| 1. |
- Banerjee, Debashish, et al.
(författare)
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Characterization of Decellularized Implants for Extracellular Matrix Integrity and Immune Response Elicitation
- 2022
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Ingår i: Tissue Engineering Part A. - : Mary Ann Liebert Inc. - 1937-3341 .- 1937-335X. ; 28:13-14, s. 621-639
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Tidskriftsartikel (refereegranskat)abstract
- 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 StatementThe 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.
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| 2. |
- Berg, Malin, 1976, et al.
(författare)
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Replacement of a Tracheal Stenosis with a Tissue-Engineered Human Trachea Using Autologous Stem Cells: A Case Report
- 2014
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Ingår i: Tissue Engineering. Part A. - 1937-3341 .- 1937-335X. ; 20:1-2, s. 389-397
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Tidskriftsartikel (refereegranskat)abstract
- Cell-based therapies, involving tissue engineering represent interesting and potentially important strategies for treatment of patients with various disorders. Here, using a detergent-enzymatic method we prepared an intact 3-dimensional scaffold of an extracellular matrix (ECM) 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 lower part of the larynx. Although the graft provided the patient with an open airway, a week after surgery, the mucous membrane of the graft was covered by a 1-2mm thick fungal infection, which was treated with local and systemic anti-fungal therapy. The airway lumen was postoperatively controlled by fiberbrandoscopy and found stable and sufficient. However, twenty-three days later the patient died due to cardiac arrest but with a patent, open, stable tracheal transplant and intact anastomoses. Histopathological results of the transplanted tracheal graft at autopsy showed a squamous but not ciliated epithelium, neovascularization, bundles of -sma positive muscle cells, serous glands and nerve fibres 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.
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| 3. |
- Methe, Ketaki, et al.
(författare)
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An alternative approach to decellularize whole porcine heart
- 2014
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Ingår i: BioResearch Open Access. - : Mary Ann Liebert Inc. - 2164-7844 .- 2164-7860. ; 3:6, s. 327-338
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Methe, Ketaki, et al.
(författare)
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Differential Activation of Immune Cells for Genetically Different Decellularized Cardiac Tissues
- 2020
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Ingår i: Tissue Engineering Part A. - : Mary Ann Liebert Inc. - 1937-3341 .- 1937-335X. ; 26:21-22, s. 1180-1198
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Nayakawde, Nikhil, et al.
(författare)
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Combined Use of Detergents and Ultrasonication for Generation of an Acellular Pig Larynx
- 2021
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Ingår i: Tissue Engineering Part A. - : Mary Ann Liebert Inc. - 1937-3341 .- 1937-335X. ; 27:5-6, s. 362-371
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Tidskriftsartikel (refereegranskat)abstract
- 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 futurein vitrorecellularization studies orin vivoimplantation studies in the clinic. Impact statement This study successfully created decellularized porcine larynx using novel method of perfusion-agitation with detergents and ultrasonication, which maintained three-dimensional architecture of the larynx scaffold. Our method is devoid of harmful enzymes, which may prevent cellular repopulation or induce inflammatory response uponin vivoimplantation. We studied important aspect of preservation of extracellular matrix proteins in different structures of the larynx. Hence, our decellularization method could be used as an alternative method to decellularize various dense tissues such as cartilage or tendon.
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| 6. |
- Nayakawde, Nikhil, et al.
(författare)
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In Vitro Regeneration of Decellularized Pig Esophagus Using Human Amniotic Stem Cells
- 2020
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Ingår i: Bioresearch Open Access. - : Mary Ann Liebert Inc. - 2164-7860. ; 9:1, s. 22-36
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Nayakawde, Nikhil
(författare)
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On tissue engineering of pig, human, and non-human primate tissues
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: Demand for donor organs for transplantation has been increasing every year more than the actual supply of suitable donor organs. One of the major problems associated with allogeneic transplantation includes lifelong immunosuppression. Tissue engineering and regenerative medicine is a growing field that uses knowledge of stem cell biology, developmental biology, immunology, and bioengineering to replace diseased and damaged tissues or organs. Tissue engineered (TE) hollow organs and tissues derived from natural extracellular matrix (ECM) have been used in several preclinical and clinical studies. More complex three-dimensional organs such as heart, liver, lungs, and kidney have been studied extensively both in-vitro and in-vivo in preclinical settings, but clinical experience is lacking. There is an increasing demand for understanding the composition of ECM, cell-ECM interaction in-vitro and in-vivo, and how tissue engineered organs behave immunologically after implantation. The current thesis focuses on investigation of decellularization methods for heart (porcine), esophagus (porcine, baboon, and human) and larynx (porcine); and recellularization of esophagus (porcine and human). It was also investigated during various time-points of recellularization if stem cells were able to synthesize ECM proteins, tissue specific proteins and growth factors, and if stem cells were able to differentiate into tissue-specific cells. Methods: In Paper I, a detergent based decellularization method was developed to create acellular whole porcine hearts. The cardiac ECM was then characterized for its structural and mechanical properties. In Paper III, physical and chemical methods were developed to decellularize porcine larynx. Decellularized larynx was analyzed microscopically for its ultrastructural changes and presence of cells. In Paper II, decellularization and recellularization (with human amniotic mesenchymal stem cells and epithelial cells) of porcine esophagus was carried out. In Paper IV, decellularization of pig, baboon, and human esophagus was performed as per the method described in Paper II. Paper IV studied the cell-ECM interaction during recellularization of human esophagus with human amniotic mesenchymal stem cells by using the stable isotope labeling with amino acids in cell culture (SILAC) technique. Results: Decellularization of heart, larynx, and esophagus was achieved successfully, with loss of cell nuclei, preservation of major ECM proteins such as collagen and elastin, preservation of growth factors, and maintaining three-dimensional structures of the tissues and organs. Decellularized esophagus was characterized by preservation of matrisome and non-matrisome proteins in the ECM using proteomics-bioinformatics analyses. Recellularization of pig and human esophagus was evidenced by stem cell proliferation, differentiation, and tissue specific protein synthesis by seeded stem cells. SILAC assay showed synthesis of newly produced proteins in the recellularized esophagus by seeded stem cells including ECM (collagens and fibronectin), cell-ECM signaling molecules (integrins), ECM regulators, secreted factors, skeletal muscle proteins, and proteins required for contraction of striated muscle. Conclusions: The decellularization protocol for heart, larynx, and esophagus was effective in removing cells while preserving ECM. Recellularization of esophagus showed the potential of human amniotic-derived stem cells for different tissue engineering applications. The SILAC based proteomics method can replace use of conventional proteomics in TE field to differentiate between cell and ECM proteins
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| 8. |
- Olausson, Michael, 1956, et al.
(författare)
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Long-term Transplant Function After Thrombolytic Treatment Ex Vivo of Donated Kidneys Retrieved 4 to 5 H After Circulatory Death
- 2022
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Ingår i: Transplantation. - : Ovid Technologies (Wolters Kluwer Health). - 0041-1337. ; 106:12, s. 2348-2359
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Tidskriftsartikel (refereegranskat)abstract
- 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 degrees C, followed by 3 h with red blood cells at 32 degrees 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 gelatinaseassociated 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.
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| 9. |
- Olausson, Michael, et al.
(författare)
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Long-term Transplant Function After Thrombolytic Treatment Ex Vivo of Donated Kidneys Retrieved 4 to 5 Hours After Circulatory Death
- 2022
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Ingår i: Transplantation. - 0041-1337. ; 106:12, s. 2348-2359
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Olausson, Michael, 1956, et al.
(författare)
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Novel Ex-Vivo Thrombolytic Reconditioning of Kidneys Retrieved 4 to 5 Hours After Circulatory Death
- 2022
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Ingår i: Transplantation. - : Ovid Technologies (Wolters Kluwer Health). - 0041-1337. ; 106:8, s. 1577-1588
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Tidskriftsartikel (refereegranskat)abstract
- 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 4-5h 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 2h, an ice slush was inserted into the abdomen. Kidneys were retrieved 4.5h 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 degrees C was continued for 3h, followed by 3h with red blood cells (RBCs) at 32 degrees 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 >100mL/100g/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.
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