Using recent advances in tissue engineering and the principles behind the prefabrication of flaps, this research will compare various methods of bone formation in a capsular flap. The ultimate goal is to provide a technique to reconstruct bony defects by using a composite tissue flap engineered at a distant location and in a specific shape and then transferred to the site of the defect, thereby minimizing donor site morbidity and avoiding the limitations presented by a finite supply of autogenous bone available for grafting.

From previous experiments on prefabrication and prelamination of different cell types, including urothelial cells, tracheal epithelial cells, and chondrocytes, we have found that transposing a vascular pedicle to a subcutaneously placed silicone block will result in a vascular capsule, that can be mobilized and transferred based solely on the pedicle. The capsule provides the necessary blood supply for cell growth. No study to date has investigated osteoinduction in a prefabricated vascularized capsular flap.

In a clinical setting, this technique may provide another option in reconstruction of bony defects of the upper and lower extremities and the mandible due to trauma, tumor resection, or congenital abnormalities. The use of the patient's own bone marrow or adipose tissue as sources for cultured mesenchymal stem cells may represent the best option for creating new bone in the prefabrication of flaps without the morbidity associated with the harvesting of autogenous bone graft and may also prove to be more reliable in the osteoinductive process than bone graft substitutes or genetically engineered products. 

Composite tissue allografts (CTAs) are our best option in reconstructing complex tissue defects, including situations of extremity loss. Previous studies have investigated the relationship between dendritic cells (DCs) and the induction of tolerance to cardiac and skin allografts. We propose to further this model, utilizing composite tissues allografts to examine the extent and flexibility of this tolerance regimen.

Previous experiments by other authors have shown that the transfer of immunodominant allopeptidepulsed host dendritic cells have tolerized recipient rats to allogeneic solid organ transplants. Garrovillo and colleagues have shown that the intravenous administration of host dendritic cells (DCs) expressing donor MHC molecules have induced tolerance to donor cardiac allografts, in a WF-to-ACI rat combination. We propose to adopt this functioning model and to extend its use to a more stringent histocompatibility barrier, utilizing a WF-to-Lewis rat combination. In addition, we propose the use of a hind-limb transplant model, to examine the protocol's potential for tolerance to the diverse elements of a composite tissue allograft (CTA). If successful, this experiment would represent a significant step towards developing an elegant preclinical model for the eventual use of composite tissue allografts in the reconstruction of complex tissue defects, including situations of traumatic limb loss and amputation.

Normal cartilage is immunologically privileged by virtue of the absence of blood and lymphatic vessels. Chondrocytes that are embedded in lacunae within the extracellular matrix obtain nutrients through diffusion from the synovial fluid in the joint cavity but are excluded from interacting with cells of the immune system by their protective extracellular matrix. Under pathologic conditions, cartilage cells are exposed and can interact with lymphocytes and other cells present in the synovial tissue and fluid. For example, human articular cartilage chondrocytes from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) express class II major histocompatibility (MHC) antigens, a potent activator of immune cells. These antigens are not known to be expressed in the absence of pathology, and their presence in RA/OA may underlie an immune response to exposed chondrocytes in these conditions.

As the potential for tissue engineering approaches expands for cartilage repair, chondrocytes can be transplanted across syngeneic, allogeneic and xenogeneic barriers. During the process of engineering cartilage, chondrocytes are removed from their native immunoprotective matrix and the cell surface antigens are exposed to immunologic or antigen-presenting cells. This may lead to immune reaction against the naked chondrocytes, which may lead to pathologic conditions like those associated with arthritis. However, little is known about the expression of MHC molecules (class I or II) in engineered cartilage and their propensity to stimulate an immune response by the host.

Our laboratory has extensive experience in the field of tissue engineering cartilage using swine chondrocytes obtained from various anatomical sites (e.g. articular joints, ribs, ears, etc.) and encapsulated in biological or synthetic hydrogels. The Transplantation Biology Research Center (TBRC) of the Massachusetts General Hospital has developed a unique population of swine in which the immunogenetics have been mapped in order to perform well-controlled organ and tissue transplantation studies in a large animals. We propose to use these swine and the numerous cell-marker antibodies available to study the immunogenicity of chondrocytes in large animals that will serve as a valuable preclinical cartilage repair model.

Since the late 1800s, many methods for reconstructing non-ossifying adult calvarial defects have been developed. Yet, the search for the source of donor materials continues. Recent findings have suggested that adipose tissue, capable of in vitro adipogenic, osteogenic, chondrogenic, and neurogenic differentiation, could contain multi potent stromal progenitor cells. These findings opened new possibilities for reconstruction of calvarial defects using tissue engineering and cell-based therapies. While much interest has been generated over the multilineage potential of these adipose-derived mesenchymal stromal cells (ADMSCs), very little is known about the effect of tissue donor age on the biology or function of ADMSCs.

We hypothesize that juvenile and adult ADMSCs have different biologic profiles and thus different abilities to re-ossify calvarial defects. In the first Specific Aim, we will determine the differences in the molecular biology of juvenile and adult ADMSCs undergoing osteogenesis differentiation. Alterations in osteogenic gene expression will be assessed using micro array technology and quantitative, real-time RT-PCR. In the Second Specific Aim, we will utilize an established mouse critical size calvarial defect model to determine the ability of juvenile and adult ADMSCs to re-ossify critical size calvarial defects.

The research proposed in this application will help to direct future strategies in cell-based therapies for calvarial healing. This will facilitate the development of novel, clinically applicable techniques with which to reconstruct non-healing calvarial defects.

Reconstruction of osseous craniofacial defects often requires bone harvest, incurring significant donor site morbidity, cost and time. Examples include autografting for cleft palate and osteocutaneous free tissue transfer for mandibular defects. Tissue engineering employing gene therapy for bone regeneration would be a highly desirable alternative. One method utilizes bioresorbable polymer scaffolding at the bony defect site containing plasmid DNA of bone growth factors. Previous studies have demonstrated bone growth in the rat calvarial critical-sized defect model with bone morphogenetic protein-4 (BMP-4) plasmid DNA. Other research suggests a synergistic effect of BMP-4 and vascular endothelial growth factor (VEGF) on bone regeneration.

Objectives: 1) To confirm bone regeneration with a polymer scaffold containing BMP-4 plasmid DNA in the rat calvarial critical-sized defect model. 2) To quantify bone regeneration with VEGF plasmid DNA in the above model. 3) To investigate a possible synergistic effect of BMP-4 and VEGF plasmid DNA in the above model. Methods: Calvarial critical sized-defects will be created in 110 male Sprague-Dawley rats. Animals will be divided into 5 groups of 22 by defect treatment: none (negative control); VEGF plasmid DNA; BMP-4 plasmid DNA; VEGF and BMP-4 plasmid DNA; and autograft (positive control). Animals will be sacrificed at 4 and 8 weeks (11 animals per group at each time point); calvaria will be radiographed and histologically sectioned.

Outcomes: Histomorphometry and radiomorphometry will measure bone growth within calvarial defects as a function of total defect area to quantify bone regeneration effects of BMP-4 and VEGF plasmid DNA alone and in combination. 

Muscle undergoes rapid atrophy after injury to its supplying peripheral nerve, and frequently fibroses before being reached by regenerating motor neurons. After 12 to 18 months, the severe atrophy and fibrotic scarring prevent any chance of recovery of function even after reinnervation. This is particularly problematic for proximal peripheral nerve injuries, where neurons regenerating at a rate of about 1mm/day may take years to cover the distance between the proximal nerve stump and the target muscle. As a result, upper brachial plexus injuries generally lead to permanent weakness or paralysis of the muscles of the shoulder, arm and wrist. We hypothesize that transplantation of neural stem cells into denervated muscle will provide trophic support to the muscle and preserve the capacity for motor recovery.

Furthermore, we hypothesize that stem cell derived motor neurons will form motor end-plates with denervated adult muscle fibers. The ability to form appropriate chemical connections with target cells would support the potential future use of neural stem cells to engineer peripheral nerve. We are encouraged by the recent demonstration of muscle innervation and motor end plate formation by transplanted stem cell derived motor neurons in an avian embryo (Wichterle et al. 2002 Cell 110: 385-97). We propose to investigate whether stem cell derived motor neuron progenitors will innervate paralyzed adult muscle in a mammalian model of peripheral nerve injury.