Christopher K. Breuer

Tissue-Engineered Lungs for in Vivo Implantation

Waiting to Exhale Lung tissue does not regenerate, so, when it is damaged by disease and/or surgically removed, lung transplantation is often the only treatment option. Because donor tissue is in short supply, there has been a long-standing interest in engineering functional and transplantable lung tissue in the laboratory. Petersen et al. (p. 538, published online 24 June; see the Perspective by Wagner and Griffith) now report an important step in this direction. After gently removing the cellular constituents of rat lungs with detergent, the residual scaffold of extracellular matrix—which retained the compliance and mechanical properties of the original lung—was re-seeded with a mixture of lung epithelial and endothelial cells and cultured in a bioreactor. Within a few days, the engineered lung tissue contained alveoli, microvessels, and small airways that were repopulated with the appropriate cell types. When transplanted into a rat for short time periods, the engineered lung showed evidence of gas exchange. Decellularized rat lungs rebuilt with new cells in vitro can function at a rudimentary level when implanted back into a rat. Because adult lung tissue has limited regeneration capacity, lung transplantation is the primary therapy for severely damaged lungs. To explore whether lung tissue can be regenerated in vitro, we treated lungs from adult rats using a procedure that removes cellular components but leaves behind a scaffold of extracellular matrix that retains the hierarchical branching structures of airways and vasculature. We then used a bioreactor to culture pulmonary epithelium and vascular endothelium on the acellular lung matrix. The seeded epithelium displayed remarkable hierarchical organization within the matrix, and the seeded endothelial cells efficiently repopulated the vascular compartment. In vitro, the mechanical characteristics of the engineered lungs were similar to those of native lung tissue, and when implanted into rats in vivo for short time intervals (45 to 120 minutes) the engineered lungs participated in gas exchange. Although representing only an initial step toward the ultimate goal of generating fully functional lungs in vitro, these results suggest that repopulation of lung matrix is a viable strategy for lung regeneration.

Stabilized polyglycolic acid fibre-based tubes for tissue engineering

Polyglycolic acid (PGA) fibre meshes are attractive candidates to transplant cells, but they are incapable of resisting significant compressional forces. To stabilize PGA meshes, atomized solutions of poly(L-lactic acid) (PLLA) and a 50/50 copolymer of poly(D,L-lactic-co-glycolic acid) (PLGA) dissolved in chloroform were sprayed over meshes formed into hollow tubes. The PLLA and PLGA coated the PGA fibres and physically bonded adjacent fibres. The pattern and extent of bonding was controlled by the concentration of polymer in the atomized solution and the total mass of polymer sprayed on the device. The compression resistance of devices increased with the extent of bonding, and PLLA bonded tubes resisted larger compressive forces than PLGA bonded tubes. Tubes bonded with PLLA degraded more slowly than devices bonded with PLGA. Implantation of PLLA bonded tubes into rats revealed that the devices maintained their structure during fibrovascular tissue ingrowth, resulting in the formation of a tubular structure with a central lumen. The potential of these devices to engineer specific tissues was exhibited by the finding that smooth muscle cells and endothelial cells seeded onto devices in vitro formed a tubular tissue with appropriate cell distribution.

Tissue-engineered vascular grafts transform into mature blood vessels via an inflammation-mediated process of vascular remodeling

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are the earliest tissue-engineered vascular grafts (TEVGs) to be used clinically. These TEVGs transform into living blood vessels in vivo, with an endothelial cell (EC) lining invested by smooth muscle cells (SMCs); however, the process by which this occurs is unclear. To test if the seeded BMCs differentiate into the mature vascular cells of the neovessel, we implanted an immunodeficient mouse recipient with human BMC (hBMC)-seeded scaffolds. As in humans, TEVGs implanted in a mouse host as venous interposition grafts gradually transformed into living blood vessels over a 6-month time course. Seeded hBMCs, however, were no longer detectable within a few days of implantation. Instead, scaffolds were initially repopulated by mouse monocytes and subsequently repopulated by mouse SMCs and ECs. Seeded BMCs secreted significant amounts of monocyte chemoattractant protein-1 and increased early monocyte recruitment. These findings suggest TEVGs transform into functional neovessels via an inflammatory process of vascular remodeling.

Late-term results of tissue-engineered vascular grafts in humans

OBJECTIVE The development of a tissue-engineered vascular graft with the ability to grow and remodel holds promise for advancing cardiac surgery. In 2001, we began a human trial evaluating these grafts in patients with single ventricle physiology. We report the late clinical and radiologic surveillance of a patient cohort that underwent implantation of tissue-engineered vascular grafts as extracardiac cavopulmonary conduits. METHODS Autologous bone marrow was obtained and the mononuclear cell component was collected. Mononuclear cells were seeded onto a biodegradable scaffold composed of polyglycolic acid and epsilon-caprolactone/L-lactide and implanted as extracardiac cavopulmonary conduits in patients with single ventricle physiology. Patients were followed up by postoperative clinic visits and by telephone. Additionally, ultrasonography, angiography, computed tomography, and magnetic resonance imaging were used for postoperative graft surveillance. RESULTS Twenty-five grafts were implanted (median patient age, 5.5 years). There was no graft-related mortality (mean follow-up, 5.8 years). There was no evidence of aneurysm formation, graft rupture, graft infection, or ectopic calcification. One patient had a partial mural thrombosis that was successfully treated with warfarin. Four patients had graft stenosis and underwent successful percutaneous angioplasty. CONCLUSION Tissue-engineered vascular grafts can be used as conduits in patients with single ventricle physiology. Graft stenosis is the primary mode of graft failure. Further follow-up and investigation for the mechanism of stenosis are warranted.

Vascular tissue engineering: towards the next generation vascular grafts.

The application of tissue engineering technology to cardiovascular surgery holds great promise for improving outcomes in patients with cardiovascular diseases. Currently used synthetic vascular grafts have several limitations including thrombogenicity, increased risk of infection, and lack of growth potential. We have completed the first clinical trial evaluating the feasibility of using tissue engineered vascular grafts (TEVG) created by seeding autologous bone marrow-derived mononuclear cells (BM-MNC) onto biodegradable tubular scaffolds. Despite an excellent safety profile, data from the clinical trial suggest that the primary graft related complication of the TEVG is stenosis, affecting approximately 16% of grafts within the first seven years after implantation. Continued investigation into the cellular and molecular mechanisms underlying vascular neotissue formation will improve our basic understanding and provide insights that will enable the rationale design of second generation TEVG.

Small-diameter biodegradable scaffolds for functional vascular tissue engineering in the mouse model.

The development of neotissue in tissue engineered vascular grafts remains poorly understood. Advances in mouse genetic models have been highly informative in the study of vascular biology, but have been inaccessible to vascular tissue engineers due to technical limitations on the use of mouse recipients. To this end, we have developed a method for constructing sub-1mm internal diameter (ID) biodegradable scaffolds utilizing a dual cylinder chamber molding system and a hybrid polyester sealant scaled for use in a mouse model. Scaffolds constructed from either polyglycolic acid or poly-l-lactic acid nonwoven felts demonstrated sufficient porosity, biomechanical profile, and biocompatibility to function as vascular grafts. The scaffolds implanted as either inferior vena cava or aortic interposition grafts in SCID/bg mice demonstrated excellent patency without evidence of thromboembolic complications or aneurysm formation. A foreign body immune response was observed with marked macrophage infiltration and giant cell formation by post-operative week 3. Organized vascular neotissue, consisting of endothelialization, medial generation, and collagen deposition, was evident within the internal lumen of the scaffolds by post-operative week 6. These results present the ability to create sub-1mm ID biodegradable tubular scaffolds that are functional as vascular grafts, and provide an experimental approach for the study of vascular tissue engineering using mouse models.

The in vitro construction of a tissue engineered bioprosthetic heart valve.

PROBLEM Heart valve replacement with either a nonliving xenograft or a mechanical prosthesis is an effective therapy for valvular heart disease. Both of these approaches have limitations, including their inability to grow, repair, and remodel. In addition, a mechanical prosthesis requires long-term anticoagulation therapy. METHODS This study demonstrates the in vitro creation of tissue engineered heart valve tissue using cardiovascular cells on degradable polymer matrices, 40 heart valve leaflets were created using this technique from two sources. Xenograft leaflets were created using human dermal fibroblasts and bovine aortic endothelial cells (n = 20) or allograft valve leaflets were created using sheep myofibroblasts and sheep endothelial cells (n = 20). A mixed sheep cell population was obtained consisting of endothelial cells and myofibroblasts. Endothelial cells were labelled with acethylated low density lipoprotein (Ac-Dil-LDL) and cells were separated into two groups using an activated cell sorter: LDL positive cells comprised of a pure endothelial cell population and LDL negative cells comprised of mixed cell population containing myofibroblasts and smooth muscle cells. The LDL negative cells were seeded on a synthetic polyglycolic acid (PGA) mesh and grown in vitro to form a tissue-like fibroblast-mesh core. Endothelial cells were then seeded onto the surface of the fibroblast-mesh core, forming a single monolayer. RESULTS Histological evaluation of these constructs revealed an inner core of LDL negative cells and outer endothelial-like cells which were factor VIII positive. There was no evidence of capillary formation from endothelial cells invading the myofibroblasts and smooth muscle matrix and the endothelial lining appeared complete. CONCLUSIONS It is feasible to construct allogenic heart valve tissue which could be used to make a valve.

A critical role for macrophages in neovessel formation and the development of stenosis in tissue-engineered vascular grafts

The primary graft‐related complication during the first clinical trial evaluating the use of tissue‐engineered vascular grafts (TEVGs) was stenosis. We investigated the role of macrophages in the formation of TEVG stenosis in a murine model. We analyzed the natural history of TEVG macrophage infiltration at critical time points and evaluated the role of cell seeding on neovessel formation. To assess the function of infiltrating macrophages, we implanted TEVGs into mice that had been macrophage depleted using clodronate liposomes. To confirm this, we used a CD11b‐diphtheria toxin‐receptor (DTR) transgenic mouse model. Monocytes infiltrated the scaffold within the first few days and initially transformed into M1 macrophages. As the scaffold degraded, the macrophage infiltrate disappeared. Cell seeding decreased the incidence of stenosis (32% seeded, 64% unseeded, P= 0.024) and the degree of macrophage infiltration at 2 wk. Unseeded TEVGs demonstrated conversion from M1 to M2 phenotype, whereas seeded grafts did not. Clodronate and DTR inhibited macrophage infiltration and decreased stenosis but blocked formation of vascular neotissue, evidenced by the absence of endothelial and smooth muscle cells and collagen. These findings suggest that macrophage infiltration is critical for neovessel formation and provides a strategy for predicting, detecting, and inhibiting stenosis in TEVGs.—Hibino, N., Yi, T., Duncan, D. R., Rathore, A., Dean, E., Naito, Y., Dardik, A., Kyriakides, T., Madri, J., Pober, J. S., Shinoka, T., Breuer, C. K. A critical role for macrophages in neovessel formation and the development of stenosis in tissue‐engineered vascular grafts. FASEB J. 25, 4253–4263 (2011). www.fasebj.org

The Treatment of Differentiated Thyroid Cancer in Children: Emphasis on Surgical Approach and Radioactive Iodine Therapy

Pediatric thyroid cancer is a rare disease with an excellent prognosis. Compared with adults, epithelial-derived differentiated thyroid cancer (DTC), which includes papillary and follicular thyroid cancer, presents at more advanced stages in children and is associated with higher rates of recurrence. Because of its uncommon occurrence, randomized trials have not been applied to test best-care options in children. Even in adults that have a 10-fold or higher incidence of thyroid cancer than children, few prospective trials have been executed to compare treatment approaches. We recognize that treatment recommendations have changed over the past few decades and will continue to do so. Respecting the aggressiveness of pediatric thyroid cancer, high recurrence rates, and the problems associated with decades of long-term follow-up, a premium should be placed on treatments that minimize risk of recurrence and the adverse effects of treatments and facilitate follow-up. We recommend that total thyroidectomy and central compartment lymph node dissection is the surgical procedure of choice for children with DTC if it can be performed by a high-volume thyroid surgeon. We recommend radioactive iodine therapy for remnant ablation or residual disease for most children with DTC. We recommend long-term follow-up because disease can recur decades after initial diagnosis and therapy. Considering the complexity of DTC management and the potential complications associated with therapy, it is essential that pediatric DTC be managed by physicians with expertise in this area.

Tissue engineered vascular grafts demonstrate evidence of growth and development when implanted in a juvenile animal model

Introduction:The development of a living, autologous vascular graft with the ability to grow holds great promise for advancing the field of pediatric cardiothoracic surgery. Objective:To evaluate the growth potential of a tissue-engineered vascular graft (TEVG) in a juvenile animal model. Methods:Polyglycolic acid nonwoven mesh tubes (3-cm length, 1.3-cm id; Concordia Fibers) coated with a 10% copolymer solution of 50:50 l-lactide and &egr;-caprolactone were statically seeded with 1 × 106 cells/cm2 autologous bone marrow derived mononuclear cells. Eight TEVGs (7 seeded, 1 unseeded control) were implanted as inferior vena cava (IVC) interposition grafts in juvenile lambs. Subjects underwent bimonthly magnetic resonance angiography (Siemens 1.5 T) with vascular image analysis (www.BioimageSuite.org). One of 7-seeded grafts was explanted after 1 month and all others were explanted 6 months after implantation. Neotissue was characterized using qualitative histologic and immunohistochemical staining and quantitative biochemical analysis. Results:All grafts explanted at 6 months were patent and increased in volume as measured by difference in pixel summation in magnetic resonance angiography at 1 month and 6 months. The volume of seeded TEVGs at explant averaged 126.9% ± 9.9% of their volume at 1 month. Magnetic resonance imaging demonstrated no evidence of aneurysmal dilation. TEVG resembled the native IVC histologically and had comparable collagen (157.9 ± 26.4 &mgr;g/mg), elastin (186.9 ± 16.7 &mgr;g/mg), and glycosaminoglycan (9.7 ± 0.8 &mgr;g/mg) contents. Immunohistochemical staining and Western blot analysis showed that Ephrin-B4, a determinant of normal venous development, was acquired in the seeded grafts 6 months after implantation. Conclusions:TEVGs demonstrate evidence of growth and venous development when implanted in the IVC of a juvenile lamb model.