Conference Agenda

The lymphatic vasculature plays a critical role in tissue homeostasis by facilitating the clearance of cellular debris, excess fluid or particles following injury, as well as orchestrating immune responses. Given their central function in immune surveillance and wound exudate clearance, lymphatic endothelial cells (LECs) inevitably interact with biomaterials employed in small diameter vascular graft (SDVG) implantation. However, the impact of various biomaterials on physiological and pathological lymphangiogenesis, and the cellular responses of LECs, remains poorly characterized. Therefore, the current study systematically investigates the effects of distinct vascular graft materials—including decellularized umbilical arteries (dUCAs), biodegradable polyurethane, and clinically used expanded polytetrafluoroethylene (ePTFE)—on LECs and the process of lymphangiogenesis. Using immortalized LECs (LEC-TERT), both direct and indirect material interactions were assessed using cell viability, proliferation, lymphvasculogenesis and two- and three-dimensional migration assays, as well as immunocytochemistry, scanning electron microscopy (SEM), and gene expression analyses. The findings demonstrated a marked reduction in LEC-TERT viability and function upon exposure to ePTFE, suggesting a mechanistic link to the suboptimal healing frequently associated with ePTFE-SDVGs in clinical settings. In contrast, both polyurethane and dUCA promoted improved LEC responses in several experimental metrics, with polyurethane exerting a particularly favorable influence on the lymphangiogenic potential. In summary, the results demonstrated that polyurethane and dUCA were superior biomaterial candidates for supporting physiological lymphatic integration and function in vascular graft applications.

Introduction: Biomaterials from decellularized human placental extracellular matrix (dhpECM) offer unique clinical potential due to their rich protein composition, anti-inflammatory properties, and accessibility. This study compares isolated placental regions to identify proteomic characteristics, compartment–specific cell–matrix interactions, and biophysical properties for the rational selection of tissue-mimetic bioinks for cardiac regeneration and soft-tissue models.

Methods: The dhpECMs, derived from four placental compartments—amnion, basal plate, chorion, and umbilical cord—were formulated as hydrogels. Mass‑spectrometry proteomics was used to assess matrisome similarity to native cardiac tissue and to predict tissue specificity through rank-based gene enrichment analysis. Cell–matrix crosstalk was evaluated in primary human cardiac fibroblasts using RT-qPCR to validate in silico predictions. Rheology quantified thermal gelation behavior, viscoelasticity, and flow properties. Printability was validated on a custom in‑gel extrusion bioprinter.

Results: Proteomics revealed distinct, compartment-specific tissue compositions and matrisomes with high batch-to-batch consistency, overlapping with cardiac ECM constituents, including collagens, glycoproteins, and other matricellular proteins. Bioinformatics ranked umbilical‑cord matrices as closest to native atrial and ventricular tissue. Gene enrichment confirmed the umbilical cord as a candidate, as cardiac terms were significantly enriched in the umbilical cord dataset (Score > 3; FDR < 0.05), with the most substantial enrichment among the hydrogels. Cardiac fibroblasts exhibited hydrogel‑dependent transcriptional responses; qPCR trends corroborated in silico rankings, confirming umbilical cord-derived matrices moderating fibroblast activation markers (aSMA, POSTN) while controlling matrix (COL1A1, COL3A1) and adhesion (ITGAV, ITGB1) expression, modulating cardiac fibrosis relative to other placental ECM groups and controls. The rheology of hydrogels was assessed by characterizing thermogelation (G′ > G″), shear thinning, and a printable yield regime, which enables continuous filament deposition and leads to stable 3D printed constructs.

Discussion: This proteomics-driven, rheology-anchored pipeline can rationally select placenta-derived ECM bioinks with high biochemical fidelity to cardiac tissue and extrusion-ready mechanics. The identified compartment-specific umbilical cord ECM supports the development of stable, cytocompatible constructs for cardiac regeneration research. The applied workflow provides a transferable framework for scaffold selection across various soft-tissue engineering applications.

Metabolic bariatric surgery leads to effective weight loss and to metabolic improvement in most cases. However, one fifth of bariatric patients experience onset of post-operative liver fibrosis with underlying mechanisms remaining unclear. Post-surgical malnutrition, including serum folic acid (FA) deficiency is commonplace. Folates have key roles in DNA-synthesis, as well as in epigenetic and immune regulations. A link between serum FA levels and liver status has been described, leading to the hypothesis that FA deficiency may contribute to post-operative liver fibrosis.

Hepatic stellate cells (HSC) are the main player in liver fibrosis. While predominantly quiescent in healthy liver, they become activated in response to damage signals, differentiating into myofibroblasts that produce high levels of extracellular matrix (ECM) proteins, leading to fibrosis. While fibrosis is reversible, targeted treatment options are currently limited, and elucidating mechanism of HSC activation is key.

In this project we are developing an in vitro model using the human immortalized HSC LX-2 line to study the role of bioavailable 5-methyltetrahydrofolate (5-MTHF) in HSC activation. Preliminary results suggest that in the LX-2 in vitro model, 5-MTHF has no effect on expression of fibrosis-associated markers. During their activation, HSC undergo metabolic reprogramming. Therefore, metabolomics studies will be performed to investigate changes in cellular metabolism in folate-depleted conditions or following 5-MTHF treatment in the presence of pro-fibrotic stimuli. Moreover, co-cultures with patient-derived CD14+ monocytes are to be carried out to examine the involvement of myeloid cells in HSC activation as well as a potential immune-modulating role for folates in this interaction.

Over the past decades, notable progress has been made in designing artificial small-diameter vascular grafts (SDVGs). However, the perivascular adipose tissue (PVAT) and its role in the healing process of synthetic SDVGs remains widely uninvestigated, despite its substantial role in blood vessel homeostasis. The current study evaluates electrospun thermoplastic polyurethane (TPU) SDVGs using combined in vitro and in vivo approaches to examine the bidirectional interaction between the biomaterial and PVAT during graft healing.

In vitro, vascular and perivascular related cells were cultured on TPU grafts for up to seven days and analyzed via cell viability assays, immunofluorescence (IF) staining, scanning electron microscopy (SEM) and qPCR. For in vivo assessment, prostheses were implanted into male Sprague Dawley rats for one week or three months (n = 6 per group), with SHAM-operated animals (n = 6) serving as controls. Cellularization of the implants was assessed using immunohistochemistry and SEM. Furthermore, qPCR was used to analyze inflammation-related genes and adipocyte subtype markers in graft-surrounding PVAT.

Viability of in vitro seeded cells increased over three days, confirming TPU’s cytocompatibility. Cell attachment to the graft as well as maintenance of their typical phenotype was demonstrated by SEM and immunofluorescence staining. Notably, IF staining revealed increased adiponectin expression in PVAT-derived cells, while endothelial cells showed reduced ICAM-1 and VCAM-1 expression compared to unchanged levels in cells seeded on tissue culture plates (TCP). These observations were supported by qPCR, which revealed elevated adiponectin levels approximately 18% higher than in the control TCP group, and downregulated TNF-alpha on TPU.

In vivo, H&E staining and SEM demonstrated rapid cell coverage of implanted grafts within one week, further validated by IF staining of CD34⁺ endothelial progenitor cells on the luminal surface. Gene expression analysis revealed a temporal divergence between compartments: anti-inflammatory markers increased within graft tissue over time, whereas pro-inflammatory markers such as TNF-alpha were elevated in adjacent PVAT by 20% at 3 months. Moreover, PVAT displayed a distinct immune cell response, with both innate (CD11c, MPO, Arg-1) and adaptive immune (FoxP3, T-bet) markers being dynamically regulated following graft implantation. Concurrently, significant alterations in adipocyte marker expression indicated a reorganization of PVAT composition, with partial transition from white-like to brown-like phenotypes during graft integration.

The present study highlights the dual role of TPU-based SDVGs, which support endothelialization while actively shaping local immune and adipose tissue response. An initial pro-inflammatory response is followed by a shift toward protective adipokine secretion, adipocyte browning, and reduced graft inflammation, accompanied by SMC stabilization and perivascular tissue formation with vasa vasorum–like features. By uncovering PVAT’s contribution to graft healing, these findings emphasize the importance of investigating PVAT responses to biomaterials when developing SDVGs to ensure long-term patency.

Background and Aim: Colorectal cancer ranks as the third most commonly diagnosed cancer and is the second leading cause of cancer-related deaths worldwide, which highlights the urgent need for the development of new treatments. One promising avenue is the use of so-called humanized mouse models to investigate the interactions between the human immune system and tumors. In this setting, the project focuses on tumor-infiltration and tumor-induced changes of human monocyte subsets: classical, intermediate, non-classical, and CD56+ monocytes.

Methods: Generation of a human immune system in immunocompromised NSG-QUAD mice was achieved by sub-lethal irradiation and intrahepatic injection of human CD34+ hematopoietic stem cells (HSCs) isolated from human cord blood. Afterwards, colorectal cancer was induced by orthotopic injection of human HCT-116 Luc2 cells and tumor growth was monitored in vivo by bioluminescent imaging. The monocyte subsets were isolated from a healthy donor, separated by their CD14, CD16 and CD56 expression in fluorescence-activated cell sorting (FACsorting), were membrane-labeled and then intravenously injected into the tumor-bearing mice. At the end of the experiment, the characterization of the generated human immune system and the monocyte distribution were assessed by flow cytometric analysis of mouse blood and bone marrow. Furthermore, membrane-labeled human monocytes were re-isolated from colorectal tumors by tissue dissociation and FACsorting. RNA isolation and sequencing were conducted of the sorted monocyte subsets before and after tumor infiltration.

Results: Humanization of NSG-QUAD mice with human CD34+ HSCs was successful, with an average human engraftment of 34% in blood and 49% in bone marrow, and a developed human immune system as characterized by flow cytometry. The orthotopic induction of colorectal cancer promoted the development of tumors in the humanized mouse model over 3 weeks, followed by adoptive cell transfer of isolated human monocyte subsets. RNA sequencing confirmed the subset identity of FACsorted classical, intermediate, non-classical and CD56+ monocytes before transfer into the tumor-bearing mice. However, the RNA integrity of the re-isolated, tumor-infiltrating monocytes did not allow further analysis of the possible changes in the gene expression of the monocyte subsets.

Conclusions: We have successfully established a xenograft model of orthotopically growing human CRC cells in humanized mice with adoptive transfer of human monocyte subsets. We are currently improving the reisolation of tumor-infiltrating monocytes for profiling by RNA sequencing.

BACKGROUND: The current state of the art to assess myocardial viability in ex-situ heart perfusion (ESHP) is serial lactate measurements. The information about myocardial function and cell damage is limited. Microdialysis is able to measure markers of cell injury and metabolites in the interstitial fluid. The study aimed to investigate the feasibility of this analysis tool in a porcine ESHP model.

METHODS: 12 german domestic pigs were used as heart and blood donors. To simulate different organ donation procedures two protocols were established: Control group (n=6) and “donation after circulatory death” (DCD) (n=6). Microdialysis catheters (CMA 20 Elite 10mm, CMA Microdialysis AB, Kista, Sweden) were inserted in the myocardium of the left ventricle. Semi-continuous perfusate samples were taken every 10 minutes. Lactate, pyruvate, and glycerol were analysed subsequently with the ISCUSflex (CMA Microdialysis AB, Kista, Sweden). The groups were compared at baseline (in-situ) and 20, 60, 180, and 360 minutes of ex-situ heart perfusion using t-test.

RESULTS: Interstitial levels of lactate, pyruvate, and glycerol did not differ at baseline.
Lactate increased significantly in the DCD group with the highest values after 20 minutes of ESHP (Control 1.28 (±0.56) mmol/L vs. DCD 7.31 (±0.11) mmol/l, p<0.001) and correlated with perfusate lactate levels. Pyruvate increased significantly faster in the DCD group (20min: Control 26.41 (±23.97) µmol/l vs. DCD 162.8 (±124.4) µmol/l, p=0.04; 360min: Control 129.58 (±72.46) µmol/l vs. DCD 455.93 (±104.59) µmol/l, p<0.001). Glycerol was stable during ESHP in the Control group, but was significantly higher in the DCD group with a rapid increase directly after reperfusion (20min: DBD 105 µmol/l (±71.73) vs. DCD 783.79 (±280.02) µmol/l, p<0.001).

CONCLUSIONS: Microdialysis in ESHP is feasible and able to detect metabolic changes in tissue. Especially, glycerol as a marker of cell injury is able to give additional information quickly.