Impact of gamma-Irradiation on Extracellular Matrix of Porcine Pulmonary Valves
ABSTRACT The extracellular matrix plays an important role in heart valve function. To improve the processing of porcine pulmonary valves for clinical use, we have studied the influence of cryopreservation, decellularization, and irradiation on extracellular matrix components.
Decellularization was carried out followed by DNAseI/RNAseA digestion and isotonic washout. Valves were cryopreserved in 10% DMSO/10% fetal bovine serum, and then subjected to 25-40 kGy γ-radiation. Extracellular matrix constituents were evaluated by histologic staining, immunohistochemistry, transmission electron microscopy, and liquid chromatography/mass spectrometry.
Histologic, immunohistochemical, ultrastructural, and biochemical analyses demonstrated a marked reduction in the expression of extracellular matrix components particularly in the valves that had been γ-irradiated following decellularization and cryopreservation. In this group, histology and immunohistochemistry showed an obvious reduction in staining for chondroitin sulphates, versican, hyaluronan, and collagens. Transmission electron microscopy revealed the smallest fibril diameter of collagen, shortest D-period, and loss of compactness of collagen fiber packaging and fragmentation of elastic fibers. Biochemical analysis showed loss of collagen and elastin crosslinks. Decellularization followed by cryopreservation showed some reduction in staining for collagens and versican, smaller diameter, shorter D-period in collagen fibers, and ridges in elastic fibers. Cryopreservation alone showed minimal changes in ECM staining intensity, collagen, and elastin ultrastructure and biochemistry.
γ-Irradiated valves that have been decellularized and cryopreserved produces significant changes in the expression of ECM components, thus providing useful information for improving valve preparation for clinical use and also some indication as to why irradiated human heart valves were not clinically successful.
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ABSTRACT: Liver disease affects millions of patients each year. The field of regenerative medicine promises alternative therapeutic approaches, including the potential to bioengineer replacement hepatic tissue. One approach combines cells with acellular scaffolds derived from animal tissue. The goal of this study was to scale up our rodent liver decellularization method to livers of a clinically relevant size. Porcine livers were cannulated via the hepatic artery, then perfused with PBS, followed by successive Triton X-100 and SDS solutions in saline buffer. After several days of rinsing, decellularized liver samples were histologically analyzed. In addition, biopsy specimens of decellularized scaffolds were seeded with hepatoblastoma cells for cytotoxicity testing or implanted s.c. into rodents to investigate scaffold immunogenicity. Histological staining confirmed cellular clearance from pig livers, with removal of nuclei and cytoskeletal components and widespread preservation of structural extracellular molecules. Scanning electron microscopy confirmed preservation of an intact liver capsule, a porous acellular lattice structure with intact vessels and striated basement membrane. Liver scaffolds supported cells over 21 days, and no increased immune response was seen with either allogeneic (rat-into-rat) or xenogeneic (pig-into-rat) transplants over 28 days, compared with sham-operated on controls. These studies demonstrate that successful decellularization of the porcine liver could be achieved with protocols developed for rat livers, yielding nonimmunogenic scaffolds for future hepatic bioengineering studies.American Journal Of Pathology 06/2013; 19(2). DOI:10.1016/j.ajpath.2013.05.002 · 4.60 Impact Factor
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ABSTRACT: Xenogeneic tissues are derived from other animal species and provide a source of material for engineering mechanically functional tissue grafts, such as heart valves, tendons, ligaments, and cartilage. Xenogeneic tissues, however, contain molecules, known as antigens, which invoke an immune reaction following implantation into a patient. Therefore, it is necessary to remove the antigens from a xenogeneic tissue to prevent immune rejection of the graft. Antigen removal can be accomplished by treating a tissue with solutions and/or physical processes that disrupt cells and solubilize, degrade, or mask antigens. However, processes used for cell and antigen removal from tissues often have deleterious effects on the extracellular matrix (ECM) of the tissue, rendering the tissue unsuitable for implantation due to poor mechanical properties. Thus, the goal of an antigen removal process should be to reduce the antigen content of a xenogeneic tissue while preserving its mechanical functionality. To expand the clinical use of antigen-removed xenogeneic tissues as biomechanically functional grafts, it is essential that researchers examine tissue antigen content, ECM composition and architecture, and mechanical properties as new antigen removal processes are developed.Journal of Biomechanics 11/2013; DOI:10.1016/j.jbiomech.2013.10.041 · 2.50 Impact Factor
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ABSTRACT: The authors compared clinical outcomes to determine whether acellular dermal matrix altered the capsular tissue architecture in irradiated and nonirradiated breasts following matrix-assisted expander reconstruction. Part I included all 27 patients who underwent bilateral tissue expander reconstruction with acellular dermal matrix between 2007 and 2012 and subsequent unilateral radiation therapy. Part II included a subset of patients with capsular biopsy specimens taken at the time of implant exchange for histologic analysis. Specimens included irradiated and nonirradiated acellular dermal matrix and irradiated and nonirradiated native capsule. Clinical outcomes were analyzed in relation to capsule architecture and acellular dermal matrix performance. In part I, mean follow-up was 28 months. Grade III/IV contractures were identified in nine patients (all on the irradiated side), and 12 developed noncontracture complications (75 percent on the irradiated side). Nine patients were unable to continue with implant reconstruction and required salvage with autologous tissue. In part II, postirradiation biopsy specimens were taken of the peri-implant capsule in six patients at the time of secondary surgery. Elastin content and the total cellular infiltrate were significantly greater in the irradiated versus nonirradiated native capsules (p = 0.0015). Conversely, the irradiated matrix capsule was composed of similar amounts of cellular infiltrate and collagen as the nonirradiated matrix capsules and nonirradiated native capsules. Irradiated acellular dermal matrix showed the least amount of alpha-smooth actin staining but a similar number of blood vessels. Acellular dermal matrix appears to limit the elastosis and chronic inflammation seen in irradiated implant reconstructions and is potentially beneficial in these patients. Therapeutic, III.Plastic and Reconstructive Surgery 02/2014; 133(2):214-21. DOI:10.1097/01.prs.0000437255.01199.42 · 3.33 Impact Factor