patients are profoundly immune suppressed and susceptible to infection. In addition, the massive skin damage sustained by these patients compromises their ability to regenerate skin quickly, which increases the infection risk and may lead to severe scarring and loss of function. Needless to say, these severely burned patients require skin quickly. Replacement of the barrier function of skin is critically important to the survival of these soldiers, while reducing scarring and improving regenerated skin function would certainly increase the likelihood of returning to active duty.

Unfortunately, this need is currently unmet. New wound healing technologies that meet both the demands of modern warfare and its unique logistical constraints are desperately needed by the U.S. military. An ideal dressing for burns meets the following criteria:

• Immediate barrier function
• Integration with no rejection
• Large, contiguous surface area coverage
• Reduced scarring
• Readily available with ease of use sometimes in austere environments

To meet these needs, a true skin replacement must be developed. Regenerative medicine holds the potential to provide a functional replacement for skin. The basis for this technology is to engineer living skin grafts using both living cells and matrices or templates. In this case, the engineered graft could be used in severe burns, because the cells within the graft would be able to regenerate new skin quickly. The WFPC has formed a team of leading burn scientists from several prominent centers that proposes to direct the development of these next generation products specifically to military burn patients, and eventually, to others with severe burns. The burn program’s projects range from new forms of tissue engineered skin to stem cell replacements for skin component cells. They include:

Projects

Human Clinical Trial with Tissue Engineered Skin Products
Delivery of Stem Cells to a Burn Wound via a Clinically-Tested Spray Device
Novel Keratin Biomaterials That Support the Survival of Damaged Cells and Tissues
Artificial Extracellular Matrix Proteins for Regenerative Medicine
In Situ Bio-Printing of Skin for Battlefield Burn Injuries
Stem Cells for Engineering Living Skin Equivalents


Human Clinical Trial with Tissue Engineered Skin Products
Principal Investigator: James Holmes, MD (Wake Forest University)
Co-Investigators: Paul Kemp, PhD (Intercytex) and Gary Cadd, PhD (Organogenesis)

At least 2 commercial companies are developing next generation products based on the premise that a cell-synthesized ECM will be better able to persist in the aggressive environment of a burn wound. The clinical evaluation of their 2nd generation engineered skin products in burn patients is the primary goal of this project. The specific aim is to assess the efficacy of these products in a prospective, side-by-side, comparative clinical study starting no later than January 2012. This clinical investigation will allow for selection of a suitable product that can be readily used by burn soldiers.

Delivery of Stem Cells to a Burn Wound via a Clinically-Tested Spray Device
Principal Investigator: Jörg Gerlach, MD, PhD (University of Pittsburgh)

This project uses a novel and clinically tested “spray gun” to coat large burned areas with skin-derived stem cells that have the ability to grow new skin. Skin-derived stem cell isolation and spraying methodologies will be optimized to prepare for clinical translation.

Healing without scar is a natural process for a fetus, but once we are born we rapidly lose this ability. Regenerative medicine research seeks to accelerate processes that essentially recapitulate fetal-like wound healing in the adult. The LSE that are available to patients today are early attempts to deliver regenerative medicine to burned patients. For either an allograft or an autograft we are essentially “sodding” something that we believe will be beneficial into a wound site. The technical and clinical challenges in this approach are centered around keeping the graft in place, minimizing damage from blistering, and nurturing the complex interaction between the surface of the graft and the wound bed. In LSE-based technology we supply a graft material that also contains cells. We know that these cells can release factors that encourage healing and repopulation of the wound site, but this effect is only transient. To better leverage the beneficial effect of allogeneic cell transplantation, we have developed a novel spray device that can “seed” the wound site in a way that mitigates some of the deleterious effects of sodding. We have established through our prior clinical work that separating cells spatially from each other during delivery minimizes many of the problems that are associated with covering the area with a graft.

Novel Keratin Biomaterials That Support the Survival of Damaged Cells and Tissues
Principal Investigator: Mark VanDyke, PhD (Wake Forest University)
Co-Investigator: Lillian Nanney, PhD (Vanderbilt University)

This project will develop new keratin biomaterials that can support the survival and regeneration of damaged skin. Keratin biomaterials derived from human hair will be extracted and configured into a biologically active form. It has been known for more than a decade that genes coding for growth factors such as bone morphogenetic protein-4 (BMP-4) and other members of the transforming growth factor-ß (TGF-ß) superfamily are expressed in developing hair follicles. In fact, more than 30 growth factors and cytokines are involved in the growth of a cycling hair follicle. Many of these molecules have a pivotal role in the regeneration of a variety of tissues.

In the experiments shown in the figure above (which were directed toward the development of ECM protein lenses for correction of refractive error), a 3.0 mm trephine was used to create a partial thickness keratotomy, approximately 100-200 microns in depth, in the central cornea. A 6.0 mm diameter protein lens was placed on the corneal surface and tucked into a grooved peripheral pocket using a spatula. Epithelialization was visualized by fluorescein staining. In all cases epithelialization initiated at the periphery of the exposed surface of the implant and progressed toward the center. The time required for full epithelialization varied from 4 to 7 days for individual animals.

We hypothesize that this milieu of keratins and other regulatory molecules is responsible for the cell survival promoting behavior of keratins. We will test this hypothesis and further develop keratin biomaterials for treating burn wounds.

Artificial Extracellular Matrix Proteins for Regenerative Medicine
Principal Investigator: David Tirrell, PhD (Cal Tech University)

The objective of the proposed work is the development of effective burn therapies that use artificial Extracellular Matrix (ECM) proteins to promote rapid regeneration of the skin. Artificial ECM proteins are designed by combining elements drawn from natural ECM proteins such as fibronectin. The needed elements are encoded into artificial genes and the corresponding proteins are expressed in bacterial cells. The modularity of the gene design allows rapid and systematic variation in mechanical and biological properties and in the rate of protein degradation by proteolytic or hydrolytic processes. Matrices can therefore be optimized individually for regenerative therapies with distinct performance requirements. Under this project, we are exploring variations in ECM protein design to optimize matrices for burn repair, and ultimately, for limb and digit reconstruction, facial reconstruction, and healing without scarring. Although we believe that artificial ECM proteins will in time contribute to all of these therapies, our initial focus will be on burn repair.

In Situ Bio-Printing of Skin for Battlefield Burn Injuries
Principal Investigator: James Yoo, MD, PhD (Wake Forest University)



This work uses a modified ink jet printer to replace skin components by “printing” cells and extracellular matrix materials in the proper configuration directly onto a burned area. This system will be designed and constructed to allow for mobility, with the ultimate aim of bringing the inkjet printing technology directly to the bedside to deliver allogeneic human skin products.

This would significantly decrease the morbidity and mortality associated with critical burn injuries. The unique advantages of the proposed in situ skin bio-printing system include:

1) The ability to treat massive burns immediately after stabilization of the wound in the battlefield.
2) The in situ bio-printing system offers the ability to deliver several dermal cell types and matrices simultaneously onto target sites to generate anatomically and functionally adequate dermal tissues.

We plan to use bioprinting techniques to develop a portable printing system that can be used to create biological skin for battlefield burn repair needs. We hypothesize that full thickness biological skin can be fabricated in situ using a portable bio-printer, and this will result in a novel treatment for battlefield burns.

Stem Cells for Engineering Living Skin Equivalents
Principal Investigator: Mark Furth, PhD (Wake Forest University)
Co-Investigator: Lillian Nanney, PhD (Vanderbilt University)

Our goal is to develop a living skin equivalent (LSE) product based on the use of broadly multipotent stem cells isolated from human amniotic fluid, termed amniotic fluid-derived stem (AFS) cells. There are several anticipated benefits if the technology if successfully translated to the clinic.

While the cellular components of existing LSE products, namely, dermal fibroblasts and keratinocytes, are crucial for integrity and function, they constitute only a small fraction of the lineages in normal skin Current products based on the expansion of committed progenitors for fibroblasts and/or keratinocytes lack cells such as melanocytes that potentially could be formed from AFS cells.