Long-term Transplant Function After Thrombolytic Treatment Ex Vivo of Donated Kidneys Retrieved 4 to 5 H After Circulatory Death.

BACKGROUND: Using a novel thrombolytic technique, we present long-term transplant function, measured by creatinine and iohexol clearance, after utilizing kidneys from porcine donors with uncontrolled donation after circulatory deaths, with 4.5-5 h of warm ischemia. METHODS: Pigs in the study group were subjected to simulated circulatory death. After 2 h, ice slush was inserted into the abdomen and 4.5 h after death, the kidneys were retrieved. Lys-plasminogen, antithrombin-III, and alteplase were injected through the renal arteries on the back table. Subsequent ex vivo perfusion was continued for 3 h at 15°C, followed by 3 h with red blood cells at 32°C, and then transplanted into pigs as an autologous graft as only renal support. Living-donor recipient pigs that did not receive ex vivo perfusion, and unilateral nephrectomized pigs served as the controls. RESULTS: Pigs in the study group (n = 13), surviving 10 d or more were included, of which 7 survived for 3 mo. Four animals in the living-donor group (n = 6) and all 5 nephrectomized animals survived for 3 mo. Creatinine levels in the plasma and urine, neutrophil gelatinase-associated lipocalin levels, Kidney Injury Marker-1 expression, and iohexol clearance at 3 mo did not differ significantly between the study and living-donor groups. Histology and transmission electron microscopy after 3 mo showed negligible fibrosis and no other damage. CONCLUSIONS: The present method salvages kidneys from extended unontrolled donation after circulatory death using thrombolytic treatment while preserving histology and enabling transplantation after ex vivo reconditioning, with clinically acceptable late function after 3 mo, as measured by creatinine and iohexol clearance.

Novel Ex-Vivo Thrombolytic Reconditioning of Kidneys Retrieved 4 to 5 Hours After Circulatory Death.

BACKGROUND: Due to organ shortage, many patients do not receive donor organs. The present novel thrombolytic technique utilizes organs from donors with uncontrolled donation after circulatory deaths (uDCD), with up to 4-5 h warm ischemia, without advanced cardiopulmonary resuscitation (aCPR) or extracorporeal circulation (EC) after death. METHODS: The study group of pigs (n = 21) underwent simulated circulatory death. After 2 h, an ice slush was inserted into the abdomen. Kidneys were retrieved 4.5 h after death. Lys-plasminogen, antithrombin-III (ATIII), and alteplase (tPA) were injected through the renal arteries on the back table. Subsequent ex vivo perfusion at 15 °C was continued for 3 h, followed by 3 h with red blood cells (RBCs) at 32 °C. Perfusion outcome and histology were compared between uDCD kidneys, receiving no thrombolytic treatment (n = 8), and live donor kidneys (n = 7). The study kidneys were then transplanted into pigs as autologous grafts with a single functioning autologous kidney as the only renal support. uDCD control pigs (n = 8), receiving no ex vivo perfusion, served as controls. RESULTS: Vascular resistance decreased to <200 mmHg/mL/min ( P < 0.0023) and arterial flow increased to >100 mL/100 g/min ( P < 0.00019) compared to controls. In total 13/21 study pigs survived for >10 days, while all uDCD control pigs died. Histology was preserved after reconditioning, and the creatinine level after 10 days was next to normal. CONCLUSIONS: Kidneys from extended uDCD, not receiving aCPR/EC, can be salvaged using thrombolytic treatment to remove fibrin thrombi while preserving histology and enabling transplantation with a clinically acceptable early function.

Characterization of Decellularized Implants for Extracellular Matrix Integrity and Immune Response Elicitation.

Biological scaffold is a popular choice for the preparation of tissue-engineered organs and has the potential to address donor shortages in clinics. However, biological scaffolds prepared by physical or chemical agents cause damage to the extracellular matrix (ECM) by potentially inducing immune responses after implantation. The current study explores the fate of the decellularized (DC) scaffolds using a cocktail of chemicals following implantation without using immunosuppressants. Using the syngeneic (Lewis male-Lewis female) and allogeneic (Brown Norway male-Lewis female) models and different tissue routes (subcutaneous vs. omentum) for implantation, we applied in-depth quantitative proteomics, genomics along with histology and quantitative image analysis tools to comprehensively describe and compare the proteins following DC and postimplantation. Our data helped to identify any alteration postdecullarization as well implantation. We could also monitor route-specific modulation of the ECM and regulation of the immune responses (macrophage and T cells) following implantation. The current approach opens up the possibility to monitor the fate of biological scaffolds in terms of the ECM and immune response against the implants. In addition, the identification of different routes helped us to identify differential immune responses against the implants. This study opens up the potential to identify the changes associated with chemical DC both pre- and postimplantation, which could further help to promote research in this direction. Impact Statement The development of a biological scaffold helps in the preparation of a functional organ in the clinics. In the current study, we develop a strategy for chemical decellularization and explored two different routes to understand the differential responses elicited postimplantation. The use of sensitive protein and genomic tools to study the changes creates a favorable environment for similar efforts to develop and characterize biological scaffolds before further trials in the clinics. The current study, which was carried out without any immunosuppressive agents, could help to establish (a) appropriate chemical strategies for preparing biological scaffolds as well as (b) identify putative implantable routes to circumvent any adverse immune reactions, which will ultimately decide the outcome for acceptance or rejection of the scaffold/implant.

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.

Replacement of a tracheal stenosis with a tissue-engineered human trachea using autologous stem cells: a case report.

Cell-based therapies involving tissue engineering represent interesting and potentially important strategies for the treatment of patients with various disorders. In this study, using a detergent-enzymatic method, we prepared an intact three-dimensional scaffold of an extracellular matrix derived from a human cadaver donor trachea, which we repopulated with autologous stem cells and implanted into a 76-year-old patient with tracheal stenosis including the lower part of the larynx. Although the graft provided the patient with an open airway, a week after the surgery, the mucous membrane of the graft was covered by a 1-2 mm thick fungal infection, which was treated with local and systemic antifungal therapy. The airway lumen was postoperatively controlled by fiber endoscopy and found stable and sufficient. However, after 23 days, the patient died due to cardiac arrest but with a patent, open, and stable tracheal transplant and intact anastomoses. Histopathological results of the transplanted tracheal graft during autopsy showed a squamous but not ciliated epithelium, neovascularization, bundles of α-sma-positive muscle cells, serous glands, and nerve fibers with S-100-positive nerve cells in the submucosa and intact chondrocytes in the cartilage. Our findings suggest that although autologous stem cells-engineered tracheal matrices may represent a tool for clinical tracheal replacement, further preclinical studies are required for generating functional airway grafts and long-term effects of such grafts.