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Quality assessment by bile composition in normothermic machine perfusion of rat livers
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Our manuscript “Quality assessment by bile composition in normothermic machine perfusion of rat livers” has been accepted for publication in Tissue Engineering Part A.
Authors are Vanessa Muth, Felix Stobl, Julian Michelotto, Jennifer A. Kirwan, Jeremy Marchand, Nathalie N. Roschke, Simon Moosburner, Johann Pratschke, Igor M. Sauer, Nathanael Raschzok, and Joseph MGV Gassner.

Due to the persistent challenge of organ scarcity in liver transplantation, there is an escalating dependence on organs obtained from extended criteria donors (ECD). Normothermic machine perfusion (NMP) can be used for improved preservation and allows quality assessment of ECD grafts. The primary objective of this study was to assess bile composition within the framework of quality analysis and explore the impact of warm ischemia on its composition in a rodent NMP model.

30 livers from male Sprague Dawley rats were divided into five distinct groups. Each group was subjected to 6 hours of NMP using either DMEM or Steen solution as perfusate, with or without a preceding 30-minute warm ischemia period. We further examined the effect of pressure-controlled perfusion on livers experiencing 30 min WIT using Steen as perfusate. We conducted regular measurements of AST, ALT, LDH, and urea levels in the perfusate at three- hour intervals. We collected bile samples at hourly intervals and assessed biliary pH, LDH, and GGT. Bile acids were measured using mass spectrometry every two hours.

Liver injury parameters were significantly higher in our DCD model. Bile production was significantly reduced in livers exposed to warm ischemia, and the bile showed a significantly more alkaline pH. This correlated with the concentration of total bile acids, which was significantly higher in livers with 30 min WIT. Taurocholic acid and its metabolites were most prominent. Secondary bile acids were significantly reduced in the course of perfusion due to the missing enterohepatic circulation. Prolonged warm ischemia-induced liver injury affects parameters we measured in bile within our small animal NMP model. We hypothesize that this phenomenon may be attributed to the cAMP-driven nature of bile secretion, potentially explaining why DCD livers produce less, yet more concentrated, bile.
A new bicornuate model of rat uterus transplantation
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Our work on a “A new bicornuate model of rat uterus transplantation” has been accepted for publication in Acta Obstetricia et Gynecologica Scandinavica.

Uterus transplantation has revolutionized reproductive medicine for women with absolute uterine factor infertility, resulting in more than 40 reported successful live births worldwide to date. Small animal models are pivotal to refine this surgical and immunological challenging procedure aiming to enhance safety for both the mother and the child.
We established a syngeneic bicornuate uterus transplantation model in young female Lewis rats. All surgical procedures were conducted by an experienced and skilled microsurgeon who organized the learning process into multiple structured steps. Animals underwent meticulous preoperative preparation and postoperative care. Transplant success was monitored by sequential biopsies, monitoring graft viability and documenting histological changes long-term. Bicornuate uterus transplantation were successfully established achieving an over 70% graft survival rate with the passage of time. The bicornuate model demonstrated safety and feasibility, yielding outcomes comparable to the unicornuate model in terms of ischemia times and complications. Longitudinal biopsies were well-tolerated, enabling comprehensive monitoring throughout the study. Our novel bicornuate rat uterus transplantation model provides a distinctive opportunity for sequential biopsies at various intervals after transplantation and therefore comprehensive monitoring of graft health, viability, and identification of potential signs of rejection. Furthermore, this model allows for different interventions in each horn for comparative studies without interobserver differences contrary to the established unicornuate model. By closely replicating the clinical setting, this model stands as a valuable tool for ongoing research in the field of uterus transplantation, promoting further innovation and deeper insights into the intricacies of the uterus transplant procedure.

Authors are Dietrich Polenz, Igor Maximilian Sauer, Friederike Martin, Anja Reutzel-Selke, Muhammad Imtiaz Ashraf , Anja Schirmeier , Steffen Lippert, Kirsten Führer, Johann Pratschke, Stefan Günther Tullius, and Simon Moosburner.
Depletion of donor dendritic cells ameliorates immunogenicity of both skin and hind limb transplants
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Our manuscript "Depletion of donor dendritic cells ameliorates immunogenicity of both skin and hind limb transplants" has been accepted for publication in Frontiers in Immunology, section Alloimmunity and Transplantation. Authors are Muhammad Imtiaz Ashraf, Joerg Mengwasser, Anja Reutzel-Selke, Dietrich Polenz, Kirsten Führer, Steffen Lippert, Peter Tang, Edward Michaelis, Rusan Catar, Johann Pratschke, Christian Witzel, Igor M. Sauer, Stefan G. Tullius, and Barbara Kern.

Acute cellular rejection remains a significant obstacle affecting successful outcomes of organ transplantation including vascularized composite tissue allografts (VCA). Donor antigen presenting cells (APC), particularly dendritic cells (DC), orchestrate early alloimmune responses by activating recipient effector T cells. Employing a targeted approach, we investigated the impact of donor-derived conventional DC (cDC) and APC on the immunogenicity of skin and skin-containing VCA grafts, using mouse models of skin and hind limb transplantation.
By post-transplantation day 6, skin grafts demonstrated severe rejections, characterized by predominance of recipient CD4 T cells. In contrast, hind limb grafts showed moderate rejection, primarily infiltrated by CD8 T cells. While donor depletion of cDC and APC reduced frequencies, maturation, and activation of DC in all analysed tissues of skin transplant recipients, reduction in DC activities was only observed in the spleen of hind limb recipients. Donor cDC and APC depletion did not impact all lymphocyte compartments but significantly affected CD8 T cells and activated CD4 T in lymph nodes of skin recipients. Moreover, both donor APC and cDC depletion attenuated the Th17 immune response, evident by significantly reduced Th17 (CD4+IL-17+) cells in the spleen of skin recipients and reduced levels of IL-17E and lymphotoxin-α in the serum samples of both skin and hind limb recipients. In conclusion, our findings underscore the highly immunogenic nature of skin component in VCA. The depletion of donor APC and cDC mitigates the immunogenicity of skin grafts while exerting minimal impact on VCA.
Optimizing environmental enrichment for Sprague Dawley rats: Exemplary insights into the liver proteome
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Considering the expected increase in the elderly population and the growing emphasis on aging-related biomedical research, the demand for aged laboratory animals has surged, challenging established husbandry practices. Our objective was to establish a cost-effective method for environmental enrichment, utilizing the liver as a representative organ to assess metabolic changes in response to differing enrichment levels.
We conducted a six-month study involving 24 male Sprague Dawley rats who were randomly assigned to four environmental enrichment groups. Two groups were housed in standard cages, while the others were placed in modified rabbit cages. Half of the groups received weekly playtime in an enriched rat housing unit. We evaluated hormone levels, playtime behavior, and subjective handling experience. Additionally, liver tissue proteomic analysis was performed.
Initial corticosterone levels and those after 3 and 6 months showed no significant differences. Yet, testosterone levels were lower in the control group by the end of the study (p=0.007). In the liver tissue, we detected 1,871 distinct proteins, with 77% of them being consistent across all groups. In gene ontology analysis, no specific pathways were overexpressed. In semiquantitative analysis, we observed differences in proteins associated in lipid metabolism such as Apolipoprotein A-I and Acyl-CoA 6-desaturase, which were lower in the control group (p= 0.024 and p=0.009). Enriched environments reduced rat distress, large cages eased handling, and conflicts between rats lessened with bi-weekly interactions.

The manuscript "Optimizing environmental enrichment for Sprague Dawley rats: Exemplary insights into the liver proteome" has been accepted for publication in PLOS ONE.
Authors are Nathalie N. Roschke, Karl H. Hillebrandt, Dietrich Polenz, Oliver Klein, Joseph MGV Gassner, Johann Pratschke, Felix Krenzien, Igor M. Sauer, Nathanael Raschzok, and Simon Moosburner.
Proteomic analysis of decellularized mice liver and kidney extracellular matrices
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Based on the collaboration between the Department of General, Visceral, and Transplant Surgery, University Hospital Münster, and Experimental Surgery, Department of Surgery, Charité – Universitätsmedizin Berlin our work on the "Proteomic analysis of decellularized mice liver and kidney extracellular matrices" has been accepted for publication in Journal of Biological Engineering.

In this study, we employed a bottom-up proteomic approach to elucidate the intricate network of proteins in the decellularized extracellular matrices of murine liver and kidney tissues. This approach involved the use of a novel, perfusion-based decellularization protocol to generate acellular whole organ scaffolds. Proteomic analysis of decellularized mice liver and kidney ECM scaffolds revealed tissue-specific differences in matrisome composition, while we found a predominantly stable composition of the core matrisome, consisting of collagens, glycoproteins, and proteoglycans. Liver matrisome analysis revealed unique proteins such as collagen type VI alpha-6, fibrillin-2 or biglycan. In the kidney, specific ECM-regulators such as cathepsin z were detected. The identification of distinct proteomic signatures provides insights into how different matrisome compositions might influence the biological properties of distinct tissues. This experimental workflow will help to further elucidate the proteomic landscape of decellularized extracellular matrix scaffolds of mice in order to decipher complex cell-matrix interactions and their contribution to a tissue-specific microenvironment.

Authors are Anna-Maria Diedrich, Assal Daneshgar, Peter Tang, Oliver Klein, Annika Mohr, Olachi A. Onwuegbuchulam, Sabine von Rueden, Kerstin Menck, Annalen Bleckmann, Mazen A. Juratli, Felix Becker, Igor M. Sauer, Karl H. Hillebrandt, Andreas Pascher, and Benjamin Struecker.
miRNA as potential biomarkers after liver transplantation: A systematic review
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The publication "miRNA as potential biomarkers after liver transplantation: A systematic review" is now available online in Transplantation Reviews. Authors are Pia F. Koch, Kristina Ludwig, Felix Krenzien, Karl H. Hillebrandt, Wenzel Schöning, Johann Pratschke, Nathanael Raschzok, Igor M. Sauer, and Simon Moosburner.

Early and accurate diagnosis of acute cellular rejection (ACR) and graft complications after liver transplantation is crucial for timely intervention and improved patient outcomes, but their diagnosis rely currently on invasive biopsy sampling, thus prompting the search for non-invasive Biomarkers. MicroRNA (miRNA) have emerged as promising biomarkers in various pathological conditions, and their potential utility in diagnosing acute cellular rejection after liver transplantation has gained significant interest.

This systematic review analyzes studies exploring miRNA as biomarkers for ACR and graft dysfunction in liver transplantation (PROSPERO ID CRD42023465278). The Cochrane Collaboration tool for assessing risk of bias was employed. Population data, identified miRNA and their dynamic regulation, as well as event prediction were compared. Data extraction and quality assessment were performed independently by two reviewers.

The results highlight the potential of miRNA as specific, non-invasive biomarkers for ACR and graft dysfunction following liver transplantation. However, further research is needed to validate these findings and establish standardized diagnostic panels to incorporate them into clinical practice and explore miRNA-based therapies in the future.
Rise of tissue- and species-specific 3D bioprinting based on decellularized extracellular matrix-derived bioinks and bioresins
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The publication "Rise of tissue- and species-specific 3D bioprinting based on decellularized extracellular matrix-derived bioinks and bioresins" is now available online in Biomaterials and Biosystems. Authors are Laura Elomaa, Ahed Almalla, Eriselda Keshi, Karl H. Hillebrandt, Igor M. Sauer, and Marie Weinhart.

Thanks to its natural complexity and functionality, decellularized extracellular matrix (dECM) serves as an excellent foundation for creating highly cell-compatible bioinks and bioresins. This enables the bioprinted cells to thrive in an environment that closely mimics their native ECM composition and offers customizable biomechanical properties. To formulate dECM bioinks and bioresins, one must first pulverize and/or solubilize the dECM into non-crosslinked fragments, which can then be chemically modified as needed. In bioprinting, the solubilized dECM-derived material is typically deposited and/or crosslinked in a layer-by-layer fashion to build 3D hydrogel structures. Since the introduction of the first liver-derived dECM-based bioinks, a wide variety of decellularized tissue have been employed in bioprinting, including kidney, heart, cartilage, and adipose tissue among others. This review aims to summarize the critical steps involved in tissue-derived dECM bioprinting, starting from the decellularization of the ECM to the standardized formulation of bioinks and bioresins, ultimately leading to the reproducible bioprinting of tissue constructs.
Moderate LMWH anticoagulation improves success rate of hind limb allotransplantation in mice
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The publication "Moderate LMWH Anticoagulation Improves Success Rate of Hind Limb Allotransplantation in Mice" is now available online in Plastic & Reconstructive Surgery-Global Open. Authors are B. Kern, M.-I. Ashraf, A. Reutzel-Selke, J. Mengwasser, D. Polenz, E. Michaels, J. Pratschke, S.G. Tullius, Ch. Witzel, and I.M. Sauer.

The mouse hind limb model represents a powerful research tool in vascularized composite tissue allotransplantation, but its applicability is limited due to poor graft survival (62%–83%). Vascular thrombosis and massive hemorrhage are the major causes for these drop-outs. We hypothesize that because of better anticoagulation effect and lower risk of thrombocytopenia, application of low molecular weight heparin (LMWH) will minimize vascular complications and enhance graft and animal survival.

Fifty allogeneic hind limb transplantations were performed (C57BL/6 to DBA/2 mice) using five different anticoagulation protocols. Bleeding and thromboembolic events were recorded macroscopically by postoperative hemorrhage and livid discoloration of the graft, respectively. Graft perfusion and survival were monitored daily by capillary-refill-time of graft toes within 2–3 seconds. Vascular congestion and tissue necrosis were examined by histological evaluation of hematoxylin-eosin-stained tissue sections.

All transplantations were technically successful. Increase in thromboembolic events and a concomitant decrease in bleeding events were observed with the decreasing concentration of heparin in the perfusion solution. Although treatment of donor and recipient with low dose of LMWH could not reduce thromboembolic events, moderate dose effectively reduced these events. Compared with the poor outcome of graft perfusion with heparin alone, additional treatment of donor and recipient with low dose of LMWH improved graft and animal survival by 18%. Interestingly, animals treated with moderate dose of LMWH demonstrated 100% graft and animal survival.
Treatment of donor and recipient mice with a moderate dose of LMWH prevents vascular complications and improves the outcome of murine hind limb transplants.
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