Plastic Surgery Research Laboratory

Principal Investigators:
James W. May, Jr., M.D.
Jonathan Winograd, M.D.
Michael J. Yaremchuk, M.D.
William G. Austen, Jr., M.D.
Mark A. Randolph, M.A.S.

Research Fellows:
Jason Burdick, Ph.D.
T. Shane Johnson, M.D
John M. Mesa, M.D.
Jamal A. Nazzal, M.D.

Other Links:
www.mgh.harvard.edu/plasticsurgerylab/

The Plastic Surgery Research Laboratory is located on the sixth floor of the Wellman Research Building (adjacent to the Knight Surgical Suite and the large animal farm) on the main campus of the Massachusetts General Hospital. The laboratory conducts investigations into four primary areas of interest: 1) tissue engineering of cartilage; 2) mechanisms of neural injury and regeneration; 3) transplantation of vascularized limb tissues; and 4) physiology of free flaps.

Tissue Engineering of Cartilage:
Tissue engineering of cartilage has been a primary focus of the Plastic Surgery Research Laboratory for more than a decade. The engineering of cartilage from autologous chondrocytes allows for the reconstruction of craniofacial and other musculoskeletal tissue deformities, including joint resurfacing, without the complications associated with frozen osteochondral allografts or prosthetic materials. In addition, by using tissue culture techniques for expanding cell numbers, only a small number of autologous cells are required, thereby eliminating the donor site morbidity associated with large cartilage autografts.

a) Injectable articular cartilage: Our early work showed that cartilage could be produced when articular chondrocytes were suspended in a three dimensional biodegradable hydrogel polymer, such as alginate, in a nude mouse model. By utilizing polymers that exist in liquid form, it is possible to introduce the polymer containing chondrocytes into various body compartments using minimally invasive techniques. Such liquid polymers could be injected through a needle into the subperiosteal space or even subcutaneously for craniofacial reconstruction. Subsequent studies have explored other polymers, namely fibrin-based gels and photocurable synthetic polymers, for improved delivery and fixation. In addition, these polymers could be injected under direct vision with the use of endoscopy into the joint space for the purpose of repairing cartilaginous defects in joints injured by osteoarthritis, rheumatoid arthritis, temporomandibular joint dysfunction, acute trauma, and a multitude of other debilitating joint disorders. Being able to create autologous cartilage in situ by employing minimally invasive technology could potentially reduce donor site morbidity, patient recuperation time, hospitalization, and overall costs. Studies recently completed in swine have demonstrated the potential for engineered cartilage to repair defects in the articular joint surface. In collaboration with chemical engineers at the Massachusetts Institute of Technology and the University of Colorado, other studies are exploring new polymer mixtures and evaluating the cartilage matrix product.

b) Craniofacial cartilage: The fibrocartilage structures of the cranium are also a focus of our tissue engineered cartilage program. Unlike the avascular nature of the joint, the cartilages in the cranium have a different composition and blood supply. Efforts are directed towards understanding the parameters for engineering craniofacial cartilage. Studies have been performed comparing the matrix products of tissue engineered cartilage formed from ear and costal-derived chondrocytes versus that from articular type cells. Because of the close interaction of the perichondrium and skin in the cranial cartilages, we are studying means to create lamination of soft tissues over the engineered cartilage as well.

c) Meniscal repair: In collaboration with the Laboratory for Musculoskeletal Tissue Engineering in the MGH Orthopaedic service, we have a program investigating repair of meniscal lesions using scaffolds and cells. Tears to the inner portion of the meniscus, referred to as the white or avascular zone, do not heal. Using a cell-seeded scaffold implanted into a lesion in a nude mouse model, we have demonstrated that this portion of the mensicus can heal. Studies are now underway exploring scaffold materials and different cell sources to repair torn menisci in situ in swine.

Mechanisms of Neural Injury and Regeneration:
The development of microsurgical techniques in the 1970’s raised the level of technical sophistication in peripheral nerve reconstruction to one that has not yet been surpassed. With proximal injuries, however, poor recovery remains the norm. Even in more favorable lesions, such as median or ulnar nerve injuries in the forearm, only 10% of patients who undergo microsurgical repair achieve an excellent recovery, i.e. full muscle strength and normal two-point discrimination. Nerve gaps and nerve deficits further complicate the regeneration process, prompting efforts to develop improved methods of neural repair. Using a rat model of peripheral nerve injury and recovery, several aspects of neural injury and repair are currently being investigated in our laboratory.

a) Stem cell transplantation: The recent experimental transplantation of neural stem cells into the central nervous system has produced favorable results. Transplanted cells have shown the ability to differentiate and incorporate into the existing neural cytoarchitecture forming synapses with native neurons. With the aid of neural stem cell transplantation, it may be possible to greatly improve the results of peripheral nerve reconstruction. First, by augmenting the numbers of regenerating axons, transplants may enlarge the magnitude of the regenerative response. Transplants could also improve the rate and quality of neuronal regeneration distal to the site of transection, allowing for a more rapid and complete recovery. Finally, by preserving motor and sensory end-organs through the "babysitting" phenomenon, stem cell transplants could help to better maintain suitable targets for the regenerating axonal growth cones. It is also possible that these different approaches will be complementary to one another, providing even further enhancement of the regeneration response. Currently in our laboratory, we are characterizing a model of neural stem cell transplantation in the rat sciatic nerve. Further efforts will be dedicated to improving the response to neural injury through stem cell transplantation.

b) Photochemical keratodesmos: An optimal nerve repair technique would be quick, atraumatic, and would minimize inflammation while maximizing axonal recovery. Currently, we are collaborating with the Massachusetts General Hospital Wellman Laboratory of Photomedicine to develop such a technique. Utilizing the process of non-thermal collagen cross-linking, we are developing alternatives to standard microsurgical anastomosis.

Transplantation of Vascularized Limb Tissues:
The limited availability of autologous tissue for reconstructing large bone and joint defects after tumor resection, traumatic injury, or congenital deformities remains a challenging clinical problem. For example, available sites for bone graft material are primarily limited to calvarium, iliac crest, rib, and fibula. Although rib and fibula grafts can be transplanted on a vascular pedicle, there is little flexibility in their dimensions and configurations and donor site morbidity can also be problematic. Current approaches to overcome autologous tissue shortage include the use of frozen cadaveric bone or osteochondral grafts. The results with large frozen allografts have been equivocal, as the lack of vascularization predisposes the grafts to nonunion and fracture, as well as soft tissue healing problems. The 20 recent hand transplants performed around the world demonstrate the technical feasibility of allotransplantation of limb tissues in humans. However, the need for chronic immunosuppression to prevent graft rejection may pose unnecessarily high risk to patients for treating non life-threatening deficiencies. Thus, our laboratory, in collaboration with the Transplantation Biology Research Center, is investigating means for inducing tolerance to vascularized limb tissue allografts.

a) In utero induction of tolerance: Along with the multiple new means for detecting congenital defects in utero, new therapeutic approaches could be developed to begin treatment during gestation. For example, fetuses identified with inborn errors of metabolism or defective organs could be inoculated in utero to induce tolerance to allogeneic transplants that could be performed after birth. During the fetal stage of immune development, it is theorized that the fetus cannot determine self from non-self. Therefore, inoculating the fetus with alloantigen could induce a state of tolerance. Our laboratory has explored this approach by injecting adult bone marrow from genetically defined miniature swine into the portal vein of fetuses in pregnant sows at mid-gestation (50-55 days). Rather than use intraperitoneal injections as other investigators have in sheep and primate models, we inject directly into the portal vein delivering the cell inoculum to the fetal liver--the primary hematopoietic organ in the developing fetus. 16 animals have been born that demonstrated the presence of donor cells in the peripheral blood after birth as determined by FACS analysis (ranging from 1-90%). This chimerism, defined as the coexistence of cells from donor and recipient, declines over time, but this group of animals has demonstrated acceptance of kidney allografts for greater than 300 days. These findings are significant in that these transplants are across a major histocompatibility barrier and immunosuppression is not required. We have recently performed vascularized limb tissue allografts into four chimeric animals. These grafts have remained viable beyond 120 days, the end of the experiment. Our attention is now focusing on the possible mechanisms involved in generating the tolerant state in these offspring.

b) Limb tissue allografts in miniature swine: Whereas recipients of organ allotransplants require life-long immunosuppressive regimens to maintain their allograft, the use of chronic immunosuppressive therapy for limb tissue transplants is far less likely. Our attention has focused on using the MGH miniature swine as a model for understanding allotransplantion of limb tissues in adults. We have demonstrated that animals receiving vascularized musculoskeletal transplants from donors that are matched for major histocompatibility antigens and having minor antigen differences can accept their grafts for greater than one year using only a 12-day course of cyclosporine postoperatively. If cyclosporine is not given, the animals can reject the grafts transplanted across this minor barrier. We are exploring transplantation across increasingly stronger histocompatibility barriers such as class I MHC only or single haplotype class I and II MHC barriers. Another approach for achieving tolerance could be to generate mixed chimerism in the adult. Work in the TBRC has developed mixed chimerism protocol to establish stable multilineage chimerism across minor and major histocompatibility barriers using non-myeloablative regimens. We have developed similar protocols that work for limb tissue allotransplantation.

Free Flap Physiology and Monitoring:
Despite improvements in the success rate of free tissue transfers since the origin of the procedure, there is still a significant complication rate. Re-exploration rates range from 17.9% to 23.6% and only 41% of those flaps that are re-explored are rendered viable. The low rate of ischemic flap salvage demonstrates the need for two approaches undertaken in this laboratory: 1) develop a clear understanding of free flap perfusion and find ways to prevent ischemia-reperfusion injury, and 2) develop reliable post operative monitoring techniques to allow for earlier detection of free flaps failing because of vessel occlusion or ischemic insult.

a) Perfusion/reperfusion studies: Understanding flap perfusion parameters and avoiding the possible injury inflicted by reperfusion are central to the clinical success of free flap transfers. Our laboratory has undertaken several studies to elucidate these perfusion parameters including flaps in which and arteriovenous fistula have been created or the flaps have been delayed prior to transfer. Such studies are valuable in designing new flaps for defect coverage in clinical practice. Because free transfers always require a period of ischemia, warm or cold, many studies have explored means for reducing or eliminating the secondary injury caused by reperfusion. One possible means explored in this laboratory is the use of antibodies that can inhibit the adhesion molecules on leukocytes as they perfuse the ischemic tissue. We have shown that by inhibiting luekocyte attachment to the endothelium skin flaps subjected to prolonged periods of warm ischemia could be salvaged.

b) Flap monitoring: Even flaps that have been successfully reperfused in the operating theater can fail during the postoperative period. Despite the numerous techniques that have been introduced for monitoring flaps including temperature, pH, visual appearance, and laser doppler, no effective real-time monitor has been introduced. In collaboration with the Wellman Laboratories for Photomedicine, our group has been investigating diffuse reflectance spectroscopy (DRS) as a possible means for noninvasive, real-time measuring of tissue oxygenation. DRS utilizes a modified diode array spectrophotometer, which emits and receives light through flexible glass fiber bundles. When in contact with skin or muscle, the concentration of oxyhemoglobin, and deoxyhemaglobin in the superficial microvasculature of the tissue can be determined. We have ongoing work evaluating the monitoring of tissue levels of oxy and deoxyhemaglobin as a means for predicting vascular pedicle occlusion in myocutaneous flaps in a swine model using the rectus flap. We are investigating the possibility for using the probe on buried muscle flaps as well.

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