Combined Use of Detergents and Ultrasonication for Generation of an Acellular Pig Larynx.

The larynx is a fairly complex organ comprised of different muscles, cartilages, mucosal membrane, and nerves. Larynx cancer is generally the most common type of head and neck cancer. Treatment options are limited in patients with total or partial laryngectomy. Tissue-engineered organs have shown to be a promising alternative treatment for patients with laryngectomy. In this report we present an alternative and simple procedure to construct a whole pig larynx scaffold consisting of complete acellular structures of integrated muscle and cartilage. Larynges were decellularized (DC) using perfusion-agitation with detergents coupled with ultrasonication. DC larynges were then characterized to investigate the extracellular matrix (ECM) proteins, residual DNA, angiogenic growth factors, and morphological and ultrastructural changes to ECM fibers. After 17 decellularization cycles, no cells were observed in all areas of the larynx as confirmed by hematoxylin and eosin and DAPI (4',6-diamidino-2-phenylindole) staining. However, DC structures of dense thyroid and cricoid cartilage showed remnants of cells. All structures of DC larynges (epiglottis [p < 0.0001], muscle [p < 0.0001], trachea [p = 0.0045], and esophagus [p = 0.0008]) showed DNA <50 ng/mg compared with native larynx. Immunohistochemistry, Masson's trichrome staining, and Luminex analyses showed preservation of important ECM proteins and angiogenic growth factors in DC larynges. Compared with other growth factors, mostly retained growth factors in DC epiglottis, thyroid muscle, and trachea include granulocyte colony-stimulating factor, Leptin, fibroblast growth factor-1, Follistatin, hepatocyte growth factor, and vascular endothelial growth factor-A. Scanning electron microscopy and transmission electron microscopy analysis confirmed the structural arrangements of ECM fibers in larynges to be well preserved after DC. Our findings suggest that larynges can be effectively DC using detergent ultrasonication. ECM proteins and angiogenic growth factors appear to be better preserved using this method when compared with the native structures of larynges. This alternative DC method could be helpful in building scaffolds from dense tissue structures such as cartilage, tendon, larynx, or trachea for future in vitro recellularization studies or in vivo implantation studies in the clinic.

Differential Activation of Immune Cells for Genetically Different Decellularized Cardiac Tissues.

The immunogenicity of the extracellular matrix (ECM) from genetically similar (syngeneic) and dissimilar (allogeneic and xenogeneic) species has puzzled the scientific community for many years. After implantation, the literature describes an absorption of ECM material since it is biodegradable. However, no clear insight really exists to substantiate how the underlying immune and biological responses result in absorption of ECM materials. In this context, it is important to characterize infiltrating cells and identify dominant cell populations in the infiltrate. We have studied the immune response in mice after implantation of decellularized (DC) cardiac scaffolds derived from pig and mouse. The polymorphism of the infiltrate into the implanted material signifies the importance of the adaptive immune response that is distinct for xenoimplants and alloimplants. Matrix resorption takes place mainly through phagocytic cells such as mast cells, dendritic cells, and macrophages. Histochemical observations show that innate CD8+ T cells develop immune tolerance, whereas proteomic analysis predicts the different T cell progenies for alloscaffolds and xenoscaffolds. The amalgamation of graft tolerance and involvement of both B and T cell populations in the vicinity of the graft could be decisive in wound remodeling and survival of the graft. This challenging area presents potential targets for the development of immune-privileged biomaterials, immune tolerant cells, and therapeutic agents in the future. Impact statement In this study, we have characterized the allogeneic and xenogeneic immune responses for decellularized (DC) cardiac scaffolds. We postulate that although the T cells are important players for immune tolerance of DC graft, the mechanism of their differentiation inside the host is donor specific. In this study, we have reported the distinct immune responses for syngeneic DC scaffolds than allogeneic and xenogeneic scaffolds. This distinct response provides the bases for the different immune responses reported for DC homografts in the literature. This study can provide the greater insight for modification of postimplant strategies to achieve host acceptance of donor extracellular matrix scaffolds.

In Vitro Regeneration of Decellularized Pig Esophagus Using Human Amniotic Stem Cells.

Decellularization of esophagus was studied using three different protocols. The sodium deoxycholate/DNase-I (SDC/DNase-I) method was the most successful as evidenced by histology and DNA quantification of the acellular scaffolds. Acellular scaffolds were further analyzed and compared with native tissue by histology, quantitative analysis of DNA, and extracellular matrix (ECM) proteins. Histologically, the SDC/DNase-I protocol effectively produced scaffold with preserved structural architecture similar to native tissue architecture devoid of any cell nucleus. ECM proteins, such as collagen, elastin, and glycosaminoglycans were present even after detergent-enzymatic decellularization. Immunohistochemical analysis of acellular scaffold showed weak expression of Gal 1, 3 Gal epitope compared with native tissue. For performing recellularization, human amnion-derived mesenchymal stem cells (MSCs) and epithelial cells were seeded onto acellular esophagus in a perfusion-rotation bioreactor. In recellularized esophagus, immunohistochemistry showed infiltration of MSCs from adventitia into the muscularis externa and differentiation of MSCs into the smooth muscle actin and few endothelial cells (CD31). Our study demonstrates successful preparation and characterization of a decellularized esophagus with reduced load of Gal 1, 3 Gal epitope with preserved architecture and ECM proteins similar to native tissue. Upon subsequent recellularization, xenogeneic acellular esophagus also supported stem cell growth and partial differentiation of stem cells. Hence, the current study offers the hope for preparing a tissue-engineered esophagus in vitro which can be transplanted further into pigs for further in vivo evaluation.

An alternative approach to decellularize whole porcine heart.

Scaffold characteristics are decisive for repopulating the acellular tissue with cells. A method to produce such a scaffold from intact organ requires a customized decellularization protocol. Here, we have decellularized whole, intact porcine hearts by serial perfusion and agitation of hypotonic solution, an ionic detergent (4% sodium deoxycholate), and a nonionic detergent (1% Triton X-100). The resultant matrix was characterized for its degree of decellularization, morphological and functional integrity. The protocol used resulted in extensive decellularization of the cardiac tissue, but the cytoskeletal elements (contractile apparatus) of cardiomyocytes remained largely unaffected by the procedure although their membranous organelles were completely absent. Further, several residual angiogenic growth factors were found to be present in the decellularized tissue.

Transplantation of an allogeneic vein bioengineered with autologous stem cells: a proof-of-concept study.

BACKGROUND: Extrahepatic portal vein obstruction can have severe health consequences. Variceal bleeding associated with this disorder causes upper gastrointestinal bleeding, leading to substantial morbidity and mortality. We report the clinical transplantation of a deceased donor iliac vein graft repopulated with recipient autologous stem cells in a patient with extrahepatic portal vein obstruction. METHODS: A 10 year old girl with extrahepatic portal vein obstruction was admitted to the Sahlgrenska University Hospital in Gothenburg, Sweden, for a bypass procedure between the superior mesenteric vein and the intrahepatic left portal vein (meso Rex bypass). A 9 cm segment of allogeneic donor iliac vein was decellularised and subsequently recellularised with endothelial and smooth muscle cells differentiated from stem cells obtained from the bone marrow of the recipient. This graft was used because the patient's umbilical vein was not suitable and other strategies (eg, liver transplantation) require lifelong immunosuppression. FINDINGS: The graft immediately provided the recipient with a functional blood supply (25-30 cm/s in the portal vein and 40 mL/s in the artery was measured intraoperatively and confirmed with ultrasound). The patient had normal laboratory values for 9 months. However, at 1 year the blood flow was low and, on exploration, the shunt was patent but too narrow due to mechanical obstruction of tissue in the mesocolon. Once the tissue causing the compression was removed the graft dilated. We therefore used a second stem-cell populated vein graft to lengthen the previous graft. After this second operation, the portal pressure was reduced from 20 mm Hg to 13 mm Hg and blood flow was 25-40 cm/s in the portal vein. With restored portal circulation the patient has substantially improved physical and mental function and growth. The patient has no anti-endothelial cell antibodies and is receiving no immunosuppressive drugs. INTERPRETATION: An acellularised deceased donor vein graft recellularised with autologous stem cells can be considered for patients in need of vascular vein shunts without the need for immunosuppression. FUNDING: Swedish Government.