Purpose: Polymicrobial biofilm complicate medical treatment, in patients with implanted medical devices (medical device associated infection ((MDAI)). In this project, we investigated the dynamics of co-colonization of staphylococci (Staphylococcus aureus and Staphylococcus epidermidis) in tissue-engineered, air-exposed, three-dimensional (3D) human skin equivalent, as a replacement for animal experiments (3R). This experimental in vitro set-up that mimic the native skin to a high degree was further developed in in vitro polymicrobial biofilm peri-implant infection model to study MDAI.

Methods: Primary human epidermal keratinocytes (HPEK) and human dermal fibroblasts (HDFp) were used together to set up the 3D human skin equivalent. After description of the 3D skin equivalent, integrating of the implant into the model follow. For the experimental infection, the tissue models were exposed on the apical side to bacterial culture to investigate host response to monospecies versus polymicrobial biofilms. Infection-related changes of the morphology, integrity, differentiation, cytokine production, proliferation and cytotoxic effect were analyzed by using immunohistochemistry, immunofluorescence, ELISA and LDH-detection.

Results: Experimental infection of 3D-skin equivalent showed destruction of St. corneum and St. granulosum with dissemination of soft tissue and accumulation of staphylococci inside of 3D equivalent on implant surface with forming of mature biofilm. The cytotoxic effect was enhanced in bi-species biofilm, suggesting a mutual support of bacteria in their virulence. Immunostaining for tight junction protein (ZO-1) demonstrated changes during infection by alterations in the distributions of ZO-1 in 3D human skin equivalent. The measurement of secreted cytokine levels (TNF-α, IL-α, MCP-1, IL-33 and IL-6) proved host–pathogen interactions in our model. In the implant model, despite the same bacterial concentration, the cytotoxicity was higher, and the proinflammatory cytokine response was more intense in the presence of an implant. The implant promoted the infection process.

Conclusions: This experimental in vitro set-up enables a structured approach to investigate MDAI with polymicrobial biofilm and its impact on skin cells in 3D skin equivalent in vitro. Our model represented a new inexpensive tool for the analysis of approval studies for antibiotics and other local therapeutic agents including antiseptic solutions as well as modern wound dressings.

Background: Nomothermic machine perfusion (NMP) allows viability assessment in livers from extended criteria donors (ECD). Additionally, NMP provides a platform for ex vivo therapeutic intervention. Nor-ursodeoxycholic acid (norUDCA) has antiinflammatory, antifibrotic, and antilipotoxic properties. We aimed to investigate the effects of norUDCA during NMP on ECD livers rejected for transplantation.

Methods: We evaluated apoptosis markers in perfusate and bile composition in samples of 18 normothermic perfused, rejected livers, over a period of 12 hours. Nine livers received treatment of 1500 mg of norUDCA after two hours. Apoptosis markers (active caspase-3, Bcl-2, cleaved PARP, Cytochrome c, p53) were measured on the Luminex platform, with a commercially available human apoptosis panel. To account for heterogeneity of donors, the relative dynamic of apoptosis markers was determined for individual grafts after application of norUDCA. Blood gas analysis of perfusate and bile, as well as additional measurements including alanine-aminotransferase (ALT), aspartate-aminotransferase (AST), and lactate dehydrogenase (LDH), were performed every hour.

Results: Apoptosis markers are detected after 5 minutes and increased over the first two hours of NMP. NorUDCA treatment after two hours lead to a significant reduction of apoptosis markers cleaved PARP (6h: p=0.0142) and p53 (6h: p=0.0106; 12h p=0.0400), additionally to a significant reduction in delta 2h-6h values compared to control livers: active caspase-3 (p=0.0188), Bcl-2 (p=0.0400), cleaved PARP (p=0.0244) and Cytochrome c (p=0.0304). Of note is the overall dynamic reduction of apoptosis markers in the treatment group, whereas the control group increased or remained constant. Further, treated grafts presented improved biliary viability, evident in higher bile bicarbonate concentration (3h: 0.026; 4h: p=0.016; 5h p=0.030), as well as lower bile transaminases AST (8h: p=0.017) and ALT (8h: p=0.004; 12 h: p=0.026). Regarding clinically used viability criteria, before treatment only 2 livers of each group met viability criteria, whereas 8 of 9 norUDCA treated livers presented transplantable after 12h of perfusion (Ctrl: 3 of 9; p=0.16).

Conclusions: The present data suggests an anti-apoptotic and bile improving effect of norUDCA during NMP of ECD liver grafts in a preclinical model. NorUDCA might be able to ameliorate the detrimental effects of bile toxicity on the ischemia-injured biliary tree. Therefore, therapeutic application of norUDCA during NMP seems promising for further improvement of outcome after liver transplantation.

Background: Ischemia during the process of liver transplantation and the concomitant reperfusion injury (IRI) both pose a threat to the graft. Resulting reactive oxygen species (ROS) especially facilitate the damages to the endothelial glycocalyx covering the vascular endothelium. The endothelial glycocalyx regulates permeability and mediates the adhesion of leukocytes, therefore playing an important role in inflammatory processes. Hypothermic oxygenated machine perfusion (HOPE) of liver grafts has been shown to reduce IRI in comparison to static cold storage (SCS). The aim of this study was to measure the degradation of the glycocalyx during and after HOPE or SCS, potentially providing a means of objective organ viability assessment during liver transplantation.

Methods: Levels of Syndecan-1 (Sdc1), the main component of the glycocalyx, were measured via ELISA in samples of 77 patients after liver transplantation. 40 grafts underwent a period of HOPE with the Organ Assist® machine perfusion system following SCS, 37 livers were transplanted without prior machine perfusion.

Results: In the graft effluent (immediately before implantation) Sdc1 concentration was significantly lower in HOPE preserved livers: 466 ng/mL (350-1073) vs. 4011 ng/mL (3382-4683), p<0.001. Furthermore, on the first postoperative day Sdc1 levels in patients receiving HOPE preserved livers already declined to baseline as opposed to SCS: 362 ng/mL (232-880) vs. 1017 ng/mL (637-1900), p<0.001. In liver grafts undergoing HOPE, Sdc1 concentrations after 60 minutes of perfusion were significantly higher in grafts that later developed early allograft dysfunction (EAD) in comparison to non-EAD livers: 896 ng/mL (419-1681) vs. 429 ng/mL (260-556), p=0.018. Further ROC analysis revealed a predictive potential of Sdc1 concentration after 60 minutes of HOPE concerning EAD using a cutoff level of 808 ng/mL, with an area under the curve (AUC) of 0.740 (p=0.018, sensitivity 0.667, specificity 0.846).

Conclusion: HOPE of liver grafts in comparison with sole SCS preservation reduces extent and duration of glycocalyx degradation, indicated by lower Sdc1 levels in effluent and postoperative sera. Furthermore, Sdc1 concentration can predict EAD after liver transplantation and therefore provides a potential option for objective viability assessment during HOPE.

Background

Limited supply and growing demand lead to a shortage of transplantable organs worldwide. Besides economic factors like increased healthcare costs, long waiting times, increased mortality and impaired quality of life are resulting from this shortage. Therefore, innovative preservation methods are explored in this context. Among these, subzero organ preservation and normothermic machine perfusion (NMP) are of great interest, as they may allow for extended viability, improved monitoring, increased quality, and better matching and transport logistics. In this study, we thus explored the feasibility of extended preservation (48hrs) of porcine kidneys with a nature-inspired anti-icing agent at -5°C followed by 6 hours of NMP.

Methods

Three porcine kidneys were retrieved using a living-donor procedure, flushed with a non-toxic peptoid based cryopreservative (XT-ViVo^, X-Therma Inc., California) and placed in a -5°C environment (TimeSeal^, X-Therma Inc., California) for 48 hours. Kidneys were subsequently flushed and subjected to 6hrs of NMP using Kidney Assist (Organ Assist, Groningen) and an autologous whole-blood based perfusate. Before, during and after NMP, biopsies were taken for histological, viability and respiratory analysis. Perfusion was assessed using hyperspectral imaging (HSI) and biochemical parameters of perfusate and urine were analyzed.

Results

Hemodynamic flow remained stable throughout perfusion, with a renal resistance index around 0.6. The kidney metabolized oxygen at all times, with a peak at two hours of reperfusion, stabilizing thereafter. Physiologic pH, moderate perfusate lactate and ion levels were detected. All kidneys instantaneously produced urine upon reperfusion and during NMP (264 ml/h [256-298]; median [IQR]). High-resolution respirometry clearly revealed one preferred substrate pathway (succinate) and showed stable oxidative phosphorylation capacity after reperfusion. Kidney parenchyma and ureter were imaged using HSI, revealing stable micro-perfusion throughout 6 hours of NMP.

Conclusions

We show that ice-free subzero preservation combined with NMP is a promising method for extended organ storage that maintains organ quality and function. This could help ease logistical hurdles and allow for better donor-recipient matching. By combining two novel technologies, subzero preservation and NMP, time could be taken out of the equation in organ transplantation to ultimately help facilitate global organ exchange in the future.

INTRODUCTION TO THE TOPIC AND BACKGROUND:

Low plasma levels of natural IgM antibodies targeting oxidation specific epitopes (OSEs), which are products of lipid peroxidation, have been shown to be associated with increased cardiovascular risk. This effect is mainly attributed to the ability of OSE-specific IgM to limit atherosclerosis progression and atherothrombotic events. However, little is known about their potential to modulate the onset and progression of abdominal aortic aneurysms (AAAs).

RESEARCH QUESTIONS AND METHODS:

AAAs were induced in two mouse models and aorta dilatation was monitored by 3D ultrasound. Aneurysms were triggered (1) in 16 ApoE KO mice by subcutaneous implantation of osmotic pumps which released angiotensin II over 28 days; (2) in 10 C57BL6/J wild-type mice by peri-adventitial elastase application to the infrarenal abdominal aorta. Circulating OSE IgM levels were determined over the time course of aneurysm development, and the local accumulation of OSEs and OSE-specific Igs was investigated in mouse AAA tissue. To evaluate the effect of OSE-specific IgM on AAA formation, mice were repeatedly administered with the malondialdehyde-specific monoclonal IgM LR04 or isotype control (n=10/group) for 28 days.

RESULTS:

The levels of circulating OSE-specific IgM increased upon aneurysm development in both mouse models of AAA. OSE-reactive IgM and B cells were detected in mouse aneurysm sections in close proximity to sites of OSE accumulation. Preliminary data indicate that treatment with OSE-specific IgM reduces AAA formation and the concomitant OSE IgM rise in the angiotensin II-based mouse model. This effect was more prominent in mice with lower levels of endogenous OSE-specific IgM.

CONCLUSION:

Processes of atherogenesis and tissue destruction may lead to the generation and accumulation of OSEs at the aneurysm site. Based on our results, we propose that resident B-cells contribute to the local release of OSE-specific Igs and to the circulating pool of OSE-specific IgM in the AAA setting. Increasing the blood levels of OSE-specific IgM may represent an effective approach to dampen AAA growth.

Over the past decades, remarkable developments have been made in research focusing on small-diameter vascular grafts (SDVG). However, challenges such as inadequate endothelialization, thrombosis, and intimal hyperplasia remain unresolved. This project aimed to enhance early reendothelialization of synthetic grafts by loading electrospun thermoplastic polyurethane (TPU) grafts with adiponectin.

Adiponectin, an adipokine primarily released by adipocytes, stimulates the phosphorylation of AMPK, thereby downregulating pro-inflammatory mediators like NF-kB and TNF-α. In addition, adiponectin enhances endothelial cell function by increasing nitric oxide levels to help prevent atherosclerosis, while also stimulating COX-2 to reduce apoptosis. By inhibiting smooth muscle cell proliferation through the suppression of growth factors, adiponectin further mitigates intimal hyperplasia, making it a promising therapeutic target in vascular graft research.

To evaluate the effects of adiponectin on the interaction between cells and TPU in vitro, the protein was immobilized on the graft surface via two different methods. First, click chemistry was performed to link adiponectin directly to the synthetic graft material, while collagen I was used to incorporate adiponectin into surface coating. Subsequently, functionalized SDVGs were seeded with rat aortic endothelial cells (RaOECs) for up to 7 days. Cytocompatibility of the modified grafts as well as their influence on cellular gene expression was evaluated on day 1, 3 and 7. Furthermore, formation of an intact endothelium was investigated via immunofluorescence staining and electron microscopy.

Cell viability assessments indicated increased endothelial cell proliferation over time, confirming the cytocompatibility of adiponectin-loaded TPU grafts. Immunofluorescence staining against VCAM-1 and ICAM-1 revealed no increased edothelial cell activation on SDVGs modified with adiponectin but showed successful reendothelialization. The formation of an intact endothelial cell monolayer as well as typical cell morphology was further confirmed by Electron Microscopy. qPCR analysis of gene expression revealed no upregulation of inflammatory-related genes, thereby indicating that the functionalized surface had no negative impact on cellular expression patterns.

The findings of this study indicate that adiponectin facilitates the formation of a healthy endothelium by enhancing cell-graft interactions. We hypothesize that immobilization of adiponectin on synthetic SDVGs could significantly improve the healing processes.

This project was funded by the Ludwig Boltzmann Institute of Cardiovascular Research.