| 1. |
- Söfteland, John M., 1977, et al.
(författare)
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Intestinal Preservation Injury: A Comparison Between Rat, Porcine and Human Intestines.
- 2019
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Ingår i: International journal of molecular sciences. - : MDPI AG. - 1422-0067. ; 20:13
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Tidskriftsartikel (refereegranskat)abstract
- Advanced preservation injury (PI) after intestinal transplantation has deleterious short- and long-term effects and constitutes a major research topic. Logistics and costs favor rodent studies, whereas clinical translation mandates studies in larger animals or using human material. Despite diverging reports, no direct comparison between the development of intestinal PI in rats, pigs, and humans is available. We compared the development of PI in rat, porcine, and human intestines. Intestinal procurement and cold storage (CS) using histidine-tryptophan-ketoglutarate solution was performed in rats, pigs, and humans. Tissue samples were obtained after 8, 14, and 24 h of CS), and PI was assessed morphologically and at the molecular level (cleaved caspase-3, zonula occludens, claudin-3 and 4, tricellulin, occludin, cytokeratin-8) using immunohistochemistry and Western blot. Intestinal PI developed slower in pigs compared to rats and humans. Tissue injury and apoptosis were significantly higher in rats. Tight junction proteins showed quantitative and qualitative changes differing between species. Significant interspecies differences exist between rats, pigs, and humans regarding intestinal PI progression at tissue and molecular levels. These differences should be taken into account both with regards to study design and the interpretation of findings when relating them to the clinical setting.
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| 2. |
- Söfteland, John M., 1977, et al.
(författare)
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Luminal polyethylene-glycol solution delays the onset of preservation injury in the human intestine.
- 2021
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Ingår i: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. - : Elsevier BV. - 1600-6143. ; 21:6, s. 2220-2230
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Tidskriftsartikel (refereegranskat)abstract
- The organ damage incurred during the cold storage (CS) of intestinal grafts has short and long-term consequences. Animal studies suggest that additional luminal preservation (LP) with polyethylene-glycol (PEG) may alleviate this damage. This study aims to validate these findings using human intestines. Ileal segments, perfused intravascularly with IGL-1 solution, were procured from 32 multiorgan donors and divided into two parts: one containing a PEG 3350-based solution introduced luminally (LP group) and another one without luminal treatment (control). Sampling was performed after 4h, 8h, 14h, and 24h of CS. Histology was assessed using the Chiu/Park score. Tight junctions (TJ), several inflammatory markers, and transcription factors were examined by immunofluorescence, ddPCR, and Western blot. Tissue water content (edema) was also measured. Apoptotic activity was assessed with caspase 2,3 and 9 assays. LP significantly lowered mucosal injury at all time points. Redistribution of TJ proteins occurred earlier and more severely in the control group. After 24h of CS, LP intestines showed an emerging unfolding protein response. Increased caspase-3 and -9 activity were found in the control group. The current results indicate that luminal PEG is safe and effective in reducing damage to the intestinal epithelium during CS.
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| 3. |
- Tsiartas, P., et al.
(författare)
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Seven days ex vivo perfusion of whole ewe ovaries with follicular maturation and oocyte retrieval: towards the development of an alternative fertility preservation method
- 2022
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Ingår i: Reproduction Fertility and Development. - : CSIRO Publishing. - 1031-3613 .- 1448-5990. ; 34:3, s. 331-342
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Tidskriftsartikel (refereegranskat)abstract
- Fertility preservation methods for prepubertal women about to undergo gonadotoxic chemo and/or radiation therapy are limited. Therefore, the aim of this study was to investigate the feasibility to develop an alternative fertility preservation method based on an ex vivo perfusion platform for whole ewe ovaries. Thirteen ewe ovaries were divided into two groups (group 1 and 2) that were perfused in a bioreactor for up to 7 days. Group 1 (n = 3) were stimulated with human menopausal gonadotropin (hMG) administered in single daily dose, while group 2 (n = 10) were stimulated continuously for 24 h. The perfused ovaries in group 1 showed no significant differences in follicular density, sub-follicular morphology and oocyte quality after ischaemia and after ex vivo perfusion compared with non-perfused control ovaries. The perfused ovaries in group 2 showed a significant decrease in the follicular reserve and oocyte quality compared with the control group. In total, 16 GV-MI oocytes were retrieved from both groups. This study describes for the first time the ex vivo maintenance of viable follicles of ewe ovaries with oocyte integrity and the retrieval of oocytes after ex vivo hormonal perfusion with two different protocols for up to 7 days.
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| 4. |
- Alshaikh, Ahmed Baker, et al.
(författare)
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Decellularization and recellularization of the ovary for bioengineering applications; studies in the mouse.
- 2020
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Ingår i: Reproductive biology and endocrinology : RB&E. - : Springer Science and Business Media LLC. - 1477-7827. ; 18:1
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Tidskriftsartikel (refereegranskat)abstract
- Fertility preservation is particularly challenging in young women diagnosed with hematopoietic cancers, as transplantation of cryopreserved ovarian cortex in these women carries the risk for re-introducing cancer cells. Therefore, the construction of a bioengineered ovary that can accommodate isolated small follicles was proposed as an alternative to minimize the risk of malignancy transmission. Various options for viable bioengineered scaffolds have been reported in the literature. Previously, we reported three protocols for producing mouse ovarian scaffolds with the decellularization technique. The present study examined these scaffolds further, specifically with regards to their extracellular composition, biocompatibility and ability to support recellularization with mesenchymal stem cells.Three decellularization protocols based on 0.5% sodium dodecyl sulfate (Protocol 1; P1), or 2% sodium deoxycholate (P2), or a combination of the two detergents (P3) were applied to produce three types of scaffolds. The levels of collagen, elastin and sulfated glycosaminoglycans (sGAGs) were quantified in the remaining extracellular matrix. Detailed immunofluorescence and scanning electron microscopy imaging were conducted to assess the morphology and recellularization efficiency of the constructs after 14days in vitro utilizing red fluorescent protein-labelled mesenchymal stem cells.All protocols efficiently removed the DNA while the elastin content was not significantly reduced during the procedures. The SDS-protocol (P1) reduced the sGAG and the collagen content more than the SDC-protocol (P2). All scaffolds were biocompatible and recellularization was successful, particularly in several P2-derived scaffolds. The cells were extensively distributed throughout the constructs, with a denser distribution observed towards the ovarian cortex. The cell density was not significantly different (400 to 550 cells/mm2) between scaffold types. However, there was a tendency towards a higher cell density in the SDC-derived constructs. Scanning electron microscope images showed fibrous scaffolds with a dense repopulated surface structure.While there were differences in the key structural macromolecules between protocols, all scaffolds were biocompatible and showed effective recellularization. The results indicate that our SDC-protocol might be better than our SDS-protocol. However, additional studies are necessary to determine their suitability for attachment of small follicles and folliculogenesis.
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| 5. |
- Alshaikh, Ahmed Baker, et al.
(författare)
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Decellularization of the mouse ovary: comparison of different scaffold generation protocols for future ovarian bioengineering.
- 2019
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Ingår i: Journal of ovarian research. - : Springer Science and Business Media LLC. - 1757-2215. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- In order to preserve fertility in young women with disseminated cancer, e.g. leukemia, an approach that has been suggested is to retransplant isolated small follicles within an ovarian matrix free from malignant cells and with no risk for contamination. The present study evaluates the first step to create a bioengineered ovarian construct that can act as growth-supporting tissue for isolated small follicles that are dependent on a stroma for normal follicular maturation. The present study used the intact mouse ovary to develop a mouse ovarian scaffold through various protocols of decellularization.Potential Immunogenic DNA and intracellular components were removed from whole mouse ovaries by agitation in a 0.5% sodium dodecyl sulfate solution (Protocol 1; P1), or in a 2% sodium deoxycholate solution (P2) or by a combination of the two (P3). The remaining decelluralized ovarian extracellular matrix structure was then assessed based on the DNA- and protein content, and was further evaluated histologically by haematoxylin and eosin-, Verhoeff's van gieson- (for elastin), Masson's trichrome- (for collagens) and Alcian blue (for glycosaminoglycans) staining. We also evaluated the decellularization efficiency using the mild detergent Triton-X100 (1%).Sodium dodecyl sulfate efficiently removed DNA and intracellular components from the ovarian tissue but also significantly reduced the integrity of the remaining ovarian extracellular matrix. Sodium deoxycholate, a considerably milder detergent compared to sodium dodecyl sulfate, preserved the ovarian extracellular matrix better, evident by a more distinct staining for glycosaminoglycan, collagen and elastic fibres. Triton-X100 was found ineffective as a decellularization reagent for mouse ovaries in our settings.The sodium dodecyl sulfate generated ovarian scaffolds contained minute amounts of DNA that may be an advantage to evade a detrimental immune response following engraftment. The sodium deoxycholate generated ovarian scaffolds had higher donor DNA content, yet, retained the extracellular composition better and may therefore have improved recellularization and other downstream bioengineering applications. These two novel types of mouse ovarian scaffolds serve as promising scaffold-candidates for future ovarian bioengineering experiments.
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| 6. |
- Bagge, Jasmine, et al.
(författare)
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Mucosal Recovery after Intestinal Transplantation in the Rat: A Sequential Histological and Molecular Assessment
- 2023
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Ingår i: European Surgical Research. - : S. Karger AG. - 0014-312X .- 1421-9921. ; 64:2, s. 201-210
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Tidskriftsartikel (refereegranskat)abstract
- Introduction Intestinal cold ischemia and subsequent reperfusion during transplantation result in various degrees of mucosal injury ranging from mild edema to extensive mucosal loss. Mucosal barrier impairment favours bacterial translocation and fluid loss and raises nutritional challenges. The injured intestine also releases proinflammatory mediators and upregulates various epitopes towards an inflammatory phenotype. We studied the process of mucosal injury and repair during the early period after intestinal transplantation from a histological and molecular standpoint.Materials and Methods Adult Sprague Dawley rats were used as donors and recipients. Donor intestines were perfused and stored in saline for 3 hours, then transplanted heterotopically using microvascular anastomoses. Intestinal graft segments were obtained after 20 minutes, 6 hours, 12 hours, and 24 hours after reperfusion. Histology studies (goblet cell count, morphometry), immunofluorescence and western blot for several tight junction proteins, apoptosis and inflammation related proteins were performed.Results Cold storage led to extensive epithelial detachment, whereas reperfusion resulted in extensive villus loss (about 60 % of the initial length) and goblet cell numbers were drastically reduced. Over the first 24 hours, gradual morphologic and molecular recovery was noted, although several molecular alterations persisted (increased apoptosis and inflammation, altered expression of several tight junctions).Conclusions The current data suggest that a near-complete morphologic recovery from a moderate mucosal injury occurs within the first 24 hours after intestinal transplantation. However, several molecular alterations persist and need to be considered when designing intestinal transplant experiments and choosing sampling and endpoints.
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| 7. |
- Kuna, Vijay Kumar, 1987, et al.
(författare)
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Significantly accelerated wound healing of full-thickness skin using a novel composite gel of porcine acellular dermal matrix and human peripheral blood cells
- 2017
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Ingår i: Cell Transplantation. - : Cognizant Communication Corporation. - 0963-6897 .- 1555-3892. ; 26:2, s. 293-307
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Tidskriftsartikel (refereegranskat)abstract
- Herein, we report the fabrication of a novel composite gel from decellularized gal-gal-knockout porcine skin and human peripheral blood mononuclear cells (hPBMC) for full-thickness skin wound healing. Decellularized skin extracellular matrix (ECM) powder was prepared via chemical treatment, freeze-drying and homogenization. The powder was mixed with culture medium containing hyaluronic acid to generate a pig skin gel (PSG). The effect of the gel in regeneration of full-thickness wound was studied in nude mice. We found significantly accelerated wound closure already on day 15 in animals treated with PSG only or PSG+hPBMC as compared to untreated and hyaluronic acid treated controls (p<0.05). Addition of the hPBMC to the gel resulted in marked increase of host blood vessels as well as the presence of human blood vessels. At day 25, histologically, the wounds in animals treated with PSG only or PSG+hPBMC were completely closed as compared to controls. Thus, the gel facilitated generation of new skin with well arranged epidermal cells and restored bilayer structure of the epidermis and dermis. These results suggest that porcine skin ECM gel together with human cells may be a novel and promising biomaterial for medical applications especially for patients with acute and chronic skin wounds.
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| 8. |
- Naeimi Kararoudi, Meisam, et al.
(författare)
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Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Gene Editing Technique in Xenotransplantation.
- 2018
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Ingår i: Frontiers in immunology. - : Frontiers Media SA. - 1664-3224. ; 9
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Forskningsöversikt (refereegranskat)abstract
- Genetically modified pigs have been considered favorable resources in xenotransplantation. Microinjection of randomly integrating transgenes into zygotes, somatic cell nuclear transfer, homologous recombination, zinc finger nucleases, transcription activator-like effector nucleases, and most recently, clustered regularly interspaced short palindromic repeats-cas9 (CRISPR/Cas9) are the techniques that have been used to generate these animals. Here, we provide an overview of the CRISPR approaches that have been used to modify genes which are vital in improving xenograft survival rate, including cytidine monophosphate-N-acetylneuraminic acid hydroxylase, B1,4N-acetylgalactosaminyltransferase, isoglobotrihexosylceramide synthase, class I MHC, von Willebrand factor, C3, and porcine endogenous retroviruses. In addition, we will mention the importance of potential candidate genes which could be targeted using CRISPR/Cas9.
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| 9. |
- Padma, Arvind M., et al.
(författare)
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Decellularization protocol-dependent damage-associated molecular patterns in rat uterus scaffolds differentially affect the immune response after transplantation.
- 2021
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Ingår i: Journal of tissue engineering and regenerative medicine. - : Hindawi Limited. - 1932-7005 .- 1932-6254. ; 15:7, s. 674-685
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Tidskriftsartikel (refereegranskat)abstract
- Scaffolds derived from decellularized tissue possess many advantages for bioengineering applications, including for novel infertility treatments. However, the decellularization process results in allogenic-independent damage-associated molecular patterns (DAMPs). This field is poorly studied, in particular for uterus bioengineering applications. An increased knowledge concerning the immune system activation after transplantation of decellularized tissue will enable safer construct development and thereby accelerate translation from research to clinic. We therefore transplanted rat uterus scaffolds produced by three different decellularization protocols based on Triton X-100 (P1 and P2) or sodium deoxycholate (P3) in a syngeneic animal model and assessed the immune response towards DAMPs exposed by the decellularization process. Biopsies were retrieved on day 5, 15, and 30 post transplantation and immunohistochemistry-stained CD45+ (leucocytes), CD4+ (T-cells), CD8a+ (cytotoxic T-cells), CD22+ (B-cells), NCR1+ (NK-cells), CD68+ (pan-macrophages), and CD163+ (M2 macrophages) cells within the grafts were quantified. The gene expression for interferon γ, interleukin (IL)-1β, IL-2, IL-6, and tumor necrosis factor (TNF) eotaxin-2, RANTES, MCP-1, MIP-1α, MIP-3α, IL-8 were also measured. Scaffolds from P1 induced a rapid cell infiltration after transplantation, presumably induced by DNA-based DAMPs. However, this response was only transient. Protocol 3 derived scaffolds induced an early pro-inflammatory cytokine response at the transcript level which remained high throughout the study. This response may be caused by the stronger decellularization detergent that could expose more extracellular matrix-related DAMPs. However, earlier proteomics analysis also identified significantly more abundant heat shook proteins-related DAMPs in this scaffold type. Protocol 2 caused the least immunogenic scaffolds and should thus be the future focus for in vivo uterus bioengineering applications.
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| 10. |
- Padma, Arvind M., et al.
(författare)
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Immune response after allogeneic transplantation of decellularized uterine scaffolds in the rat.
- 2021
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Ingår i: Biomedical Materials. - 1748-605X. ; 16:4
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Tidskriftsartikel (refereegranskat)abstract
- Data on how the immune system reacts to decellularized scaffolds after implantation is scarce and difficult to interpret due to many heterogeneous parameters such as tissue-type match, decellularization method and treatment application. The engraftment of these scaffolds must prove safe and that they remain inert to the recipient's immune system to enable successful translational approaches and potential future clinical evaluation. Herein, we investigated the immune response after the engraftment of three decellularized scaffold types that previously showed potential to repair a uterine injury in the rat. Protocol (P) 1 and P2 were based on Triton-X100 and generated scaffolds containing 820 ng mg-1and 33 ng mg-1donor DNA per scaffold weight, respectively. Scaffolds obtained with a sodium deoxycholate-based protocol (P3) contained 160 ng donor DNA per mg tissue. The total number of infiltrating cells, and the population of CD45+leukocytes, CD4+T-cells, CD8a+cytotoxic T-cells, CD22+B-cells, NCR1+NK-cells, CD68+and CD163+macrophages were quantified on days 5, 15 and 30 after a subcutaneous allogenic (Lewis to Sprague Dawley) transplantation. Gene expression for the pro-inflammatory cytokines INF-γ, IL-1β, IL-2, IL-6 and TNF were also examined. P1 scaffolds triggered an early immune response that may had been negative for tissue regeneration but it was stabilized after 30 d. Conversely, P3 initiated a delayed immune response that appeared negative for scaffold survival. P2 scaffolds were the least immunogenic and remained similar to autologous tissue implants. Hence, an effective decellularization protocol based on a mild detergent was advantageous from an immunological perspective and appears the most promising for futurein vivouterus bioengineering applications.
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| 11. |
- Padma, Arvind M., et al.
(författare)
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Protocols for Rat Uterus Isolation and Decellularization: Applications for Uterus Tissue Engineering and 3D Cell Culturing.
- 2018
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Ingår i: Decellularized Scaffolds and Organogenesis. Kursad Turksen (red.). - New York, NY : Springer. - 1940-6029. - 9781493976553 ; , s. 161-175
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Bokkapitel (refereegranskat)abstract
- Sophisticated culturing conditions are required to grow cells in a three-dimensional (3D) environment. Cells then require a type of scaffold rich in proteins, growth factors, and signaling molecules that simulates their natural environment. Tissues from all species of animals have an organ-specific extracellular matrix (ECM) structure that plays a key role in cell proliferation and migration. Hence, the scaffold composition plays a significant role for any successful 3D cell culturing system. We developed a whole rat uterus ECM scaffold by the perfusion of detergents and ionic solutions through the vascular system of an isolated normal rat uterus in a process termed "decellularization." The generated rat uterus scaffolds consist of a cell-free ECM structure similar to that of the normal rat uterus, and are thus excellent platforms on to which new cells can be added. Rat uterus 3D cell culturing systems based on these scaffolds could become valuable to decidual differentiation- and embryo implantation studies, or for investigating invasion mechanisms of endometrial cancer cells. They could also be used for the creation of tissue engineered uterine tissue, for partial or whole organogenesis developed for transplantation applications to treat absolute uterine infertility. This is a condition affecting about 1 in 500 women, and is only treatable by a uterus transplantation. This article provides valuable troubleshooting notes and describes in detail how to generate rat uterus scaffolds, including the delicate surgery required to isolate the uterus with an intact vascular tree which facilitates vascular perfusion and re-transplantation.
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| 12. |
- Padma, Arvind M., et al.
(författare)
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The development of an extended normothermic ex vivo reperfusion model of the sheep uterus to evaluate organ quality after cold ischemia in relation to uterus transplantation
- 2019
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Ingår i: Acta Obstetricia et Gynecologica Scandinavica. - : Wiley. - 0001-6349 .- 1600-0412. ; 98:9, s. 1127-1138
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Uterus transplantation has recently proved that infertility in women with uterine factor infertility can be cured. It is still an experimental procedure with numerous critical details remaining to be established, including tolerance to warm and cold ischemic insults. In preparation for human uterus transplantation trials, most teams use the sheep as a model system for research and team training, since the vasculature and the uterus is of similar size as in the human. We, therefore, aimed to develop an ex vivo sheep uterus reperfusion platform that mimics the reperfusion situation so that initial assessments and comparisons can be performed without the need for costly and labor-intensive in vivo transplantation experiments. Material and methods: Isolated sheep uteri were perfused with the preservation solution IGL-1 and were then exposed to cold ischemia for either 4(n=6) or 48hours (n=7). Uteri were then reperfused for 48hours under normothermic conditions with an oxygenated recirculating perfusate containing growth factors and synthetic oxygen carriers. Histological and biochemical analysis of the perfusate was conducted to assess reperfusion injury. Results: Quantification of cell density indicated no significant edema in the myometrium or in the endometrium of uteri exposed to 4hours cold ischemia and then a normothermic ex vivo reperfusion for 48hours. Only the outer serosa layer and the inner columnar luminal epithelial cells were affected by the reperfusion. However, a much faster and severe reperfusion damage of all uterine layers were evident during the reperfusion experiment following 48hours of cold ischemia. This was indicated by major accumulation of extracellular fluid, presence of apoptotic-labeled glandular epithelial layer and vascular endothelium. A significant accumulation of lactate was measured in the perfusate with a subsequent decrease in pH. Conclusions: We developed a novel ex vivo sheep uterus model for prolonged perfusion. This model proved to be able to distinguish reperfusion injury-related differences associated to organ preservation. The experimental setup is a platform that can be used to conduct further studies on uterine ischemia- and reperfusion injury that may lead to improved human uterus transplantation protocols. © 2019 Nordic Federation of Societies of Obstetrics and Gynecology
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| 13. |
- Padma, Arvind M.
(författare)
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Tissue engineering for novel female infertillity treatments: studies on small and large animal models
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Introduction: As with any transplantation (Tx) procedure, uterus Tx is associated with risky donor surgery and adverse side-effects from immunosuppression. With the aim to bypass these risks, this thesis investigated uterus tissue engineering strategies and the potential to develop a patient-specific uterus graft to replace the need for donor surgery and immunosuppression. A translational approach for uterus scaffold production through a process called decellularization (DC) is addressed using the rat and the sheep animal model. The immunological events following engraftment of rat uterus scaffolds was also evaluated. The thesis also assessed cellular reconstruction techniques and perfusion bioreactor protocols that can be useful to recellularize whole sheep uterus scaffolds for future uterus Tx studies. Methods: The immune response towards three different rat uterus scaffold types were evaluated after transplantation by quantifying infiltrating leucocytes and the expression of pro-inflammatory cytokines. Additionally, three novel whole sheep uterus scaffolds were produced by DC and the scaffold composition, bioactivity, mechanical strength and ability to support seeded stem cells were analyzed. Technique optimization for a perfusion bioreactor was also conducted using normal sheep uterus and a specialized perfusion medium. Results and conclusions: In Paper I, we deciphered DC protocol-dependent differences in the immune response following engraftment. A mild, yet effective DC protocol resulted in an immune-inert scaffold type. In Paper II-III, we developed three promising extracellular matrix-derived bioactive sheep uterus scaffolds that after an enzymatic pre-conditioning were able to support wide-spread cell attachment and migration during recellularization. In Paper IV, we were able to maintain normal sheep uterus ex-vivo for 48 hours using a custom made culture medium and a perfusion bioreactor. These parameters should facilitate future whole sheep uterus tissue engineering experiments so that a patient-specific tissue engineered uterus can be made to replace a donor in a uterus Tx setting.
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| 14. |
- Padma, Arvind M., et al.
(författare)
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Towards a bioengineered uterus: bioactive sheep uterus scaffolds are effectively recellularized by enzymatic preconditioning
- 2021
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Ingår i: Npj Regenerative Medicine. - : Springer Science and Business Media LLC. - 2057-3995. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- Uterine factor infertility was considered incurable until recently when we reported the first successful live birth after uterus transplantation. However, risky donor surgery and immunosuppressive therapy are factors that may be avoided with bioengineering. For example, transplanted recellularized constructs derived from decellularized tissue restored fertility in rodent models and mandate translational studies. In this study, we decellularized whole sheep uterus with three different protocols using 0.5% sodium dodecyl sulfate, 2% sodium deoxycholate (SDC) or 2% SDC, and 1% Triton X-100. Scaffolds were then assessed for bioactivity using the dorsal root ganglion and chorioallantoic membrane assays, and we found that all the uterus scaffolds exhibited growth factor activity that promoted neurogenesis and angiogenesis. Extensive recellularization optimization was conducted using multipotent sheep fetal stem cells and we report results from the following three in vitro conditions; (a) standard cell culturing conditions, (b) constructs cultured in transwells, and (c) scaffolds preconditioned with matrix metalloproteinase 2 and 9. The recellularization efficiency was improved short-term when transwells were used compared with standard culturing conditions. However, the recellularization efficiency in scaffolds preconditioned with matrix metalloproteinases was 200-300% better than the other strategies evaluated herein, independent of decellularization protocol. Hence, a major recellularization hurdle has been overcome with the improved recellularization strategies and in vitro platforms described herein. These results are an important milestone and should facilitate the production of large bioengineered grafts suitable for future in vivo applications in the sheep, which is an essential step before considering these principles in a clinical setting.
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| 15. |
- Padma, Arvind M., et al.
(författare)
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Uterus bioengineering as a future alternative to uterus transplantation
- 2022
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Ingår i: Clinical and Experimental Obstetrics & Gynecology. - : IMR Press. - 0390-6663. ; 49:3
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Tidskriftsartikel (refereegranskat)abstract
- Objective: To review the current knowledge on uterus bioengineering and discuss potential future directives. Uterus bioengineering may solve two major hurdles in organ transplantation of a uterus, organ shortage and control of rejection by immunosuppression. Mechanism: Literature search using PubMed. Findings in brief: Sixty-seven references were summarized that describe the scientific progress made on uterus bioengineering, including other studies related to the topic. Most articles describe work on rat models, including proof of-concept that uterus bioengineering can be used to restore fertility after a partial uterine injury. These promising results are currently being translated to larger and more clinically relevant animal models. In particular, uterus-specific scaffolds produced by a process called "decellularization" that were developed for the mouse, rat, rabbit, pig, goat, and sheep. These scaffolds stimulated angiogenesis and regeneration in vitro and in vivo, and successfully harbored various types of cells for an extended time in vitro. Additionally, applications for endometrial extracellular matrix-specific hydrogels derived from decellularized uterus tissue is discussed. Current challenges for uterus bioengineering are also addressed, e.g., the cellular reconstruction phase, and how they might be improved. Conclusions: Significant progress was made during the last decade with convincing evidence from multiple independent groups in experiments with small animal models. Initial steps towards large animal uterus bioengineering were made. The future continuation of such studies will provide important data required to translate these ideas to an experimental phase in the human. Partial uterus reconstruction through a bioengineered tissue transplantation is closer to a clinical reality compared to whole uterus bioengineering principles aimed to replace a donor in a UTx setting.
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| 16. |
- Simsa, Robin, et al.
(författare)
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Systematic in vitro comparison of decellularization protocols for blood vessels.
- 2018
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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 13:12
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Tidskriftsartikel (refereegranskat)abstract
- Decellularization of native blood vessels is a promising technology to generate 3D biological scaffolds for vascular grafting. Blood vessel decellularization has been performed in previous studies under various experimental conditions, that complicates comparison and optimization of suitable protocols. The goal of this work was to systematically compare the decellularization and recellularization efficacy of 5 different protocols utilizing the detergents sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), CHAPS and TritonX-100 together with DNA-removing enzymes on porcine vena cava in a perfusion bioreactor setup. Additionally, we tested the effect of DNase on the extracellular matrix (ECM) properties. We found that all protocols could efficiently decellularize blood vessels. Mechanical strength, collagen preservation and ECM integrity were similar among all tested detergents, yet TritonX protocols required long-term DNase application for complete decellularization. However, TritonX-based protocols showed the greatest recellularization efficacy with HUVECs in vitro. Furthermore, we developed a novel protocol for TritonX which improved recellularization and reduced total process time and ECM stiffness compared to previous protocols. SDS, SDC and CHAPS based protocols had a lower recellularization potential. In conclusion, decellularization of blood vessels can be achieved with all tested reagents, but TritonX treated ECM can be most efficiently recellularized with endothelial cells.
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| 17. |
- Tiemann, Tom T, et al.
(författare)
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Towards uterus tissue engineering: a comparative study of sheep uterus decellularisation
- 2020
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Ingår i: Molecular human reproduction. - : Oxford University Press (OUP). - 1460-2407 .- 1360-9947. ; 26:3, s. 167-178
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Tidskriftsartikel (refereegranskat)abstract
- © The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. Uterus tissue engineering may dismantle limitations in current uterus transplantation protocols. A uterine biomaterial populated with patient-derived cells could potentially serve as a graft to circumvent complicated surgery of live donors, immunosuppressive medication and rejection episodes. Repeated uterine bioengineering studies on rodents have shown promising results using decellularised scaffolds to restore fertility in a partially impaired uterus and now mandate experiments on larger and more human-like animal models. The aim of the presented studies was therefore to establish adequate protocols for scaffold generation and prepare for future in vivo sheep uterus bioengineering experiments. Three decellularisation protocols were developed using vascular perfusion through the uterine artery of whole sheep uteri obtained from slaughterhouse material. Decellularisation solutions used were based on 0.5% sodium dodecyl sulphate (Protocol 1) or 2% sodium deoxycholate (Protocol 2) or with a sequential perfusion of 2% sodium deoxycholate and 1% Triton X-100 (Protocol 3). The scaffolds were examined by histology, extracellular matrix quantification, evaluation of mechanical properties and the ability to support foetal sheep stem cells after recellularisation. We showed that a sheep uterus can successfully be decellularised while maintaining a high integrity of the extracellular components. Uteri perfused with sodium deoxycholate (Protocol 2) were the most favourable treatment in our study based on quantifications. However, all scaffolds supported stem cells for 2weeks in vitro and showed no cytotoxicity signs. Cells continued to express markers for proliferation and maintained their undifferentiated phenotype. Hence, this study reports three valuable decellularisation protocols for future in vivo sheep uterus bioengineering experiments.
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| 18. |
- Tsiartas, Panos, et al.
(författare)
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P–459 Ex vivo perfusion of whole ewe ovaries with follicular maturation for up to seven days: towards the development of an alternative fertility preservation method
- 2021
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Ingår i: Human Reproduction Vol 36 Issue Supplement 1. - : Oxford University Press (OUP). - 0268-1161.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Abstract Study question To develop an alternative fertility preservation method for young female cancer patients based on an ex vivo perfusion of whole ovaries serving as a platform for future ovarian stimulation studies. Summary answer It is possible to maintain viable follicles and to retrieve oocytes after ex vivo perfusion of ewe ovaries for up to 7 days. What is known already Some progress has been made in terms of follicular growth and the isolation of mature oocytes in vitro. However, full development, from early follicular stages to a viable offspring, has only been described in rodent models. The complex events controlling follicular expansion and the long time required for folliculogenesis and oocyte maturity in large mammalian species creating challenges and limitations for in vitro studies. Ex vivo perfusion of a whole ovary could potentially be a solution by exploiting the intact ovarian architecture to support folliculogenesis and oocyte maturation. Study design, size, duration Thirty-one ewe ovaries were divided into 4 groups and ex vivo perfused in a bioreactor. Group 1 (n=14) perfusion for 48hours with no hormone supplementation; Group 2 (n=4) perfusion 96–101hours with follicle stimulating hormone (FSH); Group 3 (n=3) perfusion 120–168hours with human menopausal gonadotropin (hMG); Group 4 (n=10) perfusion 72–144hours with hMG. Participants/materials, setting, methods Ewe ovaries from sexually mature ewes were ex vivo perfused in a bioreactor under normothermic conditions for up to 7 days (max total 168hours). Histomorphological, immunohistochemical, hormonal and biochemical analyses were performed to assess ovarian structure and viability after cold ischemia and after perfusion which was subsequently compared to control ovaries. Main results and the role of chance The perfused ovaries in group 2 and 3 showed no significant differences in follicular density, viability and oocyte quality after ischemia and perfusion compared to control ovaries. Estradiol and progesterone levels did not increase during the perfusion. The perfused ovaries in group 1 and 4 showed a significant decrease in the ovarian reserve and oocyte quality. In total, 16 GV-MI oocytes were retrieved from groups 3 and 4. Limitations, reasons for caution 1. Ovaries were retrieved from ewes of unknown cycle and reproductive history. 2. The perfusion medium was changed after 24hours from perfusion start to remove detrimental metabolites and this could affect the measured concentrations of hormones and metabolites in the perfusion medium. Wider implications of the findings: These results pave the way to propose ex vivo perfusion as a good platform for fertility preservation studies on whole mammalian and human ovaries to retrieve fully mature oocytes. Trial registration number Not applicable
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