The AFIRM Functional Limb and Digit Reconstruction Program

The battle mortality rate for U.S. forces has dropped from 30% in WWII to less than 10% in Afghanistan and Iraq. The untold, and perhaps more serious, implications of these figures is that our ability to decrease the mortality rate has been accompanied by an increase in the number of seriously injured soldiers who survive but are left with extraordinary injuries—especially complex and severe extremity and head/neck injuries. For example, the rate of digit and limb amputation, expressed as a percentage of total combat injuries, has doubled since the Second World War. Reasons for these changes include changes in tactical warfare, improvements in protective gear (e.g., Kevlar vests), and the recent widespread use of innovative explosive devices (IEDs). Injuries from high-velocity weapons and blast injuries are complex because of the deep penetration combined with the burning and blunt trauma that may be involved. Following traumatic soft tissue loss or amputation of a digit and limb, options for definitive treatment and rehabilitation typically involve a multidisciplinary team effort to provide comprehensive care for affected individuals. This includes the development of improved prosthetic devices and rehabilitation programs to assist affected individuals in dealing with the psychosocial aspects of lost extremity function and/or lost digits and limbs.

Amputations and digit/limb trauma are obviously not limited to combat related incidents. A recent study showed that the majority of civilian amputation injuries occur in young children, especially males. Most of these cases involve loss of fingers, and adolescents have been shown to experience a higher proportion of serious amputation injuries such as loss of limbs. There are approximately 100,000 cases annually of amputation injuries in the United States alone. Stated differently, the proposed research will not only benefit the population of soldiers injured as a result of combat, but will also have a significant impact upon the civilian population.

The restoration of functional limb and digit tissue is quite complex. It involves the orchestrated growth and differentiation of many different tissue types, and this must occur in the proper places in the injured tissue as well as in an appropriate three-dimensional pattern so that the proper shape of the limb or digit can be attained. Many types of tissue must also be restored, including muscle, nerve, skin, and blood vessels. Because digits and limbs are such multifunctional complex tissues, we have developed a multidisciplinary approach to restore the anatomical and functional components of these body parts. For example, we will explore ways to restart the tissue growth processes that occur during human development and to harness the power of the processes that occur in animals like salamanders, a species that can regenerate entire limbs when they are lost. We will also investigate new strategies that will allow transplantation of entire hands by improving the methods used to prevent rejection. Our program consists of independent but conceptually related projects:

Projects

Blastemal Approach to Digit Reconstruction
Hand Transplantation for Reconstruction of Disabling Upper Limb Battlefield Trauma Translation and Clinical Trials
Spatial and Temporal Control of Vascularization and Innervation of Composite Tissue Grafts
Peripheral Nerve Repair
Modular, Switchable, Synthetic, Extracellular Matrices for Regenerative Medicine
Oxygen Generating Biomaterials for Engineering Large Tissue Masses
High Purity Magnetophoretic Sorting for Transplant Therapies

Blastemal Approach to Digit Reconstruction
Principal Investigator: Stephen Badylak, DVM, PhD, MD (University of Pittsburgh)

This project investigates the possibility of regenerating an amputated digit using extracellular matrices (ECM) that promote and stimulate the body’s regenerative capacity.

The restoration of functional limb and digit tissue involves the orchestrated growth and differentiation of multiple tissue types in a spatially appropriate and site appropriate pattern. A logical and rational, albeit challenging, approach is the development of strategies and methods that can recapitulate the processes that occur during fetal development or the phenomenon of epimorphic regeneration that occurs in urodeles and many amphibian species. These processes involve the formation of a blastema-like structure; a structure which consists of an accumulation of multipotential progenitor cells within a primitive extracellular matrix. These cells then, in a preprogrammed fashion, proliferate, differentiate, and spatially organize into a functional tissue to replace the lost body part.

Our research team will leverage discoveries and advancements that have been made in a parallel project supported by the Defense Advance Research Program Agency (DARPA).



Hand Transplantation for Reconstruction of Disabling Upper Limb Battlefield Trauma Translational and Clinical Trials
Principal Investigator: W.P. Andrew Lee, MD (University of Pittsburgh)

Hand transplantation has recently become a clinical reality. Unfortunately, the severe adverse effects of the immunosuppressive treatment administered to prevent graft rejection have limited the use of this technology. However, recent developments now provide the opportunity to achieve long term survival of organ grafts with minimum or no immunosuppressive treatments. This project will establish a protocol that will aim to minimize the need for the life-long immunosuppression currently required to maintain survival of a hand transplant.

The proposed research will be accomplished in two phases over 5 years. Phase 1 (Translational Studies) will be performed in a large animal model and Phase 2 (Clinical Hand Transplantation) will be performed in selected amputee service members. Phase 1 and 2 studies will proceed in a concerted and simultaneous manner and findings from Phase 1 studies will be strategically implemented in the ongoing Phase 2 clinical trial to further optimize clinical outcomes and maximize safety and efficacy. We propose herein to evaluate in the setting of clinical hand transplantation promising immunomodulatory protocols that we have established as innovative therapies in solid organ transplants like liver and kidney. We hypothesize that similar protocols when complemented with local therapies targeting the highly antigenic skin component of the allograft can achieve prolonged survival of immunogenic composite tissue allotransplantations like hand transplants under minimal immunosuppression with the prospect for complete elimination of drug therapy. We will test this hypothesis in Phase 1 studies with the goal of efficient and expedient translation of innovative research findings into clinical practice (Phase 2).

Spatial and Temporal Control of Vascularization and Innervation of Composite Tissue Grafts
Principal Investigator: Barbara Boyan, PhD (Georgia Institute of Technology)

This project focuses on the restoration of complete limb function after amputation and reattachment. We will adapt strategies used by the body during limb formation to create an environment at the site of injury that is favorable for the growth and regeneration of new nerves, blood vessels, and other tissues. Our approach is to direct nerve, vascular, and bone growth in a synchronized manner.

We hypothesize that to overcome this problem, it is necessary to adapt methods used by the body during limb formation. Our approach is to provide spatial and temporal cues that direct nerve, vascular, and bone growth in a synchronized manner. This approach is based on the hypothesis that vascular development and neural development occur in tandem during fetal bone formation and post-fetal bone growth. We will use an animal model of composite tissue loss combined with novel, organized 3D presentation of tissue-specific regenerative cues for nerve, muscle, bone, and vascular repair.

We have previously established a large (8 mm) rat femur segmental bone defect model and tested the ability of sustained co-delivery of growth factors to restore limb function. Using direct and contrast-enhanced micro-CT imaging, this model features the ability to quantify 3D bone and vascular ingrowth into the defect region. In addition, the fixation plate is designed to allow torsional biomechanical testing of function restoration. The vascular perfusion imaging technique can also be used to quantify neovascularization following ischemic limb injuries. We have now reproduced this model in an athymic rat, enabling us to assess effectiveness of allograft and xenograft materials. In both immunocompetent and immunocompromised animals, the defect is critically sized, meaning that it will not heal with bone unless an interventional strategy is used.

Peripheral Nerve Repair
Principal Investigators: Kacey Marra, PhD (University of Pittsburgh), David Kaplan, PhD (Tufts University), and Tom Smith, PhD (Wake Forest University)

In parallel to the neurovascular guided tissue regeneration study led by Dr. Boyan, the present multi-institutional project will investigate peripheral nerve repair as an isolated therapy. Traumatic limb wounds frequently are limited by peripheral nerve damage and we believe that the resources devoted to developing regenerative medicine approaches to peripheral nerve repair will not only benefit those patients requiring this therapy alone but also have obvious value to all the other studies in limb and digit reconstruction. This project is highly collaborative between three laboratories at the forefront of peripheral nerve repair. All three labs have focused on a tissue engineering approach for nerve repair over long gaps, and now we are combining our efforts in a synergistic manner. The Pittsburgh group will continue developing delivery systems for neurotrophic factors using a gradient concentration of microspheres in the nerve conduits. The Tufts group will utilize their expertise in silk scaffolds to develop tubular constructs. The Wake Forest group will incorporate keratin-based gels within the conduits to enhance axonal elongation. The combination of these approaches, all using FDA-approved materials, will result in a guide that has the potential to be an off-the-shelf guide for long gap nerve repair.

Modular, Switchable, Synthetic, Extracellular Matrices for Regenerative Medicine
Principal Investigator: Matthew Tirrell, PhD (UC Santa Barbara)

The importance of understanding, and being able to control, innervation of tissues cannot be overstated. It has recently been shown that even regenerative amphibians fail to re-grow new limbs if peripheral innervation is inhibited. In fact, many of the downstream events of digit and limb regeneration are critically dependent upon the a priori formation of functional peripheral innervation. Therefore, in addition to the efforts of the other projects, we will investigate yet another approach to controlling peripheral nerve growth following traumatic amputation by modulating components of the naturally occurring ECM. The focus of this study is to create and characterize several variants of peptide amphiphiles linking a synthetic lipid tail to the model peptide ligand from bone sialoprotein: CGGNGEPRGDTYRAY, which has recently been shown (as part of a different synthetic construct) to support both self-renewal and differentiation of adult neural stem cells.Variants will include incorporation of polyethylene glycol (PEG) spacers and additional amino acids to direct assembly and secondary structures of the peptides.

Further, the research team will employ fibrous networks of the peptide amphiphiles above obtained by driving the system to assemble into long, worm-like micelles and investigate NSC attachment, proliferation, and differentiation via cell morphology as well as immunostaining of lineage-specific markers, and explore the response of these matrices to shear and build on prior experience to develop matrices that can be transformed by shear from spherical micelles to fibrous networks.

Oxygen Generating Biomaterials for Engineering Large Tissue Masses
Principal Investigator: Benjamin Harrison, PhD (Wake Forest University)

One of the significant challenges encountered in engineering clinically relevant tissues for application is the inadequate diffusion of oxygen to transplanted cells. Thus, there is the general conception that cell or tissue components may not be implanted in large volumes due to the limited oxygen diffusion which leads to tissue necrosis. Although biological factors, such as vascular endothelial growth factor (VEGF) and endothelial cells, are known to promote host angiogenic activities, this method is ineffective when larger tissues are involved. As such, supplementation of oxygen to the transplanted cells may prolong their survival in vivo. This hypothesis has been demonstrated by hyperbaric therapies in injured ischemic tissues clinically. To demonstrate the feasibility of supplying oxygen to transplanted cells for prolonged survival, we have developed a biomaterial system that releases continuous oxygen over time. This material, referred throughout as POGs, consists of an oxygen producing compound such as sodium percarbonate or calcium superoxide embedded in a catalytic matrix that allows for the controlled generation of oxygen.

In this project we plan to develop a tissue scaffolding system that would provide sustained release of oxygen to cells and tissues for prolonged survival in vivo. Successful outcome of this project would provide solutions to current challenges of limited oxygen diffusion to transplanted cells. In addition, this technology would allow us to engineer clinically relevant sized functional tissues for digit and limb tissue regeneration.

High Purity Magnetophoretic Sorting for Transplant Therapies
Principal Investigators: Hyongsok (Tom) Soh, PhD (UC Santa Barbara) and Jamie Thomson, PhD (University of Wisconsin)

This project will develop a system for isolating specific types of cells that can be used in regenerative therapies. A high performance, disposable cell separation device will be developed and then applied to the system for the purification of rare cells for cell-transplant therapy and for the study of nuclear programming in human mesenchymal cells.

Many projects in this proposal will have a high demand for purified cell populations of different types. These cells will be use locally and regionally as well as systemically. In the present study, we are developing a high performance, disposable separation device based on micromagnetics and microfluidics, and apply the system for the purification of rare cells for cell-transplant therapy, and for study of nuclear programming in human mesenchymal cells. The microfluidic system will be capable of sorting mammalian cells with unprecedented purity (> 10,000 fold enrichment), rare cell recovery (approaching ~100%) and throughput (> 30ml/hour) which is not currently available. The proposed technology has direct relevance to the needs of the DOD because, disposable, contamination-free cell sorters will open up the capability to separate and purify the cellular products in a field deployable format for cell-based therapies – a capability that does not currently exist. The successful development of the proposed device, in conjunction with advanced medical therapies such as peripheral blood stem cell transplantation (PBSCT) will enable previously unimagined possibilities in medical counter measures against radiation, nitrogen based chemicals, and bioengineered pathogens that target the human immune system.

Finally, in a collaborative effort between Soh Lab (UCSB) and Thomson Lab (Wisconsin-Madison) this system will be used to isolate extremely rare human mesenchymal cells which have gone through nuclear reprogramming to induce an “ES cell-like” state.