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There is a great need for valuable ex vivo models that allow for assessment of cartilage repair strategies to reduce the high number of animal experiments. In this paper we present three studies with our novel ex vivo osteochondral culture platform. It consists of two separated media compartments for cartilage and bone, which better represents the in vivo situation and enables supply of factors pecific to the different needs of bone and cartilage. We investigated whether separation of the cartilage and bone compartments and/or culture media results in the maintenance of viability, structural and functional properties of cartilage tissue. Next, we valuated for how long we can preserve cartilage matrix stability of osteochondral explants during long-term culture over 84 days. Finally, we determined the optimal defect size that does not show spontaneous self-healing in this culture system. It was demonstrated that separated compartments for cartilage and bone in combination with tissue-specific medium allow for long-term culture of osteochondral explants while maintaining cartilage viability, atrix tissue content, structure and mechanical properties for at least 56 days. Furthermore, we could create critical size cartilage defects of different sizes in the model. The osteochondral model represents a valuable preclinical ex vivo tool for studying clinically relevant cartilage therapies, such as cartilage biomaterials, for their regenerative potential, for evaluation of drug and cell therapies, or to study mechanisms of cartilage regeneration. It will undoubtedly reduce the number of animals needed for in vivotesting.
Purpose
Hypertrophic cartilage is an important characteristic of osteoarthritis and can often be found in patients suffering from osteoarthritis. Although the exact pathomechanism remains poorly understood, hypertrophic de-differentiation of chondrocytes also poses a major challenge in the cell-based repair of hyaline cartilage using mesenchymal stromal cells (MSCs). While different members of the transforming growth factor beta (TGF-β) family have been shown to promote chondrogenesis in MSCs, the transition into a hypertrophic phenotype remains a problem. To further examine this topic we compared the effects of the transcription growth and differentiation factor 5 (GDF-5) and the mutant R57A on in vitro chondrogenesis in MSCs.
Methods
Bone marrow-derived MSCs (BMSCs) were placed in pellet culture and in-cubated in chondrogenic differentiation medium containing R57A, GDF-5 and TGF-ß1 for 21 days. Chondrogenesis was examined histologically, immunohistochemically, through biochemical assays and by RT-qPCR regarding the expression of chondrogenic marker genes.
Results
Treatment of BMSCs with R57A led to a dose dependent induction of chondrogenesis in BMSCs. Biochemical assays also showed an elevated glycosaminoglycan (GAG) content and expression of chondrogenic marker genes in corresponding pellets. While treatment with R57A led to superior chondrogenic differentiation compared to treatment with the GDF-5 wild type and similar levels compared to incubation with TGF-ß1, levels of chondrogenic hypertrophy were lower after induction with R57A and the GDF-5 wild type.
Conclusions
R57A is a stronger inducer of chondrogenesis in BMSCs than the GDF-5 wild type while leading to lower levels of chondrogenic hypertrophy in comparison with TGF-ß1.
Transmission of Trypanosoma brucei by tsetse flies involves the deposition of the cell cycle-arrested metacyclic life cycle stage into mammalian skin at the site of the fly’s bite. We introduce an advanced human skin equivalent and use tsetse flies to naturally infect the skin with trypanosomes. We detail the chronological order of the parasites’ development in the skin by single-cell RNA sequencing and find a rapid activation of metacyclic trypanosomes and differentiation to proliferative parasites. Here we show that after the establishment of a proliferative population, the parasites enter a reversible quiescent state characterized by slow replication and a strongly reduced metabolism. We term these quiescent trypanosomes skin tissue forms, a parasite population that may play an important role in maintaining the infection over long time periods and in asymptomatic infected individuals.
Salivary gland tumors (SGTs) are a relevant, highly diverse subgroup of head and neck tumors whose entity determination can be difficult. Confocal Raman imaging in combination with multivariate data analysis may possibly support their correct classification. For the analysis of the translational potential of Raman imaging in SGT determination, a multi-stage evaluation process is necessary. By measuring a sample set of Warthin tumor, pleomorphic adenoma and non-tumor salivary gland tissue, Raman data were obtained and a thorough Raman band analysis was performed. This evaluation revealed highly overlapping Raman patterns with only minor spectral differences. Consequently, a principal component analysis (PCA) was calculated and further combined with a discriminant analysis (DA) to enable the best possible distinction. The PCA-DA model was characterized by accuracy, sensitivity, selectivity and precision values above 90% and validated by predicting model-unknown Raman spectra, of which 93% were classified correctly. Thus, we state our PCA-DA to be suitable for parotid tumor and non-salivary salivary gland tissue discrimination and prediction. For evaluation of the translational potential, further validation steps are necessary.
Adoptive cellular immunotherapy with chimeric antigen receptor (CAR) T cells is highly effective in haematological malignancies. This success, however, has not been achieved in solid tumours so far. In contrast to hematologic malignancies, solid tumours include a hostile tumour microenvironment (TME), that poses additional challenges for curative effects and consistent therapeutic outcome. These challenges manifest in physical and immunological barriers that dampen efficacy of the CAR T cells. Preclinical testing of novel cellular immunotherapies is performed mainly in 2D cell culture and animal experiments. While 2D cell culture is an easy technique for efficacy analysis, animal studies reveal information about toxicity in vivo. However, 2D cell culture cannot fully reflect the complexity observed in vivo, because cells are cultured without anchorage to a matrix and only short-term periods are feasible. Animal studies provide a more complex tissue environment, but xenografts often lack human stroma and tumour inoculation occurs mostly ectopically. This emphasises the need for standardisable and scalable tumour models with incorporated TME-aspects, which enable preclinical testing with enhanced predictive value for the clinical outcome of immunotherapies. Therefore, microphysiologic 3D tumour models based on the biological SISmuc (Small Intestinal mucosa and Submucosa) matrix with preserved basement membrane were engaged and improved in this work to serve as a modular and versatile tumour model for efficacy testing of CAR T cells. In order to reflect a variety of cancer entities, TME-aspects, long-term stability and to enhance the read-out options they were further adapted to achieve scalable and standardisable defined microphysiologic 3D tumour models. In this work, novel culture modalities (semi-static, sandwich-culture) were characterised and established that led to an increased and organised tissue generation and long-term stability. Application of the SISmuc matrix was extended to sarcoma and melanoma models and serial bioluminescence intensity (BLI)-based in vivo imaging analysis was established in the microphysiologic 3D tumour models, which represents a time-efficient read-out method for quality evaluation of the models and treatment efficacy analysis, that is independent of the cell phenotype. Isolation of cancer-associated-fibroblasts (CAFs) from lung (tumour) tissue was demonstrated and CAF-implementation further led to stromal-enriched microphysiologic 3D tumour models with in vivo-comparable tissue-like architecture. Presence of CAFs was confirmed by CAF-associated markers (FAP, α-SMA, MMP-2/-9) and cytokines correlated with CAF phenotype, angiogenesis, invasion and immunomodulation. Additionally, an endothelial cell barrier was implemented for static and dynamic culture in a novel bioreactor set-up, which is of particular interest for the analysis of immune cell diapedesis. Studies in microphysiologic 3D Ewing’s sarcoma models indicated that sarcoma cells could be sensitised for GD2-targeting CAR T cells. After enhancing the scale of assessment of the microphysiologic 3D tumour models and improving them for CAR T cell testing, the tumour models were used to analyse their sensitivity towards differently designed receptor tyrosine kinase-like orphan receptor 1 (ROR1) CAR T cells and to study the effects of the incorporated TME-aspects on the CAR T cell treatment respectively. ROR1 has been described as a suitable target for several malignancies including triple negative breast cancer (TNBC), as well as lung cancer. Therefore, microphysiologic 3D TNBC and lung cancer models were established. Analysis of ROR1 CAR T cells that differed in costimulation, spacer length and targeting domain, revealed, that the microphysiologic 3D tumour models are highly sensitive and can distinguish optimal from sub-optimal CAR design. Here, higher affinity of the targeting domain induced stronger anti-tumour efficacy and anti-tumour function depended on spacer length, respectively. Long-term treatment for 14 days with ROR1 CAR T cells was demonstrated in dynamic microphysiologic 3D lung tumour models, which did not result in complete tumour cell removal, whereas direct injection of CAR T cells into TNBC and lung tumour models represented an alternative route of application in addition to administration via the medium flow, as it induced strong anti-tumour response. Influence of the incorporated TME-aspects on ROR1 CAR T cell therapy represented by CAF-incorporation and/or TGF-β supplementation was analysed. Presence of TGF-β revealed that the specific TGF-β receptor inhibitor SD-208 improves ROR1 CAR T cell function, because it effectively abrogated immunosuppressive effects of TGF-β in TNBC models. Implementation of CAFs should provide a physical and immunological barrier towards ROR1 CAR T cells, which, however, was not confirmed, as ROR1 CAR T cell function was retained in the presence of CAFs in stromal-enriched microphysiologic 3D lung tumour models. The absence of an effect of CAF enrichment on CAR T cell efficacy suggests a missing component for the development of an immunosuppressive TME, even though immunomodulatory cytokines were detected in co-culture models. Finally, improved gene-edited ROR1 CAR T cells lacking exhaustion-associated genes (PD-1, TGF-β-receptor or both) were challenged by the combination of CAF-enrichment and TGF-β in microphysiologic 3D TNBC models. Results indicated that the absence of PD-1 and TGF-β receptor leads to improved CAR T cells, that induce strong tumour cell lysis, and are protected against the hostile TME. Collectively, the microphysiologic 3D tumour models presented in this work reflect aspects of the hostile TME of solid tumours, engage BLI-based analysis and provide long-term tissue homeostasis. Therefore, they present a defined, scalable, reproducible, standardisable and exportable model for translational research with enhanced predictive value for efficacy testing and candidate selection of cellular immunotherapy, as exemplified by ROR1 CAR T cells.
Electrochemical impedance spectroscopy (EIS) is a valuable technique analyzing electrochemical behavior of biological systems such as electrical characterization of cells and biomolecules, drug screening, and biomaterials in biomedical field. In EIS, an alternating current (AC) power signal is applied to the biological system, and the impedance of the system is measured over a range of frequencies.
In vitro culture models of endothelial or epithelial barrier tissue can be achieved by culturing barrier tissue on scaffolds made with synthetic or biological materials that provide separate compartments (apical and basal sides), allowing for further studies on drug transport. EIS is a great candidate for non-invasive and real-time monitoring of the electrical properties that correlate with barrier integrity during the tissue modeling. Although commercially available transendothelial/transepithelial electrical resistance (TEER) measurement devices are widely used, their use is particularly common in static transwell culture. EIS is considered more suitable than TEER measurement devices in bioreactor cultures that involve dynamic fluid flow to obtain accurate and reliable measurements. Furthermore, while TEER measurement devices can only assess resistance at a single frequency, EIS measurements can capture both resistance and capacitance properties of cells, providing additional information about the cellular barrier's characteristics across various frequencies. Incorporating EIS into a bioreactor system requires the careful optimization of electrode integration within the bioreactor setup and measurement parameters to ensure accurate EIS measurements. Since bioreactors vary in size and design depending on the purpose of the study, most studies have reported using an electrode system specifically designed for a particular bioreactor. The aim of this work was to produce multi-applicable electrodes and established methods for automated non-invasive and real-time monitoring using the EIS technique in bioreactor cultures. Key to the electrode material, titanium nitride (TiN) coating was fabricated on different substrates (materials and shape) using physical vapor deposition (PVD) and housed in a polydimethylsiloxane (PDMS) structure to allow the electrodes to function as independent units. Various electrode designs were evaluated for double-layer capacitance and morphology using EIS and scanning electron microscopy (SEM), respectively. The TiN-coated tube electrode was identified as the optimal choice. Furthermore, EIS measurements were performed to examine the impact of influential parameters related to culture conditions on the TiN-coated electrode system. In order to demonstrate the versatility of the electrodes, these electrodes were then integrated into in different types of perfusion bioreactors for monitoring barrier cells. Blood-brain barrier (BBB) cells were cultured in the newly developed dynamic flow bioreactor, while human umblical vascular endothelial cells (HUVECs) and Caco-2 cells were cultured in the miniature hollow fiber bioreactor (HFBR). As a result, the TiN-coated tube electrode system enabled investigation of BBB barrier integrity in long-term bioreactor culture. While EIS measurement could not detect HUVECs electrical properties in miniature HFBR culture, there was the possibility of measuring the barrier integrity of Caco-2 cells, indicating potential usefulness for evaluating their barrier function. Following the bioreactor cultures, the application of the TiN-coated tube electrode was expanded to hemofiltration, based on the hypothesis that the EIS system may be used to monitor clotting or clogging phenomena in hemofiltration. The findings suggest that the EIS monitoring system can track changes in ion concentration of blood before and after hemofiltration in real-time, which may serve as an indicator of clogging of filter membranes. Overall, our research demonstrates the potential of TiN-coated tube electrodes for sensitive and versatile non-invasive monitoring in bioreactor cultures and medical devices.
Addition of heparin binding sites strongly increases the bone forming capabilities of BMP9 in vivo
(2023)
Highlights
• Despite not being crucial for bone development BMP9 can induce bone growth in vivo.
• BMP9 induced bone formation is strongly enhanced by introduced heparin binding sites.
• BMP9s bone forming capabilities are triggered by extracellular matrix binding.
• Heparin binding BMP9 (BMP9 HB) can improve the current therapies in treating bone fractures.
Abstract
Bone Morphogenetic proteins (BMPs) like BMP2 and BMP7 have shown great potential in the treatment of severe bone defects. In recent in vitro studies, BMP9 revealed the highest osteogenic potential compared to other BMPs, possibly due to its unique signaling pathways that differs from other osteogenic BMPs. However, in vivo the bone forming capacity of BMP9-adsorbed scaffolds is not superior to BMP2 or BMP7. In silico analysis of the BMP9 protein sequence revealed that BMP9, in contrast to other osteogenic BMPs such as BMP2, completely lacks so-called heparin binding motifs that enable extracellular matrix (ECM) interactions which in general might be essential for the BMPs' osteogenic function. Therefore, we genetically engineered a new BMP9 variant by adding BMP2-derived heparin binding motifs to the N-terminal segment of BMP9′s mature part. The resulting protein (BMP9 HB) showed higher heparin binding affinity than BMP2, similar osteogenic activity in vitro and comparable binding affinities to BMPR-II and ALK1 compared to BMP9. However, remarkable differences were observed when BMP9 HB was adsorbed to collagen scaffolds and implanted subcutaneously in the dorsum of rats, showing a consistent and significant increase in bone volume and density compared to BMP2 and BMP9. Even at 10-fold lower BMP9 HB doses bone tissue formation was observed. This innovative approach of significantly enhancing the osteogenic properties of BMP9 simply by addition of ECM binding motifs, could constitute a valuable replacement to the commonly used BMPs. The possibility to use lower protein doses demonstrates BMP9 HB's high translational potential.
Lung cancer is the main cause of cancer-related deaths worldwide. Despite the availability of several targeted therapies and immunotherapies in the clinics, the prognosis for lung cancer remains poor. A major problem for the low benefit of these therapies is intrinsic and acquired resistance, asking for pre-clinical models for closer investigation of predictive biomarkers for refined personalized medicine and testing of possible combination therapies as well as novel therapeutic approaches to break resistances.
One third of all lung adenocarcinoma harbor mutations in the KRAS gene, of which 39 % are transitions from glycine to cysteine in codon 12 (KRASG12C). Being considered “undruggable” in previous decades, KRASG12C-inhibitors now paved the way into the standard-of-care for lung adenocarcinoma treatment in the clinics. Still, the overall response rates as well as overall survival of patients treated with KRASG12C-inhibitors are sobering. Therefore, 3D KRASG12C-biomarker in vitro models were developed based on a decellularized porcine jejunum (SISmuc) using commercial and PDX-derived cell lines and characterized in regards of epithelial-mesenchymal-transition (EMT), stemness, proliferation, invasion and c-MYC expression as well as the sensitivity towards KRASG12C-inhibiton. The phenotype of lung tumors harboring KRAS mutations together with a c-MYC overexpression described in the literature regarding invasion and proliferation for in vivo models was well represented in the SISmuc models. A higher resistance towards targeted therapies was validated in the 3D models compared to 2D cultures, while reduced viability after treatment with combination therapies were exclusively observed in the 3D models. In the test system neither EMT, stemness nor the c-MYC expression were directly predictive for drug sensitivity. Testing of a panel of combination therapies, a sensitizing effect of the aurora kinase A (AURKA) inhibitor alisertib for the KRASG12C-inhibitor ARS-1620 directly correlating with the level of c-MYC expression in the corresponding 3D models was observed. Thereby, the capability of SISmuc tumor models as an in vitro test system for patient stratification was demonstrated, holding the possibility to reduce animal experiments.
Besides targeted therapies the treatment of NSCLC with oncolytic viruses (OVs) is a promising approach. However, a lack of in vitro models to test novel OVs limits the transfer from bench to bedside. In this study, 3D NSCLC models based on the SISmuc were evaluated for their capability to perform efficacy and risk assessment of oncolytic viruses (OVs) in a pre-clinical setting. Hereby, the infection of cocultures of tumor cells and fibroblasts on the SISmuc with provided viruses demonstrated that in contrast to a wildtype herpes simplex virus 1 (HSV-1) based OV, the attenuated version of the OV exhibited specificity for NSCLC cells with a more advanced and highly proliferative phenotype, while fibroblasts were no longer permissive for infection. This approach introduced SISmuc tumor models as novel test system for in vitro validation of OVs.
Finally, a workflow for validating the efficacy of anti-cancer therapies in 3D tumor spheroids was established for the transfer to an automated platform based on a two-arm-robot system. In a proof-of-concept process, H358 spheroids were characterized and treated with the KRASG12C-inhibitor ARS-1620. A time- and dose-dependent reduction of the spheroid area after treatment was defined together with a live/dead-staining as easy-to-perform and cost-effective assays for automated drug testing that can be readily performed in situ in an automated system.
Diese Arbeit hatte zum Ziel quantitative Analysen histologischer Aufnahmen der Haut nach unterschiedlichen Gesichtspunkten zu etablieren. Im ersten Abschnitt wurde die bildgestützte Quantifizierung der epidermalen Histomorphologie untersucht. Nach Sichtung und Beurteilung von 2145 hochauflösenden Fotografien HE-gefärbter Epidermis- und Vollhautmodellen jeglichen Zustands, wurde der BSGC-Score als Facettenklassifikation mit seinen insgesamt 40 Beurteilungskriterien aufgestellt. Die unterschiedlichen epidermalen Strata wurden mit Wichtungsfaktoren belegt. Die Bewertungskategorien sind mit einem Ampelsystem unterlegt. Eine Befundungsformel wurde aufgestellt. Weitere Bestandteile des BSGC-Scores sind eine Anleitung mit Bildbeilage sowie Dokumentationselemente. Die Anwendung erfolgte erfolgreich im Rahmen der Qualitätssicherung an Chargentests und zur Verlaufsbeurteilung eines In-vitro-Verbrennungsmodells aus humaner Epidermis durch Schneider et al. (2021) Der BSGC-Score dient als zügig durchführbares Evaluationstool zur Befundung von In-vitro-Epidermismodellen und nicht als diagnostisches Mittel. Der zweite Abschnitt beschäftigt sich mit der Vaskularisierung als Parameter der kutanen Wundheilung. Es wurden aSMA-IF-gefärbte Abbildungen porciner Verwundungsmodelle betrachtet und nach der Entfernung drüsiger Strukturen Gefäßanschnitte zu Beginn manuell ausgezählt. Hieraus wurden die nötigen Einstellungen für die Bildbearbeitungssoftware ImageJ ermittelt und die Abbildungen dieser anschließend zugeführt. Es erfolgte die automatisierte Quantifizierung elliptischer Formationen mit einer Größe ≥ 30 Pixel. Im nächsten Schritt wurden die Abbildungen in die Bereiche Wundrand, Wundgrund und Wundheilung unterteilt. In dem Bereich Wundheilung zeigte sich eine signifikant größere Revaskularisierung als in Wundgrund. Abschließend erfolgte der Vergleich sekundärer Wundauflagen. Der Vergleich der Quotienten Wundheilung/Wundgrund nicht-okklusiver und okklusiver Wundauflagen zeigte keinen signifikanten Unterschied in der Neovaskularisierung. Die isolierte Betrachtung der Revaskularisierung als einzelner Prozess der Wundheilung kann nicht als generelles Kriterium für die Gesamtbeurteilung dienen. Hier findet die gewählte Methodik ihre Limitation. Zukünftige Anwendungsbereiche des BSGC-Scores sind die Ausweitung auf Vollhautmodelle und andere Verwundungsmodalitäten. Eine automatisierte und durch eine KI-gestützte Befundung ist ebenfalls aufgrund des zugrundeliegenden umfangreichen Datensatzes denkbar. Auch kann eine automatisierte softwaregestützte Quantifizierung der Vaskularisierung als überblickende und zügige Beurteilung der Wundheilung sinnvoll erscheinen.
Infection research largely relies on classical cell culture or mouse models. Despite having delivered invaluable insights into host-pathogen interactions, both have limitations in translating mechanistic principles to human pathologies. Alternatives can be derived from modern Tissue Engineering approaches, allowing the reconstruction of functional tissue models in vitro. Here, we combined a biological extracellular matrix with primary tissue-derived enteroids to establish an in vitro model of the human small intestinal epithelium exhibiting in vivo-like characteristics. Using the foodborne pathogen Salmonella enterica serovar Typhimurium, we demonstrated the applicability of our model to enteric infection research in the human context. Infection assays coupled to spatio-temporal readouts recapitulated the established key steps of epithelial infection by this pathogen in our model. Besides, we detected the upregulation of olfactomedin 4 in infected cells, a hitherto unrecognized aspect of the host response to Salmonella infection. Together, this primary human small intestinal tissue model fills the gap between simplistic cell culture and animal models of infection, and shall prove valuable in uncovering human-specific features of host-pathogen interplay.
Despite promising clinical results in osteochondral defect repair, a recently developed bi-layered collagen/collagen-magnesium-hydroxyapatite scaffold has demonstrated less optimal subchondral bone repair. This study aimed to improve the bone repair potential of this scaffold by adsorbing bone morphogenetic protein 2 (BMP-2) and/or platelet-derived growth factor-BB (PDGF-BB) onto said scaffold. The in vitro release kinetics of BMP-2/PDGF-BB demonstrated that PDGF-BB was burst released from the collagen-only layer, whereas BMP-2 was largely retained in both layers. Cell ingrowth was enhanced by BMP-2/PDFG-BB in a bovine osteochondral defect ex vivo model. In an in vivo semi-orthotopic athymic mouse model, adding BMP-2 or PDGF-BB increased tissue repair after four weeks. After eight weeks, most defects were filled with bone tissue. To further investigate the promising effect of BMP-2, a caprine bilateral stifle osteochondral defect model was used where defects were created in weight-bearing femoral condyle and non-weight-bearing trochlear groove locations. After six months, the adsorption of BMP-2 resulted in significantly less bone repair compared with scaffold-only in the femoral condyle defects and a trend to more bone repair in the trochlear groove. Overall, the adsorption of BMP-2 onto a Col/Col-Mg-HAp scaffold reduced bone formation in weight-bearing osteochondral defects, but not in non-weight-bearing osteochondral defects.
Einleitung: Strukturelle Defekte der gastrointestinalen Hohlorgane stellen ein allgegen-wärtiges Problem im klinischen Alltag dar. Sie entstehen meist auf dem Boden einer ent-zündlichen oder tumorösen Grunderkrankung und können außerdem traumatisch sowie durch medizinische Eingriffe hervorgerufen werden. In der Folge kommt es zur Kontami-nation des umliegenden Gewebes mit Magen- bzw. Darminhalt, wodurch deletäre Folgen wie eine systemische Infektion, also eine Sepsis mit Multiorganversagen drohen können. Vor diesem Hintergrund sind gastrointestinale Defekte immer als potenziell lebensbedroh-lich für den Patienten zu betrachten. Die adäquate und kausale Behandlung erfolgt je nach Ätiologie und Zustand des Patienten durch eine Operation oder eine endoskopische Inter-vention. Hierzu stehen zahlreiche etablierte, operative und interventionelle Therapieme-thoden zur Verfügung. In manchen Fällen stoßen die etablierten Techniken jedoch an ihre Grenzen. Bei Patienten mit schwerwiegenden Komorbiditäten oder im Rahmen neuer me-dizinischer Verfahren sind Innovationen gefragt. Die Grundidee der vorliegenden Arbeit ist die Entwicklung einer biotechnologischen Therapieoption zur Versorgung gastrointesti-naler Hohlorganperforationen.
Methoden: Zur Durchführung einer Machbarkeitsstudie wurden zehn Göttinger Mi-nischweine in zwei Gruppen mit jeweils 5 Tieren aufgeteilt. Den Tieren der Experimental-gruppe wurden Hautbiopsien entnommen und daraus Fibroblasten isoliert, welche vo-rübergehend konserviert wurden. Unter Verwendung von azellularisiertem Schweinedarm erfolgte die Herstellung von Implantaten nach den Prinzipien des Tissue Engineerings. Die Tiere beider Gruppen wurden einer Minilaparotomie und einer ca. 3cm-Inzision der Ma-genvorderwand unterzogen. Die anschließende Versorgung wurde in der Experimental-gruppe durch Implantation der neuartigen Konstrukte erzielt. In der Kontrollgruppe wur-de im Sinne des Goldstandards eine konventionelle Naht durchgeführt. Anschließend wurden die Tiere für vier Wochen beobachtet. Eine bzw. zwei Wochen nach dem pri-mären Eingriff wurde bei allen Tieren beider Gruppen eine Laparoskopie bzw. Gastrosko-pie durchgeführt. Am Ende der klinischen Observationsphase wurden die Versuchstiere getötet und die entsprechenden Magenareale zur histologischen Untersuchung explantiert.
Ergebnisse: Die Herstellung der Implantate konnte auf der Basis standardisierter zellbio-logischer Methoden problemlos etabliert werden. Alle Tiere beider Gruppen überlebten den Primäreingriff sowie das vierwöchige Nachbeobachtungsintervall und zeigten dabei keine klinischen Zeichen möglicher Komplikationen. Die durchgeführten Laparoskopien und Gastroskopien ergaben bei keinem der Tiere Hinweise auf Leckagen oder lokale Infek-tionsprozesse. Die histologische Aufarbeitung zeigte im Bereich des ursprünglichen De-fekts eine bindegewebige Überbrückung sowie ein beginnendes Remodeling der Magen-schleimhaut in beiden Gruppen.
Schlussfolgerungen: Durch die Verknüpfung von Einzelprozessen der Zellkultur und dem Großtier-OP konnte ein neues Verfahren zum Verschluss gastrointestinaler Defekt erfolgreich demonstriert und etabliert werden. Das Projekt konnte reibungslos durchge-führt werden und lieferte Ergebnisse, die dem Goldstandard nicht unterlegen waren. Auf-grund der kleinen Fallzahl und weiterer methodischer Limitationen sind jedoch nur einge-schränkt Schlussfolgerungen möglich, weshalb die Durchführung größerer und gut geplan-ter Studien notwendig ist. Die Erkenntnisse dieser Pilotstudie liefern eine solide Basis für die Planung weiterführender Untersuchungen.
Due to the wide variety of benign and malignant salivary gland tumors, classification and malignant behavior determination based on histomorphological criteria can be difficult and sometimes impossible. Spectroscopical procedures can acquire molecular biological information without destroying the tissue within the measurement processes. Since several tissue preparation procedures exist, our study investigated the impact of these preparations on the chemical composition of healthy and tumorous salivary gland tissue by Fourier-transform infrared (FTIR) microspectroscopy. Sequential tissue cross-sections were prepared from native, formalin-fixed and formalin-fixed paraffin-embedded (FFPE) tissue and analyzed. The FFPE cross-sections were dewaxed and remeasured. By using principal component analysis (PCA) combined with a discriminant analysis (DA), robust models for the distinction of sample preparations were built individually for each parotid tissue type. As a result, the PCA-DA model evaluation showed a high similarity between native and formalin-fixed tissues based on their chemical composition. Thus, formalin-fixed tissues are highly representative of the native samples and facilitate a transfer from scientific laboratory analysis into the clinical routine due to their robust nature. Furthermore, the dewaxing of the cross-sections entails the loss of molecular information. Our study successfully demonstrated how FTIR microspectroscopy can be used as a powerful tool within existing clinical workflows.
Bone morphogenetic proteins (BMPs) are involved in various aspects of cell-cell communication in complex life forms. They act as morphogens, help differentiate different cell types from different progenitor cells in development, and are involved in many instances of intercellular communication, from forming a body axis to healing bone fractures, from sugar metabolism to angiogenesis. If the same protein or protein family carries out many functions, there is a demand to regulate and fine-tune their biological activities, and BMPs are highly regulated to generate cell- and context-dependent outcomes.
Not all such instances can be explained yet. Growth/differentiation factor (GDF)5 (or BMP14) synergizes with BMP2 on chondrogenic ATDC5 cells, but antagonizes BMP2 on myoblastic C2C12 cells. Known regulators of BMP2/GDF5 signal transduction failed to explain this context-dependent difference, so a microarray was performed to identify new, cell-specific regulatory components. One identified candidate, the fibroblast growth factor receptor (FGFR)2, was analyzed as a potential new co-receptor to BMP ligands such as GDF5: It was shown that FGFR2 directly binds BMP2, GDF5, and other BMP ligands in vitro, and FGFR2 was able to positively influence BMP2/GDF5-mediated signaling outcome in cell-based assays. This effect was independent of FGFR2s kinase activity, and independent of the downstream mediators SMAD1/5/8, p42/p44, Akt, and p38. The elevated colocalization of BMP receptor type IA and FGFR2 in the presence of BMP2 or GDF5 suggests a signaling complex containing both receptors, akin to other known co-receptors of BMP ligands such as repulsive guidance molecules.
This unexpected direct interaction between FGF receptor and BMP ligands potentially opens a new category of BMP signal transduction regulation, as FGFR2 is the second receptor tyrosine kinase to be identified as BMP co-receptor, and more may follow. The integration of cell surface interactions between members of the FGF and BMP family especially may widen the knowledge of such cellular communication mechanisms which involve both growth factor families, including morphogen gradients and osteogenesis, and may in consequence help to improve treatment options in osteochodnral diseases.
Für die Verwendung von zellbasierten Therapeutika ist vor allem die korrekt Identifikation
sowohl vom Ausgangsmaterial wie auch dem produziertem Material von
zentraler Wichtigkeit. In dieser Arbeit wurde eine Methodik entwickelt, welche eine
nicht-invasive Klassifizierung von Zellen und zellulärer Entwicklung aufgrund ihrer
zweidimensionalen Magnetresonanz-Korrelationsspektren ermöglichte.
Hierzu wurde ein mobiler MR-Scanner mit einer Feldstärke von 0.5T und einem Isozentrum
von 1 cm3 verwendet. Aufgrund der kompakten und leichten Bauweise war
es möglich, das System in normalen Zellkulturlaboren zu verwenden. Von den Proben
wurde ein zweidimensionales T1/T2 -Korrelationsspektrum aufgenommen, anhand
dessen die Zellen klassifiziert werden sollten. Mithilfe von Agarose-Dotagraf® -Zell-
Phantomen konnte die Stabilität und Reproduzierbarkeit des Messsystems und der
verwendeten Sequenz validiert werden.
Aufgrund der unter Umständen recht langen Messzeiten der MR-Technologie war
auch die Handhabung und Kultur der Zellproben während des Messprozesses von
großer Bedeutung. Um hierfür den Durchsatz an Proben zu erhöhen, wurde eine kostengünstige
und ebenfalls mobile Robotikanlage entwickelt. Diese basierte auf dem
kommerziell erhältlichen Roboterarm Braccio, welcher durch einen Arduino Mega
Mikrocontroller gesteuert wurde. Mit bis zu 24 Proben pro Tag konnte durch die
Automatisierung der Durchsatz an Proben um den Faktor 3 – 4 gesteigert werden.
Durch den entwickelten Prozess war es möglich, eine umfangreiche Datenbank –
bestehend aus 362 unabhängigen Messungen (biologische Replikate) – aufzubauen.
Die Datenbank enthielt Messungen von zehn unterschiedlichen Zelllinien. Zusätzlich
wurden T1/T2 -Korrelationsspektren von mesenchymalen Stromazellen (MSCs)
vor und nach deren Differenzierung zu Adipocyten aufgenommen, um ihre zelluläre
Entwicklung nicht-invasiv charakterisieren zu können.
Die aufgenommenen Daten wurden mithilfe einer geeigneten Support Vector Machine
wie auch angepassten künstlichen neuronalen Netzwerken klassifiziert. Mithilfe
dieser Methoden konnten die Zelllinien und MSCs anhand ihrer aufgenommenen
Korrelationsspektren mit einer Genauigkeit von bis zu 98% klassifiziert werden.
Diese hohe Treffsicherheit legte den Schluss nahe, dass die Kombination aus nichtinvasiver,
zweidimensionaler T1/T2 -MR-Relaxometrie und der Verwendung von geeigneten
Methoden des machine learning und der künstlichen Intelligenz eine effiziente
Methodik für die nicht-invasive Klassifizierung von Zellen sowie zellulärer
Entwicklung darstellt.
Efficient redirection of NK cells by genetic modification with chemokine receptors CCR4 and CCR2B
(2023)
Natural killer (NK) cells are a subset of lymphocytes that offer great potential for cancer immunotherapy due to their natural anti-tumor activity and the possibility to safely transplant cells from healthy donors to patients in a clinical setting. However, the efficacy of cell-based immunotherapies using both T and NK cells is often limited by a poor infiltration of immune cells into solid tumors. Importantly, regulatory immune cell subsets are frequently recruited to tumor sites. In this study, we overexpressed two chemokine receptors, CCR4 and CCR2B, that are naturally found on T regulatory cells and tumor-resident monocytes, respectively, on NK cells. Using the NK cell line NK-92 as well as primary NK cells from peripheral blood, we show that genetically engineered NK cells can be efficiently redirected using chemokine receptors from different immune cell lineages and migrate towards chemokines such as CCL22 or CCL2, without impairing the natural effector functions. This approach has the potential to enhance the therapeutic effect of immunotherapies in solid tumors by directing genetically engineered donor NK cells to tumor sites. As a future therapeutic option, the natural anti-tumor activity of NK cells at the tumor sites can be increased by co-expression of chemokine receptors with chimeric antigen receptors (CAR) or T cell receptors (TCR) on NK cells can be performed in the future.
Das maligne Melanom nimmt als Tumorerkrankung mit hoher Metastasierungsrate und steigenden Inzidenzraten bei höchster Mortalität aller Hauttumoren eine zunehmende Bedeutung in der modernen Onkologie ein. Frühzeitige Diagnosemöglichkeiten und moderne Behandlungen konnten das Überleben der Patienten bereits erheblich verbessern. Jedoch besteht nach wie vor Bedarf an geeigneten Modellen, um die Melanomprogression vollständig zu verstehen und neue wirksame Therapien zu entwickeln. Hierfür werden häufig Tiermodelle verwendet, diese spiegeln jedoch nicht die menschliche Mikroumgebung wider. Zweidimensionalen Zellkulturen fehlen dagegen entscheidende Elemente der Tumormikroumgebung. Daher wurde in dieser Arbeit ein dreidimensionales epidermales Tumormodell des malignen Melanoms, welches aus primären humanen Keratinozyten und verschiedenen Melanomzelllinien besteht, entwickelt. Die eingesetzten Melanomzelllinien variieren in ihren Treibermutationen, wodurch das Modell in der Lage ist, Wirkstoffe zu untersuchen, die spezifisch auf diese Mutationen wirken. Mit Techniken des Tissue Engineerings konnte ein dreidimensionales Hautmodell aufgebaut werden, das alle charakteristischen Schichten der Epidermis aufweist und im Bereich des stratum basale Melanomcluster ausbildet. Diese reichen je nach Größe und Ausdehnung bis in suprabasale Epidermisschichten hinein. Die Tumor-Histopathologie, der Tumorstoffwechsel sowie tumorassoziierte Proteinsekretionen ließen sich im in vitro Modell nachweisen. Darüber hinaus konnte ein Protokoll entwickelt werden, mit dem einzelne Zellen aus den Modellen reisoliert werden können. Dies ermöglichte es, den Proliferationszustand innerhalb des jeweiligen Modells zu charakterisieren und die Wirkung von Antitumortherapien gezielt zu bewerten. Die Anwendbarkeit als Testsystem im Bereich der Tumortherapeutika wurde mit dem in der Klinik häufig verwendeten v-raf-Maus-Sarkom-Virus-Onkogen-Homolog B (BRAF)-Inhibitor Vemurafenib demonstriert. Der selektive BRAF-Inhibitor reduzierte erfolgreich das Tumorwachstum in den Modellen mit BRAF-mutierten Melanomzellen, was durch eine Verringerung der metabolischen Aktivität, der proliferierenden Zellen und des Glukoseverbrauchs gezeigt wurde. Für die Implementierung des Modells in die präklinische Therapieentwicklung wurde B-B-Dimethylacrylshikonin, ein vielversprechender Wirkstoffkandidat, welcher einen Zellzyklusarrest mit anschließender Apoptose bewirkt, im Modell getestet.
Bei einer Anwendung der Modelle im Bereich der Testung topischer Behandlungen ist eine Barrierefunktion der Modelle notwendig, die der in vivo Situation nahe kommt. Die Barriereeigenschaften der Hautäquivalente wurden durch die Melanomzellen nachweislich nicht beeinflusst, sind aber im Vergleich zur in vivo Situation noch unzureichend. Eine signifikante Steigerung der Hautbarriere konnte durch die Bereitstellung von Lipiden und die Anregung hauteigener Regenerationsprozesse erreicht werden. Über den Nachweis des transepidermalen Wasserverlusts konnte eine Messmethode zur nicht-invasiven Bestimmung der Hautbarriere etabliert und über den Vergleich zur Impedanzspektroskopie validiert werden. Hierbei gelang es, erstmals die Korrelation der Hautmodelle zur in vivo Situation über ein solches Verfahren zu zeigen. Das entwickelte epidermale Modell konnte durch die Integration eines dermalen Anteils und einer Endothelzellschicht noch weiter an die komplexe Struktur und Physiologie der Haut angepasst werden um Untersuchungen, die mit der Metastierung und Invasion zusammenhängen, zu ermöglichen. Die artifizielle Dermis basiert auf einem Kollagen-Hydrogel mit primären Fibroblasten. Eine dezellularisierte Schweinedarmmatrix ließ sich zur Erweiterung des Modells um eine Endothelzellschicht nutzen. Dabei wanderten die primären Fibroblasten apikal in die natürliche Schweindarmmatrix ein, während die Endothelzellen basolateral eine geschlossene Schicht bildeten.
Die in dieser Arbeit entwickelten Gewebemodelle sind in der Lage, die Vorhersagekraft der in vitro Modelle und die in vitro - in vivo Korrelation zu verbessern. Durch die Kombination des Melanommodells mit einer darauf abgestimmten Analytik wurde ein neuartiges Werkzeug für die präklinische Forschung zur Testung von pharmazeutischen Wirkstoffen geschaffen.
Significant advancements in the field of preclinical in vitro blood-brain barrier (BBB) models have been achieved in recent years, by developing monolayer-based culture systems towards complex multi-cellular assays. The coupling of those models with other relevant organoid systems to integrate the investigation of blood-brain barrier permeation in the larger picture of drug distribution and metabolization is still missing. Here, we report for the first time the combination of a human induced pluripotent stem cell (hiPSC)-derived blood-brain barrier model with a cortical brain and a liver spheroid model from the same donor in a closed microfluidic system (MPS). The two model compounds atenolol and propranolol were used to measure permeation at the blood–brain barrier and to assess metabolization. Both substances showed an in vivo-like permeation behavior and were metabolized in vitro. Therefore, the novel multi-organ system enabled not only the measurement of parent compound concentrations but also of metabolite distribution at the blood-brain barrier.
The host defense derived peptide was assessed in different model systems with increasing complexity employing the highly aggressive NRAS mutated melanoma metastases cell line MUG-Mel2. Amongst others, fluorescence microscopy and spectroscopy, as well as cell death studies were applied for liposomal, 2D and 3D in vitro models including tumor spheroids without or within skin models and in vivo mouse xenografts. Summarized, MUG-Mel2 cells were shown to significantly expose the negatively charged lipid phosphatidylserine on their plasma membranes, showing they are successfully targeted by RDP22. The peptide was able to induce cell death in MUG-Mel2 2D and 3D cultures, where it was able to kill tumor cells even inside the core of tumor spheroids or inside a melanoma organotypic model. In vitro studies indicated cell death by apoptosis upon peptide treatment with an LC\(_{50}\) of 8.5 µM and seven-fold specificity for the melanoma cell line MUG-Mel2 over normal dermal fibroblasts. In vivo studies in mice xenografts revealed effective tumor regression upon intratumoral peptide injection, indicated by the strong clearance of pigmented tumor cells and tremendous reduction in tumor size and proliferation, which was determined histologically. The peptide RDP22 has clearly shown high potential against the melanoma cell line MUG-Mel2 in vitro and in vivo.
A fine balance of regulatory (T\(_{reg}\)) and conventional CD4\(^+\) T cells (T\(_{conv}\)) is required to prevent harmful immune responses, while at the same time ensuring the development of protective immunity against pathogens. As for many cellular processes, sphingolipid metabolism also crucially modulates the T\(_{reg}\)/T\(_{conv}\) balance. However, our understanding of how sphingolipid metabolism is involved in T cell biology is still evolving and a better characterization of the tools at hand is required to advance the field. Therefore, we established a reductionist liposomal membrane model system to imitate the plasma membrane of mouse T\(_{reg}\) and T\(_{conv}\) with regards to their ceramide content. We found that the capacity of membranes to incorporate externally added azide-functionalized ceramide positively correlated with the ceramide content of the liposomes. Moreover, we studied the impact of the different liposomal preparations on primary mouse splenocytes in vitro. The addition of liposomes to resting, but not activated, splenocytes maintained viability with liposomes containing high amounts of C\(_{16}\)-ceramide being most efficient. Our data thus suggest that differences in ceramide post-incorporation into T\(_{reg}\) and T\(_{conv}\) reflect differences in the ceramide content of cellular membranes.
Respiratory infections are a significant health concern worldwide, and the airway epithelium plays a crucial role in regulating airway function and modulating inflammatory processes. However, most studies on respiratory infections have used cell lines or animal models, which may not accurately reflect native physiological conditions, especially regarding human pathogens. We generated human nasal mucosa (hNM) and tracheobronchial mucosa (hTM) models to address this issue using primary human airway epithelial cells and fibroblasts. We characterised these human airway tissue models (hAM) using high speed video microscopy, single cell RNA sequencing, immunofluorescence staining,
and ultrastructural analyses that revealed their complexity and cellular heterogeneity. We demonstrated that Bordetella pertussis virulence factor adenylate cyclase toxin (CyaA) elevated the intracellular production of cyclic adenosine monophosphate (cAMP) and secretion of interleukin (IL) 6, IL 8, and human beta defensin 2 (HBD2). In addition, we compared the responses of the tissue models from two different anatomical sites (the upper and lower respiratory mucosa) and are the first to report such differential susceptibility towards CyaA using 3D primary airway cell derivedmodels. The effect of toxin treatment on the epithelial barrier integrity of the tissue models was assessed by measuring the flux of fluorescein isothiocyanate (FITC)-conjugated dextran across the models. Though we observed a cell type specific response with respect to intracellular cAMP production and IL 6, IL 8, and HBD2 secretion in the models treated with CyaA on the apical side, the epithelial membrane barrier integrity was not compromised. In addition to toxin studies, using these characterised models, we established viral infection studies for Influenza A (IAV), Respiratory Syncytial Virus subtype B (RSV), and severe acute respiratory syndrome coronavirus 2. We visualised the morphological consequences of the viral infection using ultrastructural analysis
and immunofluorescence. We verified the effective infection in hAM by measuring the viral RNA using RTqPCR and detected elevated cytokine levels in response to infection using biochemical assays. In contrast to cell lines, studies on viral infection using hAM demonstrated that infected areas were localized to specific regions. This led to the formation of infection hotspots, which were more likely to occur when models derived from different donors were infected separately with all three viruses. IAV infected tissue models replicate the clinical findings of H1N1 infection, such as mucus
hypersecretion, cytokine release, and infection-associated epithelial cell damage.Finally, we paved the steps towards understanding the impact of IAV infection on disease models. We generated hTM from biopsies obtained from chronic obstructive pulmonary disease (COPD) patients. As a model to study the impact of COPD on respiratory infections, considering the increase in COPD cases in the past decade and the continued predicted increase in the future. We established the IAV infection
protocol to capture the early infection signatures in non-COPD and COPD conditions using scRNA-seq. We investigated the infection kinetics of IAV (H1N1-clinical isolate) in hTM and found that viruses were actively released approximately 24 hours post infection. The scRNA-seq data from the hTM derived from non-COPD and COPD patients, revealed lower levels of SCGB1A1 (club cell marker) gene expression in the COPD-control group compared to the non-COPD control group, consistent with previous clinical studies. Furthermore, we observed that IAV infection elevated SCGB1A1 gene expression especially in secretory cells of both the COPD and non COPD groups. This may imply the role of club cells as early responders during IAV infection providing epithelial repair, regeneration, and resistance to spread of infection. This is the first study to address the molecular diversity in COPD and non-COPD disease models infected with IAV investigating the early response (6 h) of specific cell types in the human lower airways towards infection using scRNA-seq. These findings
highlight the potential interplay between COPD, IAV infection, and altered vulnerability to other viral infections and respiratory illnesses making the hAM applicable for addressing more specific research questions and validating potential targets, such as SCGB1A1 targeted therapy for chronic lung diseases. Our findings demonstrate the potential of the hNM and hTM for investigating respiratory infections, innate immune responses, and trained immunity in non-immune cells. Our experiments show that hAM may represent a more accurate representation of the native physiological condition and improve our understanding of the disease mechanisms. Furthermore, these models promote non-animal research as they replicate clinical findings. We can further increase their complexity by incorporating dynamic flow systems and immune cells catered to the research question.
Onchocerciasis, the world's second-leading infectious cause of blindness in humans
–prevalent in Sub-Saharan Africa – is caused by Onchocerca volvulus (O. volvulus), an
obligatory human parasitic filarial worm. Commonly known as river blindness,
onchocerciasis is being targeted for elimination through ivermectin-based mass
drug administration programs. However, ivermectin does not kill adult parasites,
which can live and reproduce for more than 15 years within the human host. These
impediments heighten the need for a deeper understanding of parasite biology and
parasite-human host interactions, coupled with research into the development of
new tools – macrofilaricidal drugs, diagnostics, and vaccines. Humans are the only
definitive host for O. volvulus. Hence, no small-animal models exist for propagating
the full life cycle of O. volvulus, so the adult parasites must be obtained surgically
from subcutaneous nodules. A two-dimensional (2D) culture system allows that
O. volvulus larvae develop from the vector-derived infective stage larvae (L3) in vitro
to the early pre-adult L5 stages. As problematic, the in vitro development of
O. volvulus to adult worms has so far proved infeasible. We hypothesized that an
increased biological complexity of a three-dimensional (3D) culture system will
support the development of O. volvulus larvae in vitro. Thus, we aimed to translate
crucial factors of the in vivo environment of the developing worms into a culture
system based on human skin. The proposed tissue model should contain 1. skinspecific
extracellular matrix, 2. skin-specific cells, and 3. enable a direct contact of
larvae and tissue components. For the achievement, a novel adipose tissue model
was developed and integrated to a multilayered skin tissue comprised of epidermis,
dermis and subcutis. Challenges of the direct culture within a 3D tissue model
hindered the application of the three-layered skin tissue. However, the indirect coculture
of larvae and skin models supported the growth of fourth stage (L4) larvae in
vitro. The direct culture of L4 and adipose tissue strongly improved the larvae
survival. Furthermore, the results revealed important cues that might represent the
initial encapsulation of the developing worm within nodular tissue. These results
demonstrate that tissue engineered 3D tissues represent an appropriate in vitro
environment for the maintenance and examination of O. volvulus larvae.
Compared to cell therapy, where cells are injected into a defect region, the treatment of heart infarction with cells seeded in a vascularized scaffold bears advantages, such as an immediate nutrient supply or a controllable and persistent localization of cells. For this purpose, decellularized native tissues are a preferable choice as they provide an in vivo-like microenvironment. However, the quality of such scaffolds strongly depends on the decellularization process. Therefore, two protocols based on sodium dodecyl sulfate or sodium deoxycholate were tailored and optimized for the decellularization of a porcine heart. The obtained scaffolds were tested for their applicability to generate vascularized cardiac patches. Decellularization with sodium dodecyl sulfate was found to be more suitable and resulted in scaffolds with a low amount of DNA, a highly preserved extracellular matrix composition, and structure shown by GAG quantification and immunohistochemistry. After seeding human endothelial cells into the vasculature, a coagulation assay demonstrated the functionality of the endothelial cells to minimize the clotting of blood. Human-induced pluripotent-stem-cell-derived cardiomyocytes in co-culture with fibroblasts and mesenchymal stem cells transferred the scaffold into a vascularized cardiac patch spontaneously contracting with a frequency of 25.61 ± 5.99 beats/min for over 16 weeks. The customized decellularization protocol based on sodium dodecyl sulfate renders a step towards a preclinical evaluation of the scaffolds.
The development of novel fibrous biomaterials and further processing of medical devices is still challenging. For instance, titanium(IV) oxide is a well-established biocompatible material, and the synthesis of TiO\(_x\) particles and coatings via the sol-gel process has frequently been published. However, synthesis protocols of sol-gel-derived TiO\(_x\) fibers are hardly known. In this publication, the authors present a synthesis and fabrication of purely sol-gel-derived TiO\(_x\) fiber fleeces starting from the liquid sol-gel precursor titanium ethylate (TEOT). Here, the α-hydroxy-carboxylic acid lactic acid (LA) was used as a chelating ligand to reduce the reactivity towards hydrolysis of TEOT enabling a spinnable sol. The resulting fibers were processed into a non-woven fleece, characterized with FTIR, \(^{13}\)C-MAS-NMR, XRD, and screened with regard to their stability in physiological solution. They revealed an unexpected dependency between the LA content and the dissolution behavior. Finally, in vitro cell culture experiments proved their potential suitability as an open-mesh structured scaffold material, even for challenging applications such as therapeutic medicinal products (ATMPs).
Burns affect millions every year and a model to mimic the pathophysiology of such injuries in detail is required to better understand regeneration. The current gold standard for studying burn wounds are animal models, which are under criticism due to ethical considerations and a limited predictiveness. Here, we present a three-dimensional burn model, based on an open-source model, to monitor wound healing on the epidermal level. Skin equivalents were burned, using a preheated metal cylinder. The healing process was monitored regarding histomorphology, metabolic changes, inflammatory response and reepithelialization for 14 days. During this time, the wound size decreased from 25% to 5% of the model area and the inflammatory response (IL-1β, IL-6 and IL-8) showed a comparable course to wounding and healing in vivo. Additionally, the topical application of 5% dexpanthenol enhanced tissue morphology and the number of proliferative keratinocytes in the newly formed epidermis, but did not influence the overall reepithelialization rate. In summary, the model showed a comparable healing process to in vivo, and thus, offers the opportunity to better understand the physiology of thermal burn wound healing on the keratinocyte level.
Diabetes mellitus is an incurable, metabolic disease, which is associated with severe long-term complications. The in vitro generation of pancreatic β-cells from human induced pluripotent stem cells (hiPSCs) represent a promising strategy for a curative therapy of diabetes mellitus. However, current differentiation strategies largely fail to produce functional β-cells in vitro and require an additional in vivo transplantation to achieve terminal maturation. Previous studies demonstrated a beneficial effect of the extracellular matrix (ECM) on the survival and sustained function of adult, isolated islets of Langerhans. This raises the question whether organ-specific cell-ECM interactions might represent the missing link driving the final stage of β-cell development. In order to address this issue, this study investigated the impact of the pancreas ECM on in vitro β-cell differentiation and its use for the establishment of a pancreatic endocrine organ model.
To this purpose, a pancreas-specific ECM scaffolds (PanMa) was derived from porcine pancreata using whole organ decellularization with Sodium Deoxycholate. In a first step, the generated PanMa was thoroughly characterized using (immuno-) histological stainings, scanning electron microscopy and DNA quantification as well as perfusion and recellularization experiments with endothelial cells. Based on these data, a scoring system (PancScore) for a standardized PanMa generation was developed. Next, the generated PanMa was tested for the presence of tissue-specific ECM features. Therefore, the biophysical and physico-structural characteristics, such as rigidity, porosity and hygroscopy were analyzed using rheological measurements, particle diffusion analyses as well as a water evaporation assay and compared to the properties of ECM scaffolds derived from porcine small intestine (SISser) and lung (LungMa) to examine organ-specific scaffold cues. Following the thorough scaffold characterization, the impact of the PanMa on pluripotency and early development of hiPSC was studied. To this purpose, gene and protein expression of hiPSCs during maintenance culture and spontaneous differentiation on the PanMa were assessed. In a next step, the impact of the PanMa on the pancreatic endocrine differentiation of hiPSCs was tested. Therefore, the PanMa was used as a liquid media supplement or as a solid scaffold during the directed differentiation of hiPSC towards either pancreatic hormone-expressing cells (Rezania et al. 2012; Rezania et al. 2014) or maturing β-cells (Rezania et al. 2014). The impact of the PanMa on the generated cells was examined by gene expression analysis, immunohistochemical staining of important stage markers, as well as glucose stimulated insulin secretion assays. In a last part of this study, the potential of the PanMa for the prolonged culture of hiPSC derived endocrine cells for the establishment of an in vitro organ model of the endocrine pancreas was examined. Therefore, a PanMa-derived hydrogel was generated and used for the encapsulation and culture of hiPSC-derived hormone-expressing cells (HECs). The influence of the PanMa-hydrogel culture was analyzed on gene, protein and functional level by gene expression analysis, immunohistochemical stainings and glucose stimulated insulin secretion.
Whole organ decellularization resulted in the generation of an acellular PanMa scaffold, with low amounts of residual DNA and a preserved ECM micro- and ultrastructure, including important ECM components, such as collagen I, III and IV. Furthermore, the PanMa maintained an intact vessel system and was verified as cytocompatible as demonstrated by the successful recellularization of the arterial system with human endothelial cells. In comparison to SISser and LungMa, the PanMa was characterized as a relative soft, hygroscopic scaffold with a collagen-fiber based structure. Furthermore, the findings indicate that the ECM-specific properties have a relevant effect on the stem cell character and early multi-lineage decisions of hiPSCs. In this regard, maintenance of hiPSCs on the PanMa resulted in a slightly changed expression of pluripotency genes (OCT4, SOX2 and NANOG) and a weak immunohistochemical signal for NANOG protein, indicating a PanMa-dependent impact on hiPSC pluripotency. Strikingly, this presumption was corroborated by the finding that culture on the PanMa promoted an endodermal development of hiPSCs during spontaneous differentiation. In line with that, pancreatic differentiation of hiPSC on both the PanMa and SISser resulted in a significant decrease of glucagon and somatostatin gene expression as well as an unaltered insulin expression, suggesting an ECM-driven suppression of the development of non β-cell endocrine cells. However, this change did not result in an improved glucose stimulated insulin secretion of the generated HECs. Moreover, use of the PanMa as a hydrogel allowed prolonged culture of these cells in a defined culture system. HECs were viable after 21 days of culture, however already showed an altered islet morphology as well as a slightly decreased glucose stimulated insulin secretion.
Altogether, this study demonstrates a relevant biological effect of tissue specific ECM cues on the in vitro differentiation of hiPSCs. More specifically, the data indicate an involvement of the ECM in the endocrine commitment of hiPSC-derived pancreatic cells during directed differentiation highlighting the ECM as an important regulator of pancreatic development. Collectively, these findings emphasize the relevance of the ECM for the fabrication of functional hiPSC-derived cell types and suggest a much stronger consideration of organ specific ECM cues for tissue engineering approaches as well as clinical translation in regenerative medicine.
A balanced and moist wound environment and surface increases the effect of various growth factors, cytokines, and chemokines, stimulating cell growth and wound healing. Considering this fact, we tested in vitro and in vivo water evaporation rates from the cellulose dressing epicite\(^{hydro}\) when combined with different secondary dressings as well as the resulting wound healing efficacy in a porcine donor site model. The aim of this study was to evaluate how the different rates of water evaporation affected wound healing efficacy. To this end, epicite\(^{hydro}\) primary dressing, in combination with different secondary dressing materials (cotton gauze, JELONET\(^◊\), AQUACEL\(^®\) Extra\(^™\), and OPSITE\(^◊\) Flexifix), was placed on 3 × 3 cm-sized dermatome wounds with a depth of 1.2 mm on the flanks of domestic pigs. The healing process was analyzed histologically and quantified by morphometry. High water evaporation rates by using the correct secondary dressing, such as cotton gauze, favored a better re-epithelialization in comparison with the low water evaporation resulting from an occlusive secondary dressing, which favored the formation of a new and intact dermal tissue that nearly fully replaced all the dermis that was removed during wounding. This newly available evidence may be of great benefit to clinical wound management.
The signal modelling framework JimenaE simulates dynamically Boolean networks. In contrast to SQUAD, there is systematic and not just heuristic calculation of all system states. These specific features are not present in CellNetAnalyzer and BoolNet. JimenaE is an expert extension of Jimena, with new optimized code, network conversion into different formats, rapid convergence both for system state calculation as well as for all three network centralities. It allows higher accuracy in determining network states and allows to dissect networks and identification of network control type and amount for each protein with high accuracy. Biological examples demonstrate this: (i) High plasticity of mesenchymal stromal cells for differentiation into chondrocytes, osteoblasts and adipocytes and differentiation-specific network control focusses on wnt-, TGF-beta and PPAR-gamma signaling. JimenaE allows to study individual proteins, removal or adding interactions (or autocrine loops) and accurately quantifies effects as well as number of system states. (ii) Dynamical modelling of cell–cell interactions of plant Arapidopsis thaliana against Pseudomonas syringae DC3000: We analyze for the first time the pathogen perspective and its interaction with the host. We next provide a detailed analysis on how plant hormonal regulation stimulates specific proteins and who and which protein has which type and amount of network control including a detailed heatmap of the A.thaliana response distinguishing between two states of the immune response. (iii) In an immune response network of dendritic cells confronted with Aspergillus fumigatus, JimenaE calculates now accurately the specific values for centralities and protein-specific network control including chemokine and pattern recognition receptors.
Background
Anthocyanin-containing plant extracts and carotenoids, such as astaxanthin, have been well-known for their antiviral and anti-inflammatory activity, respectively. We hypothesised that a mixture of Ribes nigrum L. (Grossulariaceae) (common name black currant (BC)) and Vaccinium myrtillus L. (Ericaceae) (common name bilberry (BL)) extracts (BC/BL) with standardised anthocyanin content as well as single plant extracts interfered with the replication of Measles virus and Herpesviruses in vitro.
Methods
We treated cell cultures with BC/BL or defined single plant extracts, purified anthocyanins and astaxanthin in different concentrations and subsequently infected the cultures with the Measles virus (wild-type or vaccine strain Edmonston), Herpesvirus 1 or 8, or murine Cytomegalovirus. Then, we analysed the number of infected cells and viral infectivity and compared the data to non-treated controls.
Results
The BC/BL extract inhibited wild-type Measles virus replication, syncytia formation and cell-to-cell spread. This suppression was dependent on the wild-type virus-receptor-interaction since the Measles vaccine strain was unaffected by BC/BL treatment. Furthermore, the evidence was provided that the delphinidin-3-rutinoside chloride, a component of BC/BL, and purified astaxanthin, were effective anti-Measles virus compounds. Human Herpesvirus 1 and murine Cytomegalovirus replication was inhibited by BC/BL, single bilberry or black currant extracts, and the BC/BL component delphinidin-3-glucoside chloride. Additionally, we observed that BC/BL seemed to act synergistically with aciclovir. Moreover, BC/BL, the single bilberry and black currant extracts, and the BC/BL components delphinidin-3-glucoside chloride, cyanidin-3-glucoside, delphinidin-3-rutinoside chloride, and petunidin-3-galactoside inhibited human Herpesvirus 8 replication.
Conclusions
Our data indicate that Measles viruses and Herpesviruses are differentially susceptible to a specific BC/BL mixture, single plant extracts, purified anthocyanins and astaxanthin. These compounds might be used in the prevention of viral diseases and in addition to direct-acting antivirals, such as aciclovir.
Acetylsalicylic acid and salicylic acid inhibit SARS-CoV-2 replication in precision-cut lung slices
(2022)
Aspirin, with its active compound acetylsalicylic acid (ASA), shows antiviral activity against rhino- and influenza viruses at high concentrations. We sought to investigate whether ASA and its metabolite salicylic acid (SA) inhibit SARS-CoV-2 since it might use similar pathways to influenza viruses. The compound-treated cells were infected with SARS-CoV-2. Viral replication was analysed by RTqPCR. The compounds suppressed SARS-CoV-2 replication in cell culture cells and a patient-near replication system using human precision-cut lung slices by two orders of magnitude. While the compounds did not interfere with viral entry, it led to lower viral RNA expression after 24 h, indicating that post-entry pathways were inhibited by the compounds.
Oxidative stress and inflammation play a pivotal role in the development of cardiovascular diseases, an ever-growing worldwide problem. As a non-pharmacological approach, diet, especially a flavonoid-rich diet, showed promising results in the reduction of cardiovascular diseases and alleviation of their symptoms. In this study, in vitro systems based on human microvascular endothelial cells (hmvEC) and human umbilical cord endothelial cells (HUVEC) were established to determine the effect of Healthberry 865\(^®\) (HB) and ten of its relating single anthocyanins on oxidative stress. Furthermore, five metabolites were used in order to examine the effect of anthocyanin's most common breakdown molecules. The results showed an effect of HB in both models after 24 h, as well as most of its single anthocyanins. Cyanidin-rutinoside, peonidin-galactoside, and petunidin-glucoside had a model-specific effect. For the metabolites, phloroglucinaldeyhde (PGA) showed an effect in both models, while vanillic acid (VA) only had an effect in HUVEC. When combined, a combination of several anthocyanins did not have a cumulative effect, except for combining glucosides in hmvEC. The combination of PGA and VA even revealed an inhibitive behavior. Overall, the study demonstrates the antioxidative effect of HB and several of its single anthocyanins and metabolites, which are partially model specific, and coincides with animal studies.
Ovarian cancer is the second most common gynecological malignancy in women. More than 70% of the cases are diagnosed at the advanced stage, presenting as primary peritoneal metastasis, which results in a poor 5-year survival rate of around 40%. Mechanisms of peritoneal metastasis, including adhesion, migration, and invasion, are still not completely understood and therapeutic options are extremely limited. Therefore, there is a strong requirement for a 3D model mimicking the in vivo situation. In this study, we describe the establishment of a 3D tissue model of the human peritoneum based on decellularized porcine small intestinal submucosa (SIS) scaffold. The SIS scaffold was populated with human dermal fibroblasts, with LP-9 cells on the apical side representing the peritoneal mesothelium, while HUVEC cells on the basal side of the scaffold served to mimic the endothelial cell layer. Functional analyses of the transepithelial electrical resistance (TEER) and the FITC-dextran assay indicated the high barrier integrity of our model. The histological, immunohistochemical, and ultrastructural analyses showed the main characteristics of the site of adhesion. Initial experiments using the SKOV-3 cell line as representative for ovarian carcinoma demonstrated the usefulness of our models for studying tumor cell adhesion, as well as the effect of tumor cells on endothelial cell-to-cell contacts. Taken together, our data show that the novel peritoneal 3D tissue model is a promising tool for studying the peritoneal dissemination of ovarian cancer.
For the treatment of large bone defects, the commonly used technique of autologous bone grafting presents several drawbacks and limitations. With the discovery of the bone-inducing capabilities of bone morphogenetic protein 2 (BMP2), several delivery techniques were developed and translated to clinical applications. Implantation of scaffolds containing adsorbed BMP2 showed promising results. However, off-label use of this protein-scaffold combination caused severe complications due to an uncontrolled release of the growth factor, which has to be applied in supraphysiological doses in order to induce bone formation. Here, we propose an alternative strategy that focuses on the covalent immobilization of an engineered BMP2 variant to biocompatible scaffolds. The new BMP2 variant harbors an artificial amino acid with a specific functional group, allowing a site-directed covalent scaffold functionalization. The introduced artificial amino acid does not alter BMP2′s bioactivity in vitro. When applied in vivo, the covalently coupled BMP2 variant induces the formation of bone tissue characterized by a structurally different morphology compared to that induced by the same scaffold containing ab-/adsorbed wild-type BMP2. Our results clearly show that this innovative technique comprises translational potential for the development of novel osteoinductive materials, improving safety for patients and reducing costs.
Biomechanische Eigenschaften eines biomaterialbasierten Kreuzbandkonstruktes in-vivo und in-vitro
(2023)
Kreuzbandrupturen stellen nach wie vor eine Herausforderung in der klinischen Praxis hinsichtlich kurz- und langfristiger unerwünschter Nebenwirkungen dar (z.B. Reruptur und Arthrosebildung).
In der vorliegenden Arbeit wird der entwickelte Ansatz eines Kollagen-I-basierten künstlichen Kreuzbandkonstruktes hinsichtlich der Reißfestigkeit, Lagerung, Verstärkungsmöglichkeit mittels Fiber-tape und langfristigen Arthroseentstehung untersucht mittels in-vitro und in-vivo Untersuchungen unter zur Hilfe nahme des Minipig Tiermodels.
Die Ergebnisse zeigen keinen Einfluss der Lagerungstemperatur sowie des Lagerungszeitraums auf die Reißfestigkeit des Konstruktes, sowie eine mögliche initiale Verstärkung mittels Fibertape im Minipig. Darüber hinaus wurde mikroskopisch wie makroskopische Arthroseentstehung nachgewiesen. Das Ausmaß der Arthroseentstehung ist diesbezüglich mit einer Abweichung der Konstruktimplantation von der ursprünglichen Kreuzbandinsertion mittels MRT bestätigt worden.
Die Vordere Kreuzband (VKB)-Ruptur ist eine häufige Verletzung, welche eine hohe individuelle und sozioökonomische Belastung verursacht. Eine etablierte Therapie ist die VKB-Plastik, problematisch sind jedoch die hohen Rerupturraten nach operativer Versorgung. In der Annahme, dass Mesenchymale Stammzellen (MSC) eine bedeutende Rolle für die Heilung spielen, sollte in der vorliegenden Arbeit untersucht werden, ob ein Zusammenhang zwischen Zahl und Qualität der aus dem VKB isolierten MSC sowie der Latenz zwischen Ruptur und Rekonstruktion besteht und so ein optimaler Therapiezeitraum eingegrenzt werden kann.
Zunächst erfolgte die Zellisolierung aus intraoperativ gewonnenen VKB-Biopsien. Je nach Latenz zwischen Ruptur und Operation wurden drei Gruppen (akute ≙ ≤ 30 d, subakute ≙ 31-90 d, verzögerte Rekonstruktion ≙ > 90 d) gebildet. Zum Nachweis von MSC wurden die Zellen hinsichtlich ihrer Plastikadhärenz, eines multipotenten Differenzierungspotentials sowie eines spezifischen Oberflächenantigenmusters (CD73+, CD90+, CD105+, CD34-) untersucht. Zudem wurde ihr Einflusses auf die biomechanischen und histologischen Eigenschaften eines analysiert.
Der Nachweis von MSC war in allen Gruppen möglich. Das Proliferationspotential war in Gruppe II am größten, ebenso der Anteil der MSC an allen Zellen. Er war 5,4% (4,6% - 6,3%, 95% CI; p < 0,001) höher als in Gruppe I und 18,9% (18,2% - 19,6%, 95% CI; p < 0,001) höher als in Gruppe III. In den mit Zellen kultivierten Bandkonstrukten konnte im Gegensatz zu zellfreien Konstrukten humanes Kollagen I nachgewiesen werden. Die Stabilität nahm bei Kultivierung mit Zellen ab.
Die Ergebnisse legen nahe, dass das Regenerationspotential bei subakuter VKB-Rekonstruktion (31-90 d) am höchsten ist. Potenziell ursächlich sind die Regeneration hemmende Entzündungsprozesse zu Beginn sowie degenerative Prozesse im längerfristigen Verlauf. Zudem konnte gezeigt werden, dass die isolierten Zellen die Eigenschaften eines Bandkonstruktes durch Bildung von Kollagen I und Reduktion der Stabilität im kurzfristigen Verlauf verändern und dementsprechend den Therapieerfolg beeinflussen könnten. Zur Verifizierung der Ergebnisse bedarf es weiterer Untersuchungen.
Meniscal pathologies are among the most common injuries of the femorotibial joint in both human and equine patients. Pathological forces and ensuing injuries of the cranial horn of the equine medial meniscus are considered analogous to those observed in the human posterior medial horn. Biomechanical properties of human menisci are site-and depth-specific. However, the influence of equine meniscus topography and composition on its biomechanical properties is yet unknown. A better understanding of equine meniscus composition and biomechanics could advance not only veterinary therapies for meniscus degeneration or injuries, but also further substantiate the horse as suitable translational animal model for (human) meniscus tissue engineering. Therefore, the aim of this study was to investigate the composition and structure of the equine knee meniscus in a site-and age-specific manner and their relationship with potential site-specific biomechanical properties. The meniscus architecture was investigated histologically. Biomechanical testing included evaluation of the shore hardness (SH), stiffness and energy loss of the menisci. The SH was found to be subjected to both age and site-specific changes, with an overall higher SH of the tibial meniscus surface and increase in SH with age. Stiffness and energy loss showed neither site nor age related significant differences. The macroscopic and histologic similarities between equine and human menisci described in this study, support continued research in this field.
Epithelial-to-mesenchymal transition (EMT) is discussed to be centrally involved in invasion, stemness, and drug resistance. Experimental models to evaluate this process in its biological complexity are limited. To shed light on EMT impact and test drug response more reliably, we use a lung tumor test system based on a decellularized intestinal matrix showing more in vivo-like proliferation levels and enhanced expression of clinical markers and carcinogenesis-related genes. In our models, we found evidence for a correlation of EMT with drug resistance in primary and secondary resistant cells harboring KRAS\(^{G12C}\) or EGFR mutations, which was simulated in silico based on an optimized signaling network topology. Notably, drug resistance did not correlate with EMT status in KRAS-mutated patient-derived xenograft (PDX) cell lines, and drug efficacy was not affected by EMT induction via TGF-β. To investigate further determinants of drug response, we tested several drugs in combination with a KRAS\(^{G12C}\) inhibitor in KRAS\(^{G12C}\) mutant HCC44 models, which, besides EMT, display mutations in P53, LKB1, KEAP1, and high c-MYC expression. We identified an aurora-kinase A (AURKA) inhibitor as the most promising candidate. In our network, AURKA is a centrally linked hub to EMT, proliferation, apoptosis, LKB1, and c-MYC. This exemplifies our systemic analysis approach for clinical translation of biomarker signatures.
The extracellular matrix (ECM) of soft tissues in vivo has remarkable biological and structural properties. Thereby, the ECM provides mechanical stability while it still can be rearranged via cellular remodeling during tissue maturation or healing processes. However, modern synthetic alternatives fail to provide these key features among basic properties. Synthetic matrices are usually completely degraded or are inert regarding cellular remodeling. Based on a refined electrospinning process, a method is developed to generate synthetic scaffolds with highly porous fibrous structures and enhanced fiber‐to‐fiber distances. Since this approach allows for cell migration, matrix remodeling, and ECM synthesis, the scaffold provides an ideal platform for the generation of soft tissue equivalents. Using this matrix, an electrospun‐based multilayered skin equivalent composed of a stratified epidermis, a dermal compartment, and a subcutis is able to be generated without the use of animal matrix components. The extension of classical dense electrospun scaffolds with high porosities and motile fibers generates a fully synthetic and defined alternative to collagen‐gel‐based tissue models and is a promising system for the construction of tissue equivalents as in vitro models or in vivo implants.
The foreign body reaction to neuronal electrode implants limits potential applications as well as the therapeutic period. Developments in the basic electrode design might improve the tissue compatibility and thereby reduce the foreign body reaction. In this work, the approach of embedding 3D carbon nanofiber electrodes in extracellular matrix (ECM) synthesized by human fibroblasts for a compatible connection to neuronal cells was investigated. Porous electrode material was manufactured by solution coelectrospinning of polyacrylonitrile and polyamide as a fibrous porogen. Moreover, NaCl represented an additional particulate porogen. To achieve the required conductivity for an electrical interface, meshes were carbonized. Through the application of two different porogens, the electrodes' flexibility and porosity was improved. Human dermal fibroblasts were cultured on the electrode surface for ECM generation and removed afterwards. Scanning electron microscopy imaging revealed a nano fibrous ECM network covering the carbon fibers. The collagen amount of the ECM coating was quantified by hydroxyproline-assays. The modification with the natural protein coating on the electrode functionality resulted in a minor increase of the electrical capacity, which slightly improved the already outstanding electrical interface properties. Increased cell numbers of SH-SY5Y cell line on ECM-modified electrodes demonstrated an improved cell adhesion. During cell differentiation, the natural ECM enhanced the formation of neurites regarding length and branching. The conducted experiments indicated the prevention of direct cell-electrode contacts by the modification, which might help to shield temporary the electrode from immunological cells to reduce the foreign body reaction and improve the electrodes' tissue integration.
The exposure of humans to nano-and microplastic particles (NMPs) is an issue recognized as a potential health hazard by scientists, authorities, politics, non-governmental organizations and the general public. The concentration of NMPs in the environment is increasing concomitantly with global plastic production and the usage of plastic materials. NMPs are detectable in numerous aquatic organisms and also in human samples, therefore necessitating a risk assessment of NMPs for human health. So far, a comprehensive risk assessment of NMPs is hampered by limited availability of appropriate reference materials, analytical obstacles and a lack of definitions and standardized study designs. Most studies conducted so far used polystyrene (PS) spheres as a matter of availability, although this polymer type accounts for only about 7% of total plastic production. Differently sized particles, different concentration and incubation times, and various biological models have been used, yielding hardly comparable data sets. Crucial physico-chemical properties of NMPs such as surface (charge, polarity, chemical reactivity), supplemented additives and adsorbed chemicals have been widely excluded from studies, although in particular the surface of NMPs determines the interaction with cellular membranes. In this manuscript we give an overview about the critical parameters which should be considered when performing risk assessments of NMPs, including novel reference materials, taking into account surface modifications (e.g., reflecting weathering processes), and the possible role of NMPs as a substrate and/or carrier for (pathogenic) microbes. Moreover, we make suggestions for biological model systems to evaluate immediate toxicity, long-term effects and the potential of NMPs to cross biological barriers. We are convinced that standardized reference materials and experimental parameters along with technical innovations in (nano)-particle sampling and analytics are a prerequisite for the successful realization of conclusive human health risk assessments of NMPs.
Etablierung eines 3D Gewebemodells für die translationale Forschung am Malignen Pleuramesotheliom
(2022)
Einleitung:
Das maligne Pleuramesotheliom (MPM) ist ein aggressiver von den Mesothelzellen der Pleura ausgehender Tumor, der in der Regel Folge einer Exposition mit Asbest ist. Aufgrund der häufig für ein chirurgisches Vorgehen zu späten Diagnose und des nur unzureichenden Ansprechens des Tumors auf Chemotherapie und Bestrahlung ist die Prognose sehr schlecht. Die präklinische Entwicklung und Testungen neuer Wirkstoffe ist aufgrund eines Mangels an geeigneten in vivo und in vitro Modellen für die biomedizinische Forschung schwierig. Das Ziel der vorliegenden Arbeit war der Aufbau eines 3D Gewebemodells, das die physiologischen Wachstumsverhältnisse und die Tumormikroumgebung des MPM wiedergibt und das als mögliches präklinisches Testmodell eingesetzt werden kann.
Methoden:
Zwei etablierte Zelllinien des MPM, JL-1 und MSTO-211H, wurden auf in Zellkronen eingespannten Segmenten aus azellulärem porzinen Jejunum unter statischen Kulturbedingungen und unter kontinuierlicher Perfusion in einem Bioreaktorsystem kultiviert. Die 3D Gewebemodelle wurden mit 2D Kulturmodellen des Pleuramesothelioms verglichen. Aus OP-Präparaten wurden tumor-assoziierte Fibroblasten (TAF) isoliert, die zum Aufbau von Kokulturmodellen verwendet wurden. Die Modelle wurden histologisch und immunhistologisch charakterisiert (Calretinin etc.).
Ergebnisse:
Die beiden verwendeten Zelllinien bildeten in der statischen Kultur ein mehrschichtiges Gewebe auf der apikalen Oberfläche der Matrix. Im Vergleich mit der 2D Kultur war ein homogeneres Wachstumsmuster der Zellen und eine erniedrigte Proliferationsrate zu beobachten. Die unter dynamischen Bedingungen kultivierten Modelle zeigten deutlich mehr Tumorzellmasse auf der Matrix. Aus Gewebebiopsien eines malignen Pleuramesothelioms von Patienten wurden TAF isoliert und damit 3D Kokulturmodelle aufgebaut. In den Kokulturmodellen migrierten die TAF in die Matrix, während die Tumorzellen weiterhin auf der apikalen Seite wuchsen.
Diskussion
Durch die Kombination mit einem Bioreaktorsystem, das eine bessere Nährstoffversorgung und die Erzeugung von Scherstress ermöglicht, wird das Tumorzellwachstum positiv beeinflusst. Das Wachstum primärer Zellen auf und deren Migration in die Matrix zeigt das Potential für den Aufbau patienten-spezifischer Modelle auf. Die generierten Gewebemodelle stellen eine Grundlage für gewebespezifische Weiterentwicklungen der Modelle für tumorspezifische mechanistische und letztlich auch therapeutische Fragestellungen dar.
The measurement of transepithelial electrical resistance (TEER) is a common technique to determine the barrier integrity of epithelial cell monolayers. However, it is remarkable that absolute TEER values of similar cell types cultured under comparable conditions show an immense heterogeneity. Based on previous observations, we hypothesized that the heterogeneity of absolute TEER measurements can not only be explained by maturation of junctional proteins but rather by dynamics in the absolute length of cell junctions within monolayers. Therefore, we analyzed TEER in epithelial cell monolayers of Caco2 cells during their differentiation, with special emphasis on both changes in the junctional complex and overall cell morphology within monolayers. We found that in epithelial Caco2 monolayers TEER increased until confluency, then decreased for some time, which was then followed by an additional increase during junctional differentiation. In contrast, permeability of macromolecules measured at different time points as 4 kDA fluorescein isothiocyanate (FITC)-dextran flux across monolayers steadily decreased during this time. Detailed analysis suggested that this observation could be explained by alterations of junctional length along the cell borders within monolayers during differentiation. In conclusion, these observations confirmed that changes in cell numbers and consecutive increase of junctional length have a critical impact on TEER values, especially at stages of early confluency when junctions are immature.
The cornea is the most-transplanted tissue worldwide. However, the availability and quality of grafts are limited due to the current methods of corneal storage. In this study, a dynamic bioreactor system is employed to enable the control of intraocular pressure and the culture at the air-liquid interface. Thereby, in vivo-like storage conditions are achieved. Different media combinations for endothelium and epithelium are tested in standard and dynamic conditions to enhance the viability of the tissue. In contrast to culture conditions used in eye banks, the combination of the bioreactor and biochrom medium 1 allows to preserve the corneal endothelium and the epithelium. Assessment of transparency, swelling, and the trans-epithelial-electrical-resistance (TEER) strengthens the impact of the in vivo-like tissue culture. For example, compared to corneas stored under static conditions, significantly lower optical densities and significantly higher TEER values were measured (p-value <0.05). Furthermore, healing of epithelial defects is enabled in the bioreactor, characterized by re-epithelialization and initiated stromal regeneration. Based on the obtained results, an easy-to-use 3D-printed bioreactor composed of only two parts was derived to translate the technology from the laboratory to the eye banks. This optimized bioreactor facilitates noninvasive microscopic monitoring. The improved storage conditions ameliorate the quality of corneal grafts and the storage time in the eye banks to increase availability and reduce re-grafting.
Malignant melanoma (MM) is the most dangerous type of skin cancer with rising incidences worldwide. Melanoma skin models can help to elucidate its causes and formation or to develop new treatment strategies. However, most of the current skin models lack a vasculature, limiting their functionality and applicability. MM relies on the vascular system for its own supply and for its dissemination to distant body sites via lymphatic and blood vessels. Thus, to accurately study MM progression, a functional vasculature is indispensable. To date, there are no vascularized skin models to study melanoma metastasis in vitro, which is why such studies still rely on animal experimentation.
In the present thesis, two different approaches for the vascularization of skin models are employed with the aim to establish a vascularized 3D in vitro full-thickness skin equivalent (FTSE) that can serve as a test system for the investigation of the progression of MM.
Initially, endothelial cells were incorporated in the dermal part of FTSEs. The optimal seeding density, a spheroid conformation of the cells and the cell culture medium were tested. A high cell density resulted in the formation of lumen-forming shapes distributed in the dermal part of the model. These capillary-like structures were proven to be of endothelial origin by staining for the endothelial cell marker CD31. The established vascularized FTSE (vFTSE) was characterized histologically after 4 weeks of culture, revealing an architecture similar to human skin in vivo with a stratified epidermis, separated from the dermal equivalent by a basement membrane indicated by collagen type IV. However, this random capillary-like network is not functional as it cannot be perfused.
Therefore, the second vascularization approach focused on the generation of a perfusable tissue construct. A channel was molded within a collagen hydrogel and seeded with endothelial cells to mimic a central, perfusable vessel. The generation and the perfusion culture of the collagen hydrogel was enabled by the use of two custom-made, 3D printed bioreactors. Histological assessment of the hydrogels revealed the lining of the channel with a monolayer of endothelial cells, expressing the cell specific marker CD31.
For the investigation of MM progression in vitro, a 3D melanoma skin equivalent was established. Melanoma cells were incorporated in the epidermal part of FTSEs, representing the native microenvironment of the tumor. Melanoma nests grew at the dermo-epidermal junction within the well stratified epidermis and were characterized by the expression of common melanoma markers. First experiments were conducted showing the feasibility of combining the melanoma model with the vFTSE, resulting in skin models with tumors at the dermo-epidermal junction and lumen-like structures in the dermis.
Taken together, the models presented in this thesis provide further steps towards the establishment of a vascularized, perfusable melanoma model to study melanoma progression and metastasis.
In the treatment of bone non-unions, an alternative to bone autografts is the use of bone morphogenetic proteins (BMPs), e.g., BMP–2, BMP–7, with powerful osteoinductive and osteogenic properties. In clinical settings, these osteogenic factors are applied using absorbable collagen sponges for local controlled delivery. Major side effects of this strategy are derived from the supraphysiological doses of BMPs needed, which may induce ectopic bone formation, chronic inflammation, and excessive bone resorption. In order to increase the efficiency of the delivered BMPs, we designed cryostructured collagen scaffolds functionalized with hydroxyapatite, mimicking the structure of cortical bone (aligned porosity, anisotropic) or trabecular bone (random distributed porosity, isotropic). We hypothesize that an anisotropic structure would enhance the osteoconductive properties of the scaffolds by increasing the regenerative performance of the provided rhBMP–2. In vitro, both scaffolds presented similar mechanical properties, rhBMP–2 retention and delivery capacity, as well as scaffold degradation time. In vivo, anisotropic scaffolds demonstrated better bone regeneration capabilities in a rat femoral critical-size defect model by increasing the defect bridging. In conclusion, anisotropic cryostructured collagen scaffolds improve bone regeneration by increasing the efficiency of rhBMP–2 mediated bone healing.
Pilot study on the value of Raman spectroscopy in the entity assignment of salivary gland tumors
(2021)
Background
The entity assignment of salivary gland tumors (SGT) based on histomorphology can be challenging. Raman spectroscopy has been applied to analyze differences in the molecular composition of tissues. The aim of this study was to evaluate the suitability of RS for entity assignment in SGT.
Methods
Raman data were collected in deparaffinized sections of pleomorphic adenomas (PA) and adenoid cystic carcinomas (ACC). Multivariate data and chemometric analysis were completed using the Unscrambler software.
Results
The Raman spectra detected in ACC samples were mostly assigned to nucleic acids, lipids, and amides. In a principal component-based linear discriminant analysis (LDA) 18 of 20 tumor samples were classified correctly.
Conclusion
In this proof of concept study, we show that a reliable SGT diagnosis based on LDA algorithm appears possible, despite variations in the entity-specific mean spectra. However, a standardized workflow for tissue sample preparation, measurement setup, and chemometric algorithms is essential to get reliable results.
The small intestine represents a strong barrier separating the lumen from blood circulation thereby playing a major role in the absorption and the transport of pharmacological agents prior to their arrival on the respective target site. In order to gain more knowledge about specialized uptake mechanisms and risk assessment for the patient after oral admission of drugs, intestinal in vitro models demonstrating a close similarity to the in vivo situation are needed.
In the past, cell line-based in vitro models composed of Caco-2 cells cultured on synthetic cell carriers represented the “gold standard” in the field of intestinal tissue engineering. Expressive advantages of these models are a reproducible, cost-efficient and standardized model set up, but cell function can be negatively influenced by the low porosity or unwanted molecular adhesion effects of the artificial scaffold material. Natural extracellular matrices (ECM) such as the porcine decellularized small intestinal submucosa (SIS) are used as alternative to overcome some common drawbacks; however, the fabrication of these scaffolds is time- and cost-intensive, less well standardized and the 3Rs (replacement, reduction, refinement) principle is not entirely fulfilled. Nowadays, biopolymer-based scaffolds such as the bacterial nanocellulose (BNC) suggest an interesting option of novel intestinal tissue engineered models, as the BNC shows comparable features to the native ECM regarding fiber arrangement and hydrophilic properties. Furthermore, the BNC is of non-animal origin and the manufacturing process is faster as well as well standardized at low costs.
In this context, the first part of this thesis analyzed the BNC as alternative scaffold to derive standardized and functional organ models in vitro. Therefore, Caco-2 cells were cultured on two versions of BNC with respect to their surface topography, the unmodified BNC as rather smooth surface and the surface-structured BNC presenting an aligned fiber arrangement. As controls, Caco-2 in vitro models were set up on PET and SIS matrices. In this study, the BNC-based models demonstrated organ-specific properties comprising typical cellular morphologies, a characteristic tight junction protein expression profile, representative ultrastructural features and the formation of a tight epithelial barrier together with a corresponding transport activity. In summary, these results validated the high quality of the BNC-based Caco-2 models under cost-efficient conditions and their suitability for pre-clinical research purposes. However, the full functional diversity of the human intestine cannot be presented by Caco-2 cells due to their tumorigenic background and their exclusive representation of mature enterocytes.
Next to the scaffold used for the setup of in vitro models, the cellular unit mainly drives functional performance, which demonstrates the crucial importance of mimicking the cellular diversity of the small intestine in vitro. In this context, intestinal primary organoids are of high interest, as they show a close similarity to the native epithelium regarding their cellular diversity comprising enterocytes, goblet cells, enteroendocrine cells, paneth cells, transit amplifying cells and stem cells. In general, such primary organoids grow in a 3D Matrigel® based environment and a medium formulation supplemented with a variety of growth factors to maintain stemness, to inhibit differentiation and to stimulate cell migration supporting long term in vitro culture.
Intestinal primary spheroid/organoid cultures were set up as Transwell®-like models on both BNC variants, which resulted in a fragmentary cell layer and thereby unfavorable properties of these scaffold materials under the applied circumstances. As the BNC manufacturing process is highly flexible, surface properties could be adapted in future studies to enable a good cell adherence and barrier formation for primary intestinal cells, too. However, the application of these organoid cultures in pre-clinical research represents an enormous challenge, as the in vitro culture is complex and additionally time- and cost-intensive.
With regard to the high potential of primary intestinal spheroids/organoids and the necessity of a simplified but predictive model in pre-clinical research purposes, the second part of this thesis addressed the establishment of a primary-derived immortalized intestinal cell line, which enables a standardized and cost-efficient culture (including in 2D), while maintaining the cellular diversity of the organoid in vitro cultures. In this study, immortalization of murine and human intestinal primary organoids was induced by ectopic expression of a 10- (murine) or 12 component (human) pool of genes regulating stemness and the cell cycle, which was performed in cooperation with the InSCREENeX GmbH in a 2D- and 3D-based transduction strategy. In first line, the established cell lines (cell clones) were investigated for their cell culture prerequisites to grow under simplified and cost-efficient conditions. While murine cell clones grew on uncoated plastic in a medium formulation supplemented with EGF, Noggin, Y-27632 and 10% FCS, the human cell clones demonstrated the necessity of a Col I pre coating together with the need for a medium composition commonly used for primary human spheroid/organoid cultures. Furthermore, the preceding analyses resulted in only one human cell clone and three murine cell clones for ongoing characterization. Studies regarding the proliferative properties and the specific gene as well as protein expression profile of the remaining cell clones have shown, that it is likely that transient amplifying cells (TACs) were immortalized instead of the differentiated cell types localized in primary organoids, as 2D, 3D or Transwell®-based cultures resulted in slightly different gene expression profiles and in a dramatically reduced mRNA transcript level for the analyzed marker genes representative for the differentiated cell types of the native epithelium. Further, 3D cultures demonstrated the formation of spheroid-like structures; however without forming organoid-like structures due to prolonged culture, indicating that these cell populations have lost their ability to differentiate into specific intestinal cell types. The Transwell®-based models set up of each clone exhibit organ-specific properties comprising an epithelial-like morphology, a characteristic protein expression profile with an apical mucus-layer covering the villin-1 positive cell layer, thereby representing goblet cells and enterocytes, together with representative tight junction complexes indicating an integer epithelial barrier. The proof of a functional as well as tight epithelial barrier in TEER measurements and in vivo-like transport activities qualified the established cell clones as alternative cell sources for tissue engineered models representing the small intestine to some extent. Additionally, the easy handling and cell expansion under more cost-efficient conditions compared to primary organoid cultures favors the use of these newly generated cell clones in bioavailability studies.
Altogether, this work demonstrated new components, structural and cellular, for the establishment of alternative in vitro models of the small intestinal epithelium, which could be used in pre-clinical screenings for reproducible drug delivery studies.
Objective
Cartilage defect treatment strategies are dependent on the lesion size and severity. Osteochondral explant models are a platform to test cartilage repair strategies ex vivo. Current models lack in mimicking the variety of clinically relevant defect scenarios. In this controlled laboratory study, an automated device (artificial tissue cutter, ARTcut®) was implemented to reproducibly create cartilage defects with controlled depth. In a pilot study, the effect of cartilage defect depth and oxygen tension on cartilage repair was investigated.
Design
Osteochondral explants were isolated from porcine condyles. 4 mm chondral and full thickness defects were treated with either porcine chondrocytes (CHON) or co-culture of 20% CHON and 80% MSCs (MIX) embedded in collagen hydrogel. Explants were cultured with tissue specific media (without TGF-β) under normoxia (20% O\(_2\)) and physiological hypoxia (2% O\(_2\)). After 28 days, immune-histological stainings (collagen II and X, aggrecan) were scored (modified Bern score, 3 independent scorer) to quantitatively compare treatment outcome.
Results
ARTcut® represents a software-controlled device for creation of uniform cartilage defects. Comparing the scoring results of the MIX and the CHON treatment, a positive relation between oxygen tension and defect depth was observed. Low oxygen tension stimulated cartilaginous matrix deposition in MIX group in chondral defects and CHON treatment in full thickness defects.
Conclusion
ARTcut® has proved a powerful tool to create cartilage defects and thus opens a wide range of novel applications of the osteochondral model, including the relation between oxygen tension and defect depth on cartilage repair.
Mit jährlich circa 11 Millionen Fällen weltweit, stellen schwere Brandwunden bis heute einen großen Anteil an Verletzungen dar, die in Kliniken behandelt werden müssen. Während leichte Verbrennungen meist problemlos heilen, bedarf die Behandlung tieferer Verbrennungen medizinischer Intervention. Zellbasierte Therapeutika zeigen hier bereits große Erfolge, aufgrund der eingeschränkten Übertragbarkeit von Ergebnissen aus Tiermodellen ist jedoch sowohl die Testung neuer Produkte, als auch die Erforschung der Wundheilung bei Brandwunden noch immer schwierig.
Aufgrund dessen wurden in dieser Arbeit zwei Ziele verfolgt: Die Etablierung von Methoden, um ein zellbasiertes Therapeutikum produzieren zu können und die Entwicklung eines Modells zur Untersuchung von Verbrennungswunden. Zunächst wurden hierfür die Kulturbedingungen und -protokolle zur Isolation und Expansion von Keratinozyten so angepasst, dass sie gängigen Regularien zur Produktion medizinischer Produkte entsprechen. Hier zeigten die Zellen auch in anschließenden Analysen, dass charakteristische Merkmale nicht verloren hatten. Darüber hinaus gelang es, die Zellen mithilfe verschiedener protektiver Substanzen erfolgreich einzufrieren und zu konservieren.
Des Weiteren konnte ein Modell etabliert werden, das eine Verbrennung ersten Grades widerspiegelt. Über einen Zeitraum von zwei Wochen wurde seine Regeneration hinsichtlich verschiedener Aspekte, wie der Histomorphologie, dem Metabolismus und der Reepithelialisierungsrate, untersucht. Die Modelle zeigten hier viele Parallelen zur Wundheilung in vivo auf. Um die Eignung der Modelle zur Testung von Wirkstoffen zu ermitteln wurde außerdem eine Behandlung mit 5% Dexpanthenol getestet. Sie resultierte in einer verbesserten Histomorphologie und einer erhöhten Anzahl an proliferativen Zellen in den Modellen, beschleunigte jedoch die Reepithelialisierung nicht. Zusammengefasst konnten in dieser Arbeit zunächst Methoden etabliert werden, um ein medizinisches Produkt aus Keratinozyten herzustellen und zu charakterisieren. Außerdem wurde ein Modell entwickelt, anhand dessen die Wundheilung und Behandlung von Verbrennungen ersten Grades untersucht werden kann und welches als Basis zur Entwicklung von Modellen von tieferen Verbrennungen dienen kann.
To circumvent time-consuming clinical trials, testing whether existing drugs are effective inhibitors of SARS-CoV-2, has led to the discovery of Remdesivir. We decided to follow this path and screened approved medications "off-label" against SARS-CoV-2. Fluoxetine inhibited SARS-CoV-2 at a concentration of 0.8 mu g/ml significantly in these screenings, and the EC50 was determined with 387 ng/ml. Furthermore, Fluoxetine reduced viral infectivity in precision-cut human lung slices showing its activity in relevant human tissue targeted in severe infections. Fluoxetine treatment resulted in a decrease in viral protein expression. Fluoxetine is a racemate consisting of both stereoisomers, while the S-form is the dominant serotonin reuptake inhibitor. We found that both isomers show similar activity on the virus, indicating that the R-form might specifically be used for SARS-CoV-2 treatment. Fluoxetine inhibited neither Rabies virus, human respiratory syncytial virus replication nor the Human Herpesvirus 8 or Herpes simplex virus type 1 gene expression, indicating that it acts virus-specific. Moreover, since it is known that Fluoxetine inhibits cytokine release, we see the role of Fluoxetine in the treatment of SARS-CoV-2 infected patients of risk groups.
Gonorrhea, a sexually transmitted disease caused by the bacteria Neisseria gonorrhoeae, is characterized by a large number of neutrophils recruited to the site of infection. Therefore, proper modeling of the N. gonorrhoeae interaction with neutrophils is very important for investigating and understanding the mechanisms that gonococci use to evade the immune response. We have used a combination of a unique human 3D tissue model together with a dynamic culture system to study neutrophil transmigration to the site of N. gonorrhoeae infection. The triple co-culture model consisted of epithelial cells (T84 human colorectal carcinoma cells), human primary dermal fibroblasts, and human umbilical vein endothelial cells on a biological scaffold (SIS). After the infection of the tissue model with N. gonorrhoeae, we introduced primary human neutrophils to the endothelial side of the model using a perfusion-based bioreactor system. By this approach, we were able to demonstrate the activation and transmigration of neutrophils across the 3D tissue model and their recruitment to the site of infection. In summary, the triple co-culture model supplemented by neutrophils represents a promising tool for investigating N. gonorrhoeae and other bacterial infections and interactions with the innate immunity cells under conditions closely resembling the native tissue environment.
Here, a postpolymerization modification method for an α-terminal functionalized poly-(N-methyl-glycine), also known as polysarcosine, is introduced. 4-(Methylthio)phenyl piperidine-4-carboxylate as an initiator for the ring-opening polymerization of N-methyl-glycine-N-carboxyanhydride followed by oxidation of the thioester group to yield an α-terminal reactive 4-(methylsulfonyl)phenyl piperidine-4-carboxylate polymer is utilized. This represents an activated carboxylic acid terminus, allowing straightforward modification with nucleophiles under mild reaction conditions and provides the possibility to introduce a wide variety of nucleophiles as exemplified using small molecules, fluorescent dyes, and model proteins. The new initiator yielded polymers with well-defined molar mass, low dispersity, and high end-group fidelity, as observed by gel permeation chromatography, nuclear magnetic resonance spectroscopy, and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy. The introduced method can be of great interest for bioconjugation, but requires optimization, especially for protein conjugation.
To study the interaction of human pathogens with their host target structures, human tissue models based on primary cells are considered suitable. Complex tissue models of the human airways have been used as infection models for various viral and bacterial pathogens. The Gram-negative bacterium Bordetella pertussis is of relevant clinical interest since whooping cough has developed into a resurgent infectious disease. In the present study, we created three-dimensional tissue models of the human ciliated nasal and tracheo-bronchial mucosa. We compared the innate immune response of these models towards the B. pertussis virulence factor adenylate cyclase toxin (CyaA) and its enzymatically inactive but fully pore-forming toxoid CyaA-AC\(^-\). Applying molecular biological, histological, and microbiological assays, we found that 1 µg/ml CyaA elevated the intracellular cAMP level but did not disturb the epithelial barrier integrity of nasal and tracheo-bronchial airway mucosa tissue models. Interestingly, CyaA significantly increased interleukin 6, interleukin 8, and human beta defensin 2 secretion in nasal tissue models, whereas tracheo-bronchial tissue models were not significantly affected compared to the controls. Subsequently, we investigated the interaction of B. pertussis with both differentiated primary nasal and tracheo-bronchial tissue models and demonstrated bacterial adherence and invasion without observing host cell type-specific significant differences. Even though the nasal and the tracheo-bronchial mucosa appear similar from a histological perspective, they are differentially susceptible to B. pertussis CyaA in vitro. Our finding that nasal tissue models showed an increased innate immune response towards the B. pertussis virulence factor CyaA compared to tracheo-bronchial tissue models may reflect the key role of the nasal airway mucosa as the first line of defense against airborne pathogens.
Advanced Therapy Medicinal Products (ATMP) provide promising treatment options particularly for unmet clinical needs, such as progressive and chronic diseases where currently no satisfying treatment exists. Especially from the ATMP subclass of Tissue Engineered Products (TEPs), only a few have yet been translated from an academic setting to clinic and beyond. A reason for low numbers of TEPs in current clinical trials and one main key hurdle for TEPs is the cost and labor-intensive manufacturing process. Manual production steps require experienced personnel, are challenging to standardize and to scale up. Automated manufacturing has the potential to overcome these challenges, toward an increasing cost-effectiveness. One major obstacle for automation is the control and risk prevention of cross contaminations, especially when handling parallel production lines of different patient material. These critical steps necessitate validated effective and efficient cleaning procedures in an automated system. In this perspective, possible technologies, concepts and solutions to existing ATMP manufacturing hurdles are discussed on the example of a late clinical phase II trial TEP. In compliance to Good Manufacturing Practice (GMP) guidelines, we propose a dual arm robot based isolator approach. Our novel concept enables complete process automation for adherent cell culture, and the translation of all manual process steps with standard laboratory equipment. Moreover, we discuss novel solutions for automated cleaning, without the need for human intervention. Consequently, our automation concept offers the unique chance to scale up production while becoming more cost-effective, which will ultimately increase TEP availability to a broader number of patients.
Background: Human bone marrow-derived mesenchymal stromal cells (hBMSCs) provide a promising therapeutic approach in the cell-based therapy of osteoarthritis (OA). However, several disadvantages evolved recently, including immune responses of the host and regulatory hurdles, making it necessary to search for alternative treatment options. Extracellular vesicles (EVs) are released by multiple cell types and tissues into the extracellular microenvironment, acting as message carriers during intercellular communication. Here, we investigate putative protective effects of hBMSC-derived EVs as a cell-free approach, on IL-1β-stimulated chondrocytes obtained from OA-patients.
Methods: EVs were harvested from the cell culture supernatant of hBMSCs by a sequential ultracentrifugation process. Western blot, scanning electron microscopy (SEM), and nanoparticle tracking analysis (NTA) were performed to characterize the purified particles as EVs. Intracellular incorporation of EVs, derived from PHK26-labeled hBMSCs, was tested by adding the labeled EVs to human OA chondrocytes (OA-CH), followed by fluorescence microscopy. Chondrocytes were pre-stimulated with IL-1β for 24 h, followed by EVs treatment for 24 h. Subsequently, proliferation, apoptosis, and migration (wound healing) were analyzed via BrdU assay, caspase 3/7 assay, and scratch assay, respectively. With qRT-PCR, the relative expression level of anabolic and catabolic genes was determined. Furthermore, immunofluorescence microscopy and western blot were performed to evaluate the protein expression and phosphorylation levels of Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB as components of pro-inflammatory signaling pathways in OA-CH.
Results: EVs from hBMSCs (hBMSC-EVs) promote proliferation and reduce apoptosis of OA-CH and IL-1β-stimulated OA-CH. Moreover, hBMSC-EVs attenuate IL-1β-induced reduction of chondrocyte migration. Furthermore, hBMSC-EVs increase gene expression of PRG4, BCL2, and ACAN (aggrecan) and decrease gene expression of MMP13, ALPL, and IL1ß in OA-CH. Notably, COL2A1, SOX9, BCL2, ACAN, and COMP gene expression levels were significantly increased in IL-1β+ EV groups compared with those IL-1β groups without EVs, whereas the gene expression levels of COLX, IL1B, MMP13, and ALPL were significantly decreased in IL-1β+ EV groups compared to IL-1β groups without EVs. In addition, the phosphorylation status of Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB signaling molecules, induced by IL-1β, is prevented by hBMSC- EVs.
Conclusion: EVs derived from hBMSCs alleviated IL-1β-induced catabolic effects on OA-CH via promoting proliferation and migration and reducing apoptosis, probably via downregulation of IL-1ß-activated pro-inflammatory Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB signaling pathways. EVs released from BMSCs may be considered as promising cell-free intervention strategy in cartilage regenerative medicine, avoiding several adverse effects of cell-based regenerative approaches.
Background: Studies with extracellular vesicles (EVs), including exosomes, isolated from mesenchymal stem cells (MSC) indicate benefits for the treatment of musculoskeletal pathologies as osteoarthritis (OA) and osteoporosis (OP). However, little is known about intercellular effects of EVs derived from pathologically altered cells that might influence the outcome by counteracting effects from “healthy” MSC derived EVs. We hypothesize, that EVs isolated from osteoblasts of patients with hip OA (coxarthrosis/CA), osteoporosis (OP), or a combination of both (CA/OP) might negatively affect metabolism and osteogenic differentiation of bone-marrow derived (B)MSCs.
Methods: Osteoblasts, isolated from bone explants of CA, OP, and CA/OP patients, were compared regarding growth, viability, and osteogenic differentiation capacity. Structural features of bone explants were analyzed via μCT. EVs were isolated from supernatant of naïve BMSCs and CA, OP, and CA/OP osteoblasts (osteogenic culture for 35 days). BMSC cultures were stimulated with EVs and subsequently, cell metabolism, osteogenic marker gene expression, and osteogenic differentiation were analyzed.
Results: Trabecular bone structure was different between the three groups with lowest number and highest separation in the CA/OP group. Viability and Alizarin red staining increased over culture time in CA/OP osteoblasts whereas growth of osteoblasts was comparable. Alizarin red staining was by trend higher in CA compared to OP osteoblasts after 35 days and ALP activity was higher after 28 and 35 days. Stimulation of BMSC cultures with CA, OP, and CA/OP EVs did not affect proliferation but increased caspase 3/7-activity compared to unstimulated BMSCs. BMSC viability was reduced after stimulation with CA and CA/OP EVs compared to unstimulated BMSCs or stimulation with OP EVs. ALP gene expression and activity were reduced in BMSCs after stimulation with CA, OP, and CA/OP EVs. Stimulation of BMSCs with CA EVs reduced Alizarin Red staining by trend.
Conclusion: Stimulation of BMSCs with EVs isolated from CA, OP, and CA/OP osteoblasts had mostly catabolic effects on cell metabolism and osteogenic differentiation irrespective of donor pathology and reflect the impact of tissue microenvironment on cell metabolism. These catabolic effects are important for understanding differences in effects of EVs on target tissues/cells when harnessing them as therapeutic drugs.
Elastic fibers are essential for the proper function of organs including cardiovascular tissues such as heart valves and blood vessels. Although (tropo)elastin production in a tissue-engineered construct has previously been described, the assembly to functional elastic fibers in vitro using human cells has been highly challenging. In the present study, we seeded primary isolated human vascular smooth muscle cells (VSMCs) onto 3D electrospun scaffolds and exposed them to defined laminar shear stress using a customized bioreactor system. Increased elastin expression followed by elastin deposition onto the electrospun scaffolds, as well as on newly formed fibers, was observed after six days. Most interestingly, we identified the successful deposition of elastogenesis-associated proteins, including fibrillin-1 and -2, fibulin-4 and -5, fibronectin, elastin microfibril interface located protein 1 (EMILIN-1) and lysyl oxidase (LOX) within our engineered constructs. Ultrastructural analyses revealed a developing extracellular matrix (ECM) similar to native human fetal tissue, which is composed of collagens, microfibrils and elastin. To conclude, the combination of a novel dynamic flow bioreactor and an electrospun hybrid polymer scaffold allowed the production and assembly of an elastic fiber-containing ECM.
The incidence of cardiovascular and metabolic diseases has increased over the last decades and is an important cause of death worldwide. An upcoming ingredient on the nutraceutical market are anthocyanins, a flavonoid subgroup, abundant mostly in berries and fruits. Epidemiological studies have suggested an association between anthocyanin intake and improved cardiovascular risk, type 2 diabetes and myocardial infarct. Clinical studies using anthocyanins have shown a significant decrease in inflammation markers and oxidative stress, a beneficial effect on vascular function and hyperlipidemia by decreasing low-density lipoprotein and increasing high-density lipoprotein. They have also shown a potential effect on glucose homeostasis and cognitive decline. This review summarizes the effects of anthocyanins in in-vitro, animal and human studies to give an overview of their application in medical prevention or as a dietary supplement.
Irritable bowel syndrome (IBS) is a gut-brain disorder involving alterations in intestinal sensitivity and motility. Serotonin 5-HT4 receptors are promising candidates in IBS pathophysiology since they regulate gut motor function and stool consistency, and targeted 5-HT4R selective drug intervention has been proven beneficial in subgroups of patients. We identified a single nucleotide polymorphism (SNP) (rs201253747) c.*61 T > C within the 5-HT4 receptor gene \(HTR4\) to be predominantly present in diarrhoea-IBS patients (IBS-D). It affects a binding site for the miR-16 family and miR-103/miR-107 within the isoforms \({HTR4b/i}\) and putatively impairs \(HTR4\) expression. Subsequent miRNA profiling revealed downregulation of miR-16 and miR-103 in the jejunum of IBS-D patients correlating with symptoms. \(In\) \(vitro\) assays confirmed expression regulation via three 3′UTR binding sites. The novel isoform \(HTR4b\_2\) lacking two of the three miRNA binding sites escapes miR-16/103/107 regulationin SNP carriers. We provide the first evidence that \(HTR4\) expression is fine-tuned by miRNAs, and that this regulation is impaired either by the SNP c.*61 T > C or bydiminished levels of miR-16 and miR-103 suggesting that \(HTR4\) might be involved in the development of IBS-D.
Translating basic biological knowledge into applications remains a key issue for effectively tackling neurodegenerative, neuroinflammatory, or neuroendocrine disorders. Efficient delivery of therapeutics across the neuroprotective blood‐brain barrier (BBB) still poses a demanding challenge for drug development targeting central nervous system diseases. Validated in vitro models of the BBB could facilitate effective testing of drug candidates targeting the brain early in the drug discovery process during lead generation. We here review the potential of mono‐ or (isogenic) co‐culture BBB models based on brain capillary endothelial cells (BCECs) derived from human‐induced pluripotent stem cells (hiPSCs), and compare them to several available BBB in vitro models from primary human or non‐human cells and to rodent in vivo models, as well as to classical and widely used barrier models [Caco‐2, parallel artificial membrane permeability assay (PAMPA)]. In particular, we are discussing the features and predictivity of these models and how hiPSC‐derived BBB models could impact future discovery and development of novel CNS‐targeting therapeutics.
Da in den letzten Jahrzehnten nur geringfügige Verbesserungen der Überlebensraten bei an einem Pankreaskarzinom erkrankten Patienten erzielt wurden, besteht ein dringender klinischer Bedarf für die Entwicklung wirksamer therapeutischer Strategien. Dreidimensionale in vitro Modelle sind für das Screening und die Validierung von Therapeutika essenziell.
In der vorliegenden Arbeit konnte mittels der Methoden des Tissue Engineerings ein biolumineszenzbasiertes dreidimensionales in vitro Testsystem des pankreatischen Karzinoms aufgebaut und charakterisiert werden. Für die Detektion von LumineszenzIntensitäten wurde die pankreatische Krebszelllinie PANC-1 zuvor mit firefly luciferase (FLUC) transduziert. PANC-1 FLUC Zellen wurden auf porziner Pankreasmatrix (PanMa) und Dünndarmmatrix (SISser) kultiviert, um den Einfluss unterschiedlicher Matrizen auf das Verhalten der Zellen im Tumormodell zu untersuchen. Darüber hinaus wurden in dieser Arbeit die PANC-1 FLUC mit einem Standardtherapeutikum der Pankreaskarzinomtherapie, Gemcitabin, behandelt und die Wirkung mittels biolumineszenbasierter Bildgebung detektiert.
Es konnte gezeigt werden, dass die Lumineszenz-Intensität von PANC-1 FLUC Zellen einer bestimmten Zellzahl durch biolumineszenzbasierte Messverfahren zugeordnet werden kann. Weiter wurde nachgewiesen, dass die Extrazellulärmatrix einen Einfluss auf die Expression tumorspezifischer Marker hat und PANC-1 FLUC Zellen ein unterschiedlich invasives Wachstum auf organspezifischen Matrizen aufweisen. Die Wirkung von Gemcitabin auf die Tumorzellen kann durch das hier vorgestellte biolumineszenzbasierte Messverfahren detektiert werden. Die in dieser Arbeit vorgestellten Ergebnisse sind die Grundlage für die weitere Validierung eines biolumineszenzbasierten dreidimemsionalen in vitro Testystems des pankreatischen Karzinoms für die präklinische Erforschung neuartiger Therapiestrategien.
Aufgrund der sich umkehrenden Alterspyramide in Deutschland leiden bereits jetzt immer mehr Menschen an Gelenkknorpelschäden. Doch nicht nur das Alter, sondern auch Unfälle und Sportverletzungen und Übergewicht können zu irreversiblen Knorpeldefekten führen. Obwohl es diverse Behandlungsmöglichkeiten gibt, können die bisherige Methoden nicht als dauerhafte Heilung betrachtet werden. Im Rahmen des internationalen Forschungsprojektes BIO-CHIP sollte eine vielsprechende Behandlungsmethode mit neuartigen Arzneimitteln untersucht werden.
Als Ausgangsmaterial des Arzneimittels, ein hergestelltes Knorpelimplantat, dienen patienteneigene Knorpelzellen aus der Nase. Diese werden isoliert, vermehrt und letztlich auf einer Matrix zu einem Knorpelimplantat kultiviert. Wesentliche Voraussetzung für die Implantatfreigabe stellt neben toxikologischen und biologischen Unbedenklichkeitstests die Beurteilung der Viabilität dar. Diese wurde bisher anhand von Histologieschnitten von der Pathologie durchgeführt.
Ziel der vorliegenden Arbeit war die Entwicklung und Validierung eines standardisierten und objektiven Viabilitätstests für die Chondrozyten innerhalb der Knorpelmatrix. Hierfür wurde die LDH als Marker für irreversibel geschädigte Zellen verwendet. Die LDH Konzentration konnte mit dem CyQuant LDH-Assay durch die Messung der Absorption gemessen werden. Es konnte nachgewiesen werden, dass LDH die erforderliche Stabilität und Nachweisbarkeit im Medium besitzt. Mithilfe der Lyse, analog zum Herstellungsprozess, gezüchteter Mini-Knorpelimplantate, konnten die maximal erreichbaren LDH Konzentrationen ermittelt werden. Mithilfe dieser Konzentrationen wurde eine Eichkurve generiert. Diese dient als Beurteilung der Viabilität zukünftig gemessener Absorptionen des Überstandmediums.
Das entwickelte Verfahren erfordert keine invasiven Eingriffe am Implantat und zeichnet sich durch eine einfache Durchführung aus, da nur der Überstand gemessen werden muss. Die durchgeführte Validierung der Methode bescheinigte eine hohe Robustheit, Linearität, Genauigkeit und Präzision.
Alzheimer′s disease (AD) is a neurological disorder with still no preventive or curative treatment. Flavonoids are phytochemicals with potential therapeutic value. Previous studies described the flavanone sterubin isolated from the Californian plant Eriodictyon californicum as a potent neuroprotectant in several in vitro assays. Herein, the resolution of synthetic racemic sterubin (1) into its two enantiomers, (R)‐1 and (S)‐1, is described, which has been performed on a chiral chromatographic phase, and their stereochemical assignment online by HPLC‐ECD coupling. (R)‐1 and (S)‐1 showed comparable neuroprotection in vitro with no significant differences. While the pure stereoisomers were configurationally stable in methanol, fast racemization was observed in the presence of culture medium. We also established the occurrence of extracted sterubin as its pure (S)‐enantiomer. Moreover, the activity of sterubin (1) was investigated for the first time in vivo, in an AD mouse model. Sterubin (1) showed a significant positive impact on short‐ and long‐term memory at low dosages.
In this study we aimed to assess the effects of continuous formalin fixation on diffusion and relaxation metrics of the ex vivo porcine heart at 7 T. Magnetic resonance imaging was performed on eight piglet hearts using a 7 T whole body system. Hearts were measured fresh within 3 hours of cardiac arrest followed by immersion in 10% neutral buffered formalin. T\(_{2}\)* and T\(_{2}\) were assessed using a gradient multi‐echo and multi‐echo spin echo sequence, respectively. A spin echo and a custom stimulated echo sequence were employed to assess diffusion time‐dependent changes in metrics of cardiac diffusion tensor imaging. SNR was determined for b = 0 images. Scans were performed for 5 mm thick apical, midcavity and basal slices (in‐plane resolution: 1 mm) and repeated 7, 15, 50, 100 and 200 days postfixation. Eigenvalues of the apparent diffusion coefficient (ADC) and fractional anisotropy (FA) decreased significantly (P < 0.05) following fixation. Relative to fresh hearts, FA values 7 and 200 days postfixation were 90% and 80%, while respective relative ADC values at those fixation stages were 78% and 92%. Statistical helix and sheetlet angle distributions as well as respective mean and median values showed no systematic influence of continuous formalin fixation. Similar to changes in the ADC, values for T\(_{2}\), T\(_{2}\)* and SNR dropped initially postfixation. Respective relative values compared with fresh hearts at day 7 were 64%, 79% and 68%, whereas continuous fixation restored T\(_{2}\), T\(_{2}\)* and SNR leading to relative values of 74%, 100%, and 81% at day 200, respectively. Relaxation parameters and diffusion metrics are significantly altered by continuous formalin fixation. The preservation of microstructure metrics following prolonged fixation is a key finding that may enable future studies of ventricular remodeling in cardiac pathologies.
Critical size bone defects and nonunion fractures remain difficult to treat. Although cell‐loaded bone substitutes have improved bone ingrowth and formation, the lack of methods for achieving viability and the uniform distribution of cells in the scaffold limits their use as bone grafts. In addition, the predominant mechanical stimulus that drives early osteogenic cell maturation has not been clearly identified. Further, it is challenging to evaluate mechanical stimuli (i.e., deformation and fluid–flow-induced shear stress) because they are interdependent. This thesis compares different mechanical stimuli applied to cell-seeded scaffolds to develop bone grafts efficiently for the treatment of critical size bone defects. It also seeks to understand how deformation strain and interstitial fluid–flow-induced shear stress promote osteogenic lineage commitment. In this thesis, different scaffolds were seeded with primary human bone marrow mesenchymal stem cells (BM-MSCs) from different donors and subjected to static and dynamic culture conditions. In contrast with the static culture conditions, homogenous cell distributions were accomplished under dynamic culture conditions. Additionally, the induction of osteogenic lineage commitment without the addition of soluble factors was observed in the bioreactor system after one week of cell culture. To determine the role of mechanical stimuli, a bioreactor was developed to apply mechanical deformation force to a mesenchymal stem sell (MSC) line (telomerase reverse transcriptase (TERT)) expressing a strain-responsive AP-1 luciferase reporter construct on porous scaffolds. Increased luciferase expression was observed in the deformation strain compared with the shear stress strain. Furthermore, the expression of osteogenic lineage commitment markers such as osteonectin, osteocalcin (OC), osteopontin, runt-related transcription factor 2 (RUNX2), alkaline phosphate (AP), and collagen type 1 was significantly downregulated in the shear stress strain compared with the deformation strain. These findings establish that the deformation strain was the predominant stimulus causing skeletal precursors to undergo osteogenesis in earlier stages of osteogenic cell maturation. Finally, these findings were used to develop a bioreactor in vitro test system in which the effect of medication on osteoporosis could be tested. Primary human BM-MSCs from osteoporotic donors were subjected to strontium ranelate (an osteoporotic drug marketed as Protelos®). Increased expression of collagen type 1 and calcification was seen in the drugtreated osteoporotic stem cells compared with the nondrug-treated osteoporotic stem cells. Thus, this bioreactor technology can easily be adapted into an in vitro osteoporotic drug testing system.
The fine-tuning of glucose uptake mechanisms is rendered by various glucose transporters with distinct transportcharacteristics. In the pancreatic islet, facilitative diffusion glucose transporters (GLUTs), and sodium-glucosecotransporters (SGLTs) contribute to glucose uptake and represent important components in the glucose-stimulatedhormone release from endocrine cells, therefore playing a crucial role in blood glucose homeostasis. This reviewsummarizes the current knowledge aboutcell type-specific expression profiles as well as proven and putative functionsof distinct GLUT and SGLT family members in the human and rodent pancreatic islet and further discusses their possibleinvolvement in onset and progression ofdiabetes mellitus. In context of GLUTs, we focus on GLUT2, characterizing themain glucose transporter in insulin-secretingβ-cells in rodents. In addition, we discuss recent data proposing that otherGLUT family members, namely GLUT1 and GLUT3, render this task in humans. Finally, we summarize latest infor-mation about SGLT1 and SGLT2 as representatives of the SGLT family that have been reported to be expressed predominantly in the α-cell population with a suggested functional role in the regulation of glucagon release
High attrition-rates entailed by drug testing in 2D cell culture and animal models stress the need for improved modeling of human tumor tissues. In previous studies our 3D models on a decellularized tissue matrix have shown better predictivity and higher chemoresistance. A single porcine intestine yields material for 150 3D models of breast, lung, colorectal cancer (CRC) or leukemia. The uniquely preserved structure of the basement membrane enables physiological anchorage of endothelial cells and epithelial-derived carcinoma cells. The matrix provides different niches for cell growth: on top as monolayer, in crypts as aggregates and within deeper layers. Dynamic culture in bioreactors enhances cell growth. Comparing gene expression between 2D and 3D cultures, we observed changes related to proliferation, apoptosis and stemness. For drug target predictions, we utilize tumor-specific sequencing data in our in silico model finding an additive effect of metformin and gefitinib treatment for lung cancer in silico, validated in vitro. To analyze mode-of-action, immune therapies such as trispecific T-cell engagers in leukemia, as well as toxicity on non-cancer cells, the model can be modularly enriched with human endothelial cells (hECs), immune cells and fibroblasts. Upon addition of hECs, transmigration of immune cells through the endothelial barrier can be investigated. In an allogenic CRC model we observe a lower basic apoptosis rate after applying PBMCs in 3D compared to 2D, which offers new options to mirror antigen-specific immunotherapies in vitro. In conclusion, we present modular human 3D tumor models with tissue-like features for preclinical testing to reduce animal experiments.
3D printing is a rapidly evolving field for biological (bioprinting) and non-biological applications. Due to a high degree of freedom for geometrical parameters in 3D printing, prototype printing of bioreactors is a promising approach in the field of Tissue Engineering. The variety of printers, materials, printing parameters and device settings is difficult to overview both for beginners as well as for most professionals. In order to address this problem, we designed a guidance including test bodies to elucidate the real printing performance for a given printer system. Therefore, performance parameters such as accuracy or mechanical stability of the test bodies are systematically analysed. Moreover, post processing steps such as sterilisation or cleaning are considered in the test procedure. The guidance presented here is also applicable to optimise the printer settings for a given printer device. As proof of concept, we compared fused filament fabrication, stereolithography and selective laser sintering as the three most used printing methods. We determined fused filament fabrication printing as the most economical solution, while stereolithography is most accurate and features the highest surface quality. Finally, we tested the applicability of our guidance by identifying a printer solution to manufacture a complex bioreactor for a perfused tissue construct. Due to its design, the manufacture via subtractive mechanical methods would be 21-fold more expensive than additive manufacturing and therefore, would result in three times the number of parts to be assembled subsequently. Using this bioreactor we showed a successful 14-day-culture of a biofabricated collagen-based tissue construct containing human dermal fibroblasts as the stromal part and a perfusable central channel with human microvascular endothelial cells. Our study indicates how the full potential of biofabrication can be exploited, as most printed tissues exhibit individual shapes and require storage under physiological conditions, after the bioprinting process.
Objective
As native cartilage consists of different phenotypical zones, this study aims to fabricate different types of neocartilage constructs from collagen hydrogels and human mesenchymal stromal cells (MSCs) genetically modified to express different chondrogenic factors.
Design
Human MSCs derived from bone-marrow of osteoarthritis (OA) hips were genetically modified using adenoviral vectors encoding sex-determining region Y-type high-mobility-group-box (SOX)9,transforming growth factor beta (TGFB) 1or bone morphogenetic protein (BMP) 2cDNA, placed in type I collagen hydrogels and maintained in serum-free chondrogenic media for three weeks. Control constructs contained unmodified MSCs or MSCs expressing GFP. The respective constructs were analyzed histologically, immunohistochemically, biochemically, and by qRT-PCR for chondrogenesis and hypertrophy.
Results
Chondrogenesis in MSCs was consistently and strongly induced in collagen I hydrogels by the transgenesSOX9,TGFB1andBMP2as evidenced by positive staining for proteoglycans, chondroitin-4-sulfate (CS4) and collagen (COL) type II, increased levels of glycosaminoglycan (GAG) synthesis, and expression of mRNAs associated with chondrogenesis. The control groups were entirely non-chondrogenic. The levels of hypertrophy, as judged by expression of alkaline phosphatase (ALP) and COL X on both the protein and mRNA levels revealed different stages of hypertrophy within the chondrogenic groups (BMP2>TGFB1>SOX9).
Conclusions
Different types of neocartilage with varying levels of hypertrophy could be generated from human MSCs in collagen hydrogels by transfer of genes encoding the chondrogenic factorsSOX9,TGFB1andBMP2. This technology may be harnessed for regeneration of specific zones of native cartilage upon damage.
The investigation of the biodistribution profile of a cell-based medicinal product is a pivotal prerequisite to allow a factual benefit-risk assessment within the non-clinical to clinical translation in product development. Here, a qPCR-based method to determine the amount of human DNA in mouse DNA was validated according to the guidelines of the European Medicines Agency and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. Furthermore, a preclinical worst-case scenario study was performed in which this method was applied to investigate the biodistribution of 2 x 10\(^6\) intravenously administered, genetically modified, blood outgrowth endothelial cells from hemophilia A patients after 24 h and 7 days. The validation of the qPCR method demonstrated high accuracy, precision, and linearity for the concentration interval of 1:1 x 10\(^3\) to 1:1 x 10\(^6\) human to mouse DNA. The application of this method in the biodistribution study resulted in the detection of human genomes in four out of the eight investigated organs after 24 h. After 7 days, no human DNA was detected in the eight organs analyzed. This biodistribution study provides mandatory data on the toxicokinetic safety profile of an actual candidate cell-based medicinal product. The extensive evaluation of the required validation parameters confirms the applicability of the qPCR method for non-clinical biodistribution studies.
Due to the rapidly increasing development and use of cellular products, there is a rising demand for non-animal-based test platforms to predict, study and treat undesired immunity. Here, we generated human organotypic skin models from human biopsies by isolating and expanding keratinocytes, fibroblasts and microvascular endothelial cells and seeding these components on a collagen matrix or a biological vascularized scaffold matrix in a bioreactor. We then were able to induce inflammation-mediated tissue damage by adding pre-stimulated, mismatched allogeneic lymphocytes and/or inflammatory cytokine-containing supernatants histomorphologically mimicking severe graft versus host disease (GvHD) of the skin. This could be prevented by the addition of immunosuppressants to the models. Consequently, these models harbor a promising potential to serve as a test platform for the prediction, prevention and treatment of GvHD. They also allow functional studies of immune effectors and suppressors including but not limited to allodepleted lymphocytes, gamma-delta T cells, regulatory T cells and mesenchymal stromal cells, which would otherwise be limited to animal models. Thus, the current test platform, developed with the limitation that no professional antigen presenting cells are in place, could greatly reduce animal testing for investigation of novel immune therapies.
The Gram-negative Epsilonproteobacterium Campylobacter jejuni is currently the most prevalent bacterial foodborne pathogen. Like for many other human pathogens, infection studies with C. jejuni mainly employ artificial animal or cell culture models that can be limited in their ability to reflect the in-vivo environment within the human host. Here, we report the development and application of a human three-dimensional (3D) infection model based on tissue engineering to study host-pathogen interactions. Our intestinal 3D tissue model is built on a decellularized extracellular matrix scaffold, which is reseeded with human Caco-2 cells. Dynamic culture conditions enable the formation of a polarized mucosal epithelial barrier reminiscent of the 3D microarchitecture of the human small intestine. Infection with C. jejuni demonstrates that the 3D tissue model can reveal isolate-dependent colonization and barrier disruption phenotypes accompanied by perturbed localization of cell-cell junctions. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D model deviated from those obtained with 2D-monolayers, but recapitulated phenotypes previously observed in animal models. Moreover, we demonstrate the involvement of a small regulatory RNA pair, CJnc180/190, during infections and observe different phenotypes of CJnc180/190 mutant strains in 2D vs. 3D infection models. Hereby, the CJnc190 sRNA exerts its pathogenic influence, at least in part, via repression of PtmG, which is involved in flagellin modification. Our results suggest that the Caco-2 cell-based 3D tissue model is a valuable and biologically relevant tool between in-vitro and in-vivo infection models to study virulence of C. jejuni and other gastrointestinal pathogens.
A major obstacle in infection biology is the limited ability to recapitulate human disease trajectories in traditional cell culture and animal models, which impedes the translation of basic research into clinics. Here, we introduce a three-dimensional (3D) intestinal tissue model to study human enteric infections at a level of detail that is not achieved by conventional two-dimensional monocultures. Our model comprises epithelial and endothelial layers, a primary intestinal collagen scaffold, and immune cells. Upon Salmonella infection, the model mimics human gastroenteritis, in that it restricts the pathogen to the epithelial compartment, an advantage over existing mouse models. Application of dual transcriptome sequencing to the Salmonella-infected model revealed the communication of epithelial, endothelial, monocytic, and natural killer cells among each other and with the pathogen. Our results suggest that Salmonella uses its type III secretion systems to manipulate STAT3-dependent inflammatory responses locally in the epithelium without accompanying alterations in the endothelial compartment. Our approach promises to reveal further human-specific infection strategies employed by Salmonella and other pathogens.
IMPORTANCE Infection research routinely employs in vitro cell cultures or in vivo mouse models as surrogates of human hosts. Differences between murine and human immunity and the low level of complexity of traditional cell cultures, however, highlight the demand for alternative models that combine the in vivo-like properties of the human system with straightforward experimental perturbation. Here, we introduce a 3D tissue model comprising multiple cell types of the human intestinal barrier, a primary site of pathogen attack. During infection with the foodborne pathogen Salmonella enterica serovar Typhimurium, our model recapitulates human disease aspects, including pathogen restriction to the epithelial compartment, thereby deviating from the systemic infection in mice. Combination of our model with state-of-the-art genetics revealed Salmonella-mediated local manipulations of human immune responses, likely contributing to the establishment of the pathogen's infection niche. We propose the adoption of similar 3D tissue models to infection biology, to advance our understanding of molecular infection strategies employed by bacterial pathogens in their human host.
Bevor ein zellbasiertes GTMP erstmalig beim Menschen angewendet werden kann, müssen verschiedene notwendige nicht-klinische Studien durchgeführt werden. Wichtig ist hier u.a. die Untersuchung der Biodistribution im Tiermodel. Diese umfasst die Verteilung, das Engraftment, die Persistenz, die Eliminierung und gegebenenfalls die Expansion der humanen Zellen in verschiedenen Organen, meistens im Mausmodel. Deshalb wurde eine qPCR-basierte Analysenmethode entwickelt, mit der humane genomische DNA innerhalb von muriner genomischer DNA bestimmt werden kann, und entsprechend den regulatorischen Richtlinien der European Medicines Agency und des International Council for Harmonisation validiert. Anschließend wurde diese Methode innerhalb einer präklinischen worst-case Szenario Biodistributionsstudie angewendet. Das Ziel dieser Studie war die Untersuchung des Biodistributionsprofils von genetisch modifizierten Blood Outgrowth Endothelial Cells von Hämophilie A Patienten 24 Stunden und sieben Tage nach intravenöser Applikation einer Dosis von 2x106 Zellen. Die Isolation, genetische Modifikation und die Expansion der Zellen sollte entsprechend den Richtlinien der Guten Herstellungspraxis durchgeführt werden. Hierbei ist die Auswahl und Anwendung geeigneter und essentieller Rohstoffe wichtig. Gleichermaßen ist die Durchführung einer definierten Qualitätskontrollstrategie notwendig und die Patientenzellen sollten nur innerhalb von nicht-klinischen Studien eingesetzt werden, wenn alle Akzeptanzkriterien erfüllt wurden. Die Validierung der qPCR-Methode zeigte eine hohe Genauigkeit, Präzision und Linearität innerhalb des Konzentrationsintervalls von 1:1x103 bis 1:1x106 humanen zu murinen Genomen. Bei Anwendung dieser Methode für die Biodistributionsstudie konnten nach 24 Stunden humane Genome in vier der acht untersuchten Mausorgane bestimmt werden. Nach sieben Tagen konnten in keinem der acht Organe humane Genome nachgewiesen werden...
Development of predictable in vitro tumor models is a challenging task due to the enormous complexity of tumors in vivo. The closer the resemblance of these models to human tumor characteristics, the more suitable they are for drug-development and –testing. In the present study, we generated a complex 3D lung tumor test system based on acellular rat lungs. A decellularization protocol was established preserving the architecture, important ECM components and the basement membrane of the lung. Human lung tumor cells cultured on the scaffold formed cluster and exhibited an up-regulation of the carcinoma-associated marker mucin1 as well as a reduced proliferation rate compared to respective 2D culture. Additionally, employing functional imaging with 2-deoxy-2-[\(^{18}\)F]fluoro-D-glucose positron emission tomography (FDG-PET) these tumor cell cluster could be detected and tracked over time. This approach allowed monitoring of a targeted tyrosine kinase inhibitor treatment in the in vitro lung tumor model non-destructively. Surprisingly, FDG-PET assessment of single tumor cell cluster on the same scaffold exhibited differences in their response to therapy, indicating heterogeneity in the lung tumor model. In conclusion, our complex lung tumor test system features important characteristics of tumors and its microenvironment and allows monitoring of tumor growth and -metabolism in combination with functional imaging. In longitudinal studies, new therapeutic approaches and their long-term effects can be evaluated to adapt treatment regimes in future.
The human pathogen Bordetella pertussis targets the respiratory epithelium and causes whooping cough. Its virulence factor adenylate cyclase toxin (CyaA) plays an important role in the course of infection. Previous studies on the impact of CyaA on human epithelial cells have been carried out using cell lines derived from the airways or the intestinal tract. Here, we investigated the interaction of CyaA and its enzymatically inactive but fully pore-forming toxoid CyaA-AC– with primary human airway epithelial cells (hAEC) derived from different anatomical sites (nose and tracheo-bronchial region) in two-dimensional culture conditions. To assess possible differences between the response of primary hAEC and respiratory cell lines directly, we included HBEC3-KT in our studies. In comparative analyses, we studied the impact of both the toxin and the toxoid on cell viability, intracellular cAMP concentration and IL-6 secretion. We found that the selected hAEC, which lack CD11b, were differentially susceptible to both CyaA and CyaA-AC–. HBEC3-KT appeared not to be suitable for subsequent analyses. Since the nasal epithelium first gets in contact with airborne pathogens, we further studied the effect of CyaA and its toxoid on the innate immunity of three-dimensional tissue models of the human nasal mucosa. The present study reveals first insights in toxin–cell interaction using primary hAEC.
Activity of Tracheal Cytotoxin of Bordetella pertussis in a Human Tracheobronchial 3D Tissue Model
(2021)
Bordetella pertussis is a highly contagious pathogen which causes whooping cough in humans. A major pathophysiology of infection is the extrusion of ciliated cells and subsequent disruption of the respiratory mucosa. Tracheal cytotoxin (TCT) is the only virulence factor produced by B. pertussis that has been able to recapitulate this pathology in animal models. This pathophysiology is well characterized in a hamster tracheal model, but human data are lacking due to scarcity of donor material. We assessed the impact of TCT and lipopolysaccharide (LPS) on the functional integrity of the human airway mucosa by using in vitro airway mucosa models developed by co-culturing human tracheobronchial epithelial cells and human tracheobronchial fibroblasts on porcine small intestinal submucosa scaffold under airlift conditions. TCT and LPS either alone and in combination induced blebbing and necrosis of the ciliated epithelia. TCT and LPS induced loss of ciliated epithelial cells and hyper-mucus production which interfered with mucociliary clearance. In addition, the toxins had a disruptive effect on the tight junction organization, significantly reduced transepithelial electrical resistance and increased FITC-Dextran permeability after toxin incubation. In summary, the results indicate that TCT collaborates with LPS to induce the disruption of the human airway mucosa as reported for the hamster tracheal model.
Implants elicit an immunological response after implantation that results in the worst case in a complete implant rejection. This biomaterial-induced inflammation is modulated by macrophages and can be influenced by nanotopographical surface structures such as titania nanotubes or fractal titanium nitride (TiN) surfaces. However, their specific impact on a distinct macrophage phenotype has not been identified. By using two different levels of nanostructures and smooth samples as controls, the influence of tubular TiO2 and fractal TiN nanostructures on primary human macrophages with M1 or M2-phenotype was investigated. Therefore, nanotopographical coatings were either, directly generated by physical vapor deposition (PVD) or by electrochemical anodization of titanium PVD coatings. The cellular response of macrophages was quantitatively assessed to demonstrate a difference in biocompatibility of nanotubes in respect to human M1 and M2-macrophages. Depending on the tube diameter of the nanotubular surfaces, low cell numbers and impaired cellular activity, was detected for M2-macrophages, whereas the impact of nanotubes on M1-polarized macrophages was negligible. Importantly, we could confirm this phenotypic response on the fractal TiN surfaces. The results indicate that the investigated topographies specifically impact the macrophage M2-subtype that modulates the formation of the fibrotic capsule and the long-term response to an implant.
Bei der zystischen Fibrose (CF) sowie der primären Ziliendyskinesie (PCD) handelt es sich um zwei seltene Erkrankungen, die unter anderem den mukoziliären Transport beeinträchtigen. CF gehört hierbei zu den am häufigsten vorkommenden angeborenen Stoffwechselerkrankungen, wobei Betroffene unter einem Defekt des Cystic Fibrosis Transmembrane Conductor Regulator (CFTR)-Gens leiden, der durch die Produktion von hochviskosem Sekret in muzinproduzierenden Organen, wie dem gastrointestinalen Trakt und der Lunge, gekennzeichnet ist. Patienten, die an PCD leiden, weisen Defekte in, zum jetzigen Zeitpunkt, ca. 38 bekannten und PCD-assoziierten Genen auf, die in strukturellen Defekten des ziliären Apparats und somit in dysfunktionalen Kinozilien resultieren. Da aktuell weder für die CF noch für die PCD eine Heilung möglich ist, steht bei der Therapie vor allem die Linderung der Symptome im Fokus. Grundlegendes Ziel ist der langfristige Erhalt der Lungenfunktion sowie die Prävention bakterieller Infekte. Als bisherige Modellsysteme zur Erforschung möglicher Therapeutika gelten Tiermodelle, die den humanen Phänotyp aufgrund von Speziesdiversität nicht vollständig abbilden können. Als vielversprechende Testsysteme für die zystische Fibrose gelten humane intestinale Organoidkulturen. Nachdem allerdings vorwiegend respiratorische Symptome für die Mortalität der Patienten verantwortlich sind, stellen CF-Atemwegsmodelle bessere Testsysteme für zukünftige Therapeutika dar. Atmungsorganoidkulturen wurden verwendet, um die CFTR-Funktionalität zu untersuchen, repräsentieren aber nicht vollständig die in vivo Situation. Deshalb werden zur Entwicklung neuer Therapiestrategien patientenspezifische 3D in vitro Testsysteme der humanen Atemwege benötigt, die insbesondere im Hinblick auf personalisierte Medizin ihren Einsatz finden. In der vorliegenden Arbeit wurde eine für den Lehrstuhl neue Methode zur Zellgewinnung aus nasalen Schleimhautabstrichen etabliert, die eine standardisierte Versorgung mit humanem Primärmaterial garantiert. Zur Generierung einer krankheitsspezifischen Zelllinie, wie beispielsweise einer PCD-Zelllinie mit Hilfe des CRISPR/Cas9-Systems, ist eine Atemwegszelllinie erforderlich, die die in vivo Situation vollständig repräsentiert. So wurden vier verschiedene respiratorische Epithelzelllinien (HBEC3-KT, Calu-3, VA10 und Cl-huAEC) auf ihren mukoziliären Phänotyp hin untersucht, wobei lediglich die Zelllinie HBEC3-KT in zilientragende Zellen differenzierte. Diese zeigten jedoch nur auf ca. 5 % der Modelloberfläche Kinozilien, wodurch die humane respiratorische Mukosa nicht komplett abgebildet werden konnte und die HBEC3-KT-Zelllinie keine geeignete Zelllinie zur Generierung einer PCD-Zelllinie darstellte. Mit Hilfe des Tissue Engineering war es möglich, 3D in vitro Testsysteme basierend auf zwei unterschiedlichen Matrices, der biologischen SIS (small intestinal submucosa) und der synthetischen Polyethylenterephthalat (PET)-Membran, aufzubauen. Es wurden 3D Atemwegstestsysteme mit humanen primären nasalen und tracheobronchialen Epithelzellen generiert. Ergänzend zu histologischen Untersuchungen und zur Charakterisierung spezifischer Marker des respiratorischen Systems mittels Immunfluoreszenz, wurde die Ultrastruktur der Modelle, mit speziellem Fokus auf ziliäre Strukturen, analysiert. Um Rückschlüsse auf die ziliäre Funktionalität ziehen zu können und somit eine hohe in vivo Korrelation zu bestätigen, wurde im Rahmen dieser Arbeit am Lehrstuhl für Tissue Engineering und Regenerative Medizin die Methode der Hochgeschwindigkeitsvideomikroskopie etabliert, welche die Analyse der Zilienschlagfrequenz sowie des mukoziliären Transports ermöglicht. Ebenfalls wurde der Einfluss von isotoner Kochsalzlösung und des � 2-adrenergen Agonisten Salbutamol, das vor allem als Bronchodilatator bei Asthmapatienten eingesetzt wird, auf die Zilienschlagfrequenz analysiert. Es konnte gezeigt werden, dass beide Substanzen den Zilienschlag im Atemwegsmodell erhöhen. Zur Generierung der Testsysteme der beiden seltenen Erkrankungen CF und PCD wurden Epithelzellen der betroffenen Patienten zunächst mittels nicht-invasiver Raman-Spektroskopie auf einen potentiellen Biomarker untersucht, welcher Einsatz in der Diagnostik der beiden Krankheiten finden könnte. Es konnte jedoch weder für die CF noch für die PCD ein Biomarker aufgedeckt werden. Jedoch zeigten PCD-Zellen eine geringe Auftrennung gegenüber nicht-PCD Zellen. Anschließend wurden 3D-Atemwegstestsysteme basierend auf Patientenzellen aufgebaut. Der Phänotyp der CF-Modelle wurde mittels immunhistologischer Färbung und der Analyse des gestörten mukoziliären Transports verifiziert. Strukturelle ziliäre Defekte konnten durch die ultrastrukturelle Analyse von Zilienquerschnitten in drei donorspezifischen PCD-Modellen identifiziert werden. Darüber hinaus konnte die ziliäre Funktionalität mit Hilfe der Hochgeschwindigkeitsvideomikroskopie nicht nachgewiesen werden. Zusammenfassend ist es in dieser Arbeit gelungen, eine neue Methode zur vollständigen Charakterisierung von 3D-Atemwegstestsystemen zu etablieren, die die Analyse der Zilienschlagfrequenz sowie des mukoziliären Transports ermöglicht. Es konnte erstmalig gezeigt werden, dass mit Hilfe des Tissue Engineering ein personalisiertes Krankheitsmodell für die PCD auf Segmenten eines dezellularisierten porzinen Jejunums generiert werden kann, das zukünftig ein Testsystem für potentielle Therapeutika darstellen kann.
Surgical implantation of a biomaterial triggers foreign-body-induced fibrous encapsulation. Two major mechanisms of this complex physiological process are (I) chemotaxis of fibroblasts from surrounding tissue to the implant region, followed by (II) tissue remodeling. As an alternative to animal studies, we here propose a process-aligned \({in}\) \({vitro}\) test platform to investigate the material dependency of fibroblast chemotaxis and tissue remodeling mediated by material-resident macrophages.
Embedded in a biomimetic three-dimensional collagen hydrogel, chemotaxis of fibroblasts in the direction of macrophage-material-conditioned cell culture supernatant was analyzed by live cell imaging. A combination of statistical analysis with a complementary parameterized random walk model allowed quantitative and qualitative characterization of the cellular walk process. We thereby identified an increasing macrophage-mediated chemotactic potential ranking of biomaterials from glass over polytetrafluorethylene to titanium. To address long-term effects of biomaterial-resident macrophages on fibroblasts in a three-dimensional microenvironment, we further studied tissue remodeling by applying macrophage-material-conditioned medium on fibrous \({in}\) \({vitro}\) tissue models. A high correlation of the \({in}\) \({vitro}\) tissue model to state of the art \({in}\) \({vivo}\) study data was found. Titanium exhibited a significantly lower tissue remodeling capacity compared to polytetrafluorethylene. With this approach, we identified a material dependency of both chemotaxis and tissue remodeling processes, strengthening knowledge on their specific contribution to the foreign body reaction.
Gegenstand dieser Arbeit war die Etablierung eines dreidimensionalen in vitro Tumormodells, welches ein orales in vivo Plattenepithelkarzinom nachbilden sollte. Dabei standen Aufbau, Reproduzierbarkeit und Reliabilität an vorderster Stelle. Als Zellquelle sollten sowohl Tumorzellen aus den Zelllinien FaDu, HLaC79 und HLaC79 Clone 1 als auch primäre Zellen aus karzinogenem Primärgewebe dienen. Als Referenz wurden dabei stets Modelle aus primär isolierten Zellen herangezogen, die ein Äquivalent zur gesunden Mundschleimhaut bildeten. Während der Isolationsvorgang von pathologischen Zellen primärer Plattenepithelkarzinomen aus der Mundhöhle und dem Pharynx aufgrund zahlreicher Kontaminationen und Stagnationen des Zellwachstums keinen Erfolg erzielte und der Versuch eingestellt wurde, war es mit den Tumorzelllinien FaDu und HLaC79 möglich, dreidimensionale in vitro Tumormodelle herzustellen. Ihre Malignität wurde durch die besonderen histologischen Architekturstörungen wie die geringere Epitheldicke, das Fehlen einer Parakeratinisierung im Stratum corneum und die Invasion von Tumorzellen in die Submukosa verdeutlicht. Um einen eindeutigen Vergleich zu den Mukosaäquivalenten zu ziehen, fand eine Immunhistochemie mit unterschiedlichen Markern statt, die vor allem den gestörten Epithelaufbau des Tumormodells verdeutlichte. Als Maß für die Zell-Zell-Kontakte, die im Laufe der Kultivierung entstanden, diente der transepitheliale elektrische Widerstand. Die Behandlung der Tumorzellen und Tumormodelle mit dem klinisch bewährten Zytostatikum Paclitaxel und dem neuen Polyether-Antibiotikum Salinomycin erzielte vor allem in der zweidimensionalen Kultivierung große Erfolge. Hier wurde verdeutlicht, dass Paclitaxel toxisch auf die HLaC79 Tumorzellen wirkt, während die paclitaxelresistenten HLaC79 Clone 1 Tumorzellen immun gegen dieses Medikament sind. Salinomycin hingegen sorgte für eine Verringerung der Zellviabilität bei beiden Zelllinien. Die histologischen Untersuchungen nach der 24-stündigen Medikamentenapplikation mit Paclitaxel bei den Tumormodellen zeigten keine signifikanten Unterschiede, während der transepitheliale elektrische Widerstand stieg und auf eine verstärkte Barriere nach Paclitaxelgabe schließen ließ.
Bisherige per Tissue Engineering hergestellte Testsysteme der Mundschleimhaut basieren in der Regel auf allogenen und teils dysplastischen Keratinozyten. Dies schmälert die Aussagekraft der gewonnenen Ergebnisse hinsichtlich des Anspruchs, Nativgewebe bestmöglich nachzubilden.
In der vorliegenden Arbeit sollte daher ein am Lehrstuhl für Tissue Engineering und Regenerative Medizin entwickeltes Protokoll zur Herstellung dreidimensionaler epidermaler Oralmukosaäquivalente auf Basis autologer Keratinozyten auf seine Eigenschaften und Einsatzmöglichkeit als in-vitro Testsystem untersucht werden.
Nach erfolgreicher Isolierung und Kultivierung im Monolayer konnten insgesamt 420 Modelle zu drei verschiedenen Zeitpunkten (Passagen) aufgebaut werden. Die Untersuchung von Histologie, Viabilität und Barrierefunktion mittels MTT, TEER und Natriumfluoresceinpermeabilität konnte einen suffizienten Aufbau von verhorntem, mehrschichtigen oralen Plattenepithel nachweisen. Gleichzeitig konnte eine Abnahme der Epithelqualität mit steigendem Keratinozytenalter festgestellt werden.
Eine sich anschließende Untersuchung von 14 Cytokeratinen sowie Apoptosemarkern per effizienzkorrigierter und normalisierter RT-qPCR konnte die Überlegenheit der dreidimensionalen autologen Oralmukosaäquivalente gegenüber der zweidimensionalen Monolayerkultur auf Genebene zeigen.
The aim is to evaluate the effect of modifying poly[(L-lactide)-co-(epsilon-caprolactone)] scaffolds (PLCL) with nanodiamonds (nDP) or with nDP+physisorbed BMP-2 (nDP+BMP-2) on in vivo host tissue response and degradation. The scaffolds are implanted subcutaneously in Balb/c mice and retrieved after 1, 8, and 27 weeks. Molecular weight analysis shows that modified scaffolds degrade faster than the unmodified. Gene analysis at week 1 shows highest expression of proinflammatory markers around nDP scaffolds; although the presence of inflammatory cells and foreign body giant cells is more prominent around the PLCL. Tissue regeneration markers are highly expressed in the nDP+BMP-2 scaffolds at week 8. A fibrous capsule is detectable by week 8, thinnest around nDP scaffolds and at week 27 thickest around PLCL scaffolds. mRNA levels of ALP, COL1 alpha 2, and ANGPT1 are signifi cantly upregulating in the nDP+BMP-2 scaffolds at week 1 with ectopic bone seen at week 8. Even when almost 90% of the scaffold is degraded at week 27, nDP are observable at implantation areas without adverse effects. In conclusion, modifying PLCL scaffolds with nDP does not aggravate the host response and physisorbed BMP-2 delivery attenuates infl ammation while lowering the dose of BMP-2 to a relatively safe and economical level.
Die Stammzellforschung beschäftigt sich bereits seit Jahren mit der Frage, wie Gewebe oder sogar Organe im Labor hergestellt werden können. Als besonders vielversprechend erscheinen hierfür humane Mesenchymale Stammzellen (hMSC), da diese in vielen Fällen direkt vom Empfänger gewonnen werden können und so keine Organ- oder Gewebeabstoßung durch Abwehrreaktionen zu erwarten ist. Für die weitere Erforschung des Verhaltens von Stammzellen in vivo ist es notwendig, diese nicht-invasiv darstellen zu können. Dies ist zum Beispiel mittels Magnetischer Partikel Bildgebung (MPI) möglich. Hierfür müssen die Stammzellen mit einer geeigneten Substanz markiert werden. Eine solche sind beispielsweise superparamagnetische Eisenoxidnanopartikel (SPION). Derzeit gibt es keine von den medizinischen Zulassungsbehörden zugelassenen SPION die ohne TA in hMSC aufgenommen werden. In der hier vorliegenden Arbeit sollte also im Rahmen des
EU-weiten „IDEA-Projekts“ ein geeigneter SPION identifiziert werden, der eine optimale Zell-Partikel-Interaktion aufweist und sowohl mittels MPI als auch mit Raman-Spektroskopie nachweisbar ist. Zudem sollte die Nachweisbarkeit des SPION über einen längeren Zeitraum gegeben und kein Einfluss auf die hMSC feststellbar sein. Es wurden hMSC mit den Eisenoxidnanopartikeln M4E, M4F, M4F2 und M3A-PDL in unterschiedlichen Konzentrationen markiert. Für M3A-PDL und M4E erfolgten bei einer Konzentration von 0,5 mg/ml Untersuchungen in Zellkultur sowie auf SIS-ser als Matrix im 3D-Modell. Desweiteren wurde das Differenzierungsverhalten der mit M4E markierten hMSC bei chondrogener Differenzierung untersucht. Außerdem kamen Magnetische Partikel Spektroskopie (MPS) und Raman-Spektroskopie als nicht-invasive Nachweisverfahren zum Einsatz. Der SPION-Nachweis erfolgte histologisch mittels Berliner Blau Färbung. Untersuchungen zu Zellviabilität und Proliferation erfolgten durch Trypanblau sowie Ki67-Antikörper-Färbung. Um Nachzuweisen ob auch markierte Zellen proliferieren wurde eigens ein kombiniertes Färbeprotokoll zur Kombination von Berliner Blau und immunhistochemischer Färbung
etabliert. Der Erfolg der chrondrogenen Differenzierung wurde mittels Alcianblau, Aggrecan- und Kollagen-II-Antikörper Färbung überprüft. Es konnte gezeigt werden, dass M4E bei der Markierung von hMSC eine sehr gute Zell-Partikel-Interaktion aufweist und im Gegensatz zu M3A auch ohne TA in die Zellen aufgenommen wird. Durch beide Partikel werden Zellviabilität und Proliferation nicht beeinflusst. M4F sowie M4F2 ist zur Markierung nicht geeignet. Die Markierung ließ sich im 3D-Modell mit vier Wochen deutlich länger nachweisen als in 2D Zellkultur mit maximal zwei Wochen. Die chondrogene Differenzierung wird durch die Markierung mit 0,5 mg/ml M4E beeinflusst. M3A-PDL sind durch MPS nachweisbar. Die Raman-Spektroskopie eignet sich zur Differenzierung zwischen mit M3A-PDL markierten und unmarkierten hMSC. Es ist im Rahmen dieser Arbeit gelungen, einen
Eisenoxidnanopartikel mit hervorragender Zell-Partikel-Interaktion zu identifizieren, der ohne zusätzliches TA eine intensive Markierung der hMSC ermöglicht und mit MPS nachweisbar ist. Für M4E konnte in weiteren Arbeiten am Institut bereits gezeigt werden, dass auch eine Differenzierung zwischen markierten und unmarkierten Zellen mittels Raman-Spektroskopie möglich ist. Die chondrogene Differenzierung der hMSC wurde in der vorliegenden Arbeit allerdings beeinträchtigt. In der Literatur finden sich Hinweise auf eine dosisabhängige Inhibition der Differenzierung. Es sind daher weitere Versuche notwendig, um herauszufinden, ob die Inhibition der Differenzierung möglicherweise bei geringerer SPION-Konzentration weniger ausgeprägt ist. Zudem sollte untersucht werden, ob auch geringere Konzentrationen in den Zellen über mehrere Wochen mittels MPS nachweisbar bleiben. Desweiteren sollten Untersuchungen in, der in vivo Situation ähnlicheren,
Systemen, wie dem dynamischen Umfeld einer BioVaSc-TERM® durchgeführt werden um bessere Vorhersagen zum Verhalten markierter hMSC in vivo treffen zu können.
Feasibility Study on a Microwave-Based Sensor for Measuring Hydration Level Using Human Skin Models
(2016)
Tissue dehydration results in three major types of exsiccosis—hyper-, hypo-, or isonatraemia. All three types entail alterations of salt concentrations leading to impaired biochemical processes, and can finally cause severe morbidity. The aim of our study was to demonstrate the feasibility of a microwave-based sensor technology for the non-invasive measurement of the hydration status. Electromagnetic waves at high frequencies interact with molecules, especially water. Hence, if a sample contains free water molecules, this can be detected in a reflected microwave signal. To develop the sensor system, human three-dimensional skin equivalents were instituted as a standardized test platform mimicking reproducible exsiccosis scenarios. Therefore, skin equivalents with a specific hydration and density of matrix components were generated and microwave measurements were performed. Hydration-specific spectra allowed deriving the hydration state of the skin models. A further advantage of the skin equivalents was the characterization of the impact of distinct skin components on the measured signals to investigate mechanisms of signal generation. The results demonstrate the feasibility of a non-invasive microwave-based hydration sensor technology. The sensor bears potential to be integrated in a wearable medical device for personal health monitoring.
Induction of ectopic bone formation by site directed immobilized BMP2 variants \(in\) \(vivo\)
(2020)
In contrast to common bone fractures, critical size bone defects are unable to self-regenerate and therefore external sources for bone replacement are needed. Currently, the gold standard to treat critical size bone fractures, resulting from diseases, trauma or surgical interventions, is the use of autologous bone transplantation that is associated with several drawbacks such as postoperative pain, increased loss of blood during surgery and extended operative time.
The field of bone tissue engineering focuses on the combination of biomaterials and growth factors to circumvent these adverse events and thereby to improve critical size bone defects treatment.
To this aim, a promising approach is represented by using a collagen sponge soaked with one of the most powerful osteoinductive proteins, the bone morphogenetic protein 2 (BMP2). After the approval by the Food and Drug Administration (FDA), BMP2 was used to successfully treat several severe bone defects. However, the use of BMP2 delivery systems is associated with severe side effects such as inflammation, swelling, ectopic bone formation outside of the site of implantation and breathing problems if implanted in the area of the cervical spine. The occurrence of severe side effects is related to the supraphysiological amounts of the applied protein at the implantation site. The BMP2 is typically adsorbed into the scaffold and diffuses rapidly after implantation. Therefore, intensive research has been conducted to improve the protein’s retention ability, since a prolonged entrapment of the BMP2 at the implantation site would induce superior bone formation in vivo due to a minimized protein release. By controlling the release from newly designed materials or changing the protein immobilization methods, it seems possible to improve the osteoinductive properties of the resulting BMP2-functionalized scaffolds.
The combination of biocompatible and biodegradable scaffolds functionalized with a covalently immobilized protein such as BMP2 would constitute a new alternative in bone tissue engineering by eliminating the aforementioned severe side effects. One of the most common immobilization techniques is represented by the so-called EDC/NHS chemistry. This coupling technique allows covalent biding of the growth factor but in a non-site direct manner, thus producing an implant with uncontrollable and unpredictable osteogenic activities. Therefore, the generation of BMP2 variants harboring functional groups that allow a site-directed immobilization to the scaffold, would enable the production of implants with reproducible osteogenic activity.
The new BMP2 variants harbor an artificial amino acid at a specific position of the mature polypeptide sequence. The presence of the unnatural amino acid allows to use particular covalent immobilization techniques in a highly specific and site directed manner. The two selected BMP2 variants, BMP2 E83Plk and BMP2 E83Azide, were expressed in E. coli, renatured and purified by cation exchange chromatography. The final products were intensively analyzed in terms of purity and biological activity in vitro. The two BMP2 variants enabled the application of different coupling techniques and verify the possible options for site directed immobilization to the scaffold.
Intensive analyses on the possible side effects caused by the coupling reactions and on the quantification of the coupled protein were performed. Both click chemistry reactions showed high reaction efficacies when the BMP2 variants were coupled to functionalized fluorophores. Quantification by ELISA and scintillation counting of radioactively labeled protein revealed different outcomes. Moreover, the amounts of protein detected for the BMP2 variants coupled to microspheres were similar to that of the wild type protein. Therefore, it was not possible to conclude whether the BMP2 variants were covalently coupled or just adsorbed.
BMP2 variants being immobilized to various microspheres induced osteogenic differentiation of C2C12 cells in vitro, but only in those cells that were located in close proximity to the functionalized beads. This selectivity strongly indicates that the protein is for a great portion covalently coupled and not just adsorbed. Moreover, the difference between the covalently coupled BMP2 variants and the adsorbed BMP2 WT was confirmed in vivo. Injection of the BMP2-functionalized microspheres in a rat model induced subcutaneous bone formation.
The main aim of the animal experiment was to prove whether covalently coupled BMP2 induces bone formation at significant lower doses if compared to the amount being required if the protein is simply adsorbed. To this aim, several BMP2 concentrations were tested in this animal experiment. The BMP2 variants, being covalently immobilized, were hypothesized to be retained and therefore bio-available at the site of implantation for a prolonged time. However, in the animal experiments, lower doses of either coupled or adsorbed protein were unable to induce any bone formation within the 12 weeks.
In contrast, the highest doses induced bone formation that was first detected at week 4. During the 12 weeks of the experiment, an increase in bone density and a steady state bone volume was observed. These results were obtained only for the covalently coupled BMP2 E83Azide but not for BMP2 E83Plk that did not induce bone formation in any condition. The negative outcome after application of BMP2 E83Plk suggested that the coupling reaction might have provoked changes in the protein structure that extremely influenced its osteogenic capabilities in vivo.
However, the histological examination of the different ossicles induced either by BMP2 WT or BMP2 E83Azide, revealed clear morphological differences. BMP2 WT induced a bone shell-like structure, while the covalently coupled protein induced uniform bone formation also throughout the inner part. The differences between the two newly formed bones can be clearly associated with the different protein delivery mechanisms. Thus, the developed functionalized microspheres constitute a new interesting strategy that needs further investigations in order to be able to be used as replacement of the currently used BMP2 WT loaded medical devices.
Despite advancements of modern medicine, the number of patients with the the end-stage kidney disease keeps growing, and surgical procedures to establish and maintain a vascular access for hemodialysis are rising accordingly. Surgical access of choice remains autogenous arteriovenous fistula, whereas approach “fistula first at all costs” leads to failure in certain subgroups of patients. Modern synthetic vascular grafts fail to deliver long-term results comparable with AV fistula. With all that in mind, this work has an aim of developing a new alternative vascular graft, which can be used for hemodialysis access using the methods of TE, especially electrospinning technique. It is hypothesized that electrospun scaffold, made of PCL and collagen type I may assemble mechanical properties similar to native blood vessels. Seeding such electrospun scaffolds with human microvascular endothelial cells (hmvECs) and preconditioning with shear stress and continuous flow might achieve sufficient endothelial lining being able to resist acute thrombosis. One further topic considered on-site infections, which represents one of the most spread complications of dialysis therapy due to continuous needle punctures. The main hypothesis was that during electrospinning process, polymers can be blended with antibiotics with the aim of producing scaffolds with antimicrobial properties, which could lead to reducing the risk of on-site infection on one side, while not affecting the cell viability.
Tumor models based on cancer cell lines cultured two-dimensionally (2D) on plastic lack histological complexity and functionality compared to the native microenvironment. Xenogenic mouse tumor models display higher complexity but often do not predict human drug responses accurately due to species-specific differences. We present here a three-dimensional (3D) in vitro colon cancer model based on a biological scaffold derived from decellularized porcine jejunum (small intestine submucosa+mucosa, SISmuc). Two different cell lines were used in monoculture or in coculture with primary fibroblasts. After 14 days of culture, we demonstrated a close contact of human Caco2 colon cancer cells with the preserved basement membrane on an ultrastructural level as well as morphological characteristics of a well-differentiated epithelium. To generate a tissue-engineered tumor model, we chose human SW480 colon cancer cells, a reportedly malignant cell line. Malignant characteristics were confirmed in 2D cell culture: SW480 cells showed higher vimentin and lower E-cadherin expression than Caco2 cells. In contrast to Caco2, SW480 cells displayed cancerous characteristics such as delocalized E-cadherin and nuclear location of beta-catenin in a subset of cells. One central drawback of 2D cultures-especially in consideration of drug testing-is their artificially high proliferation. In our 3D tissue-engineered tumor model, both cell lines showed decreased numbers of proliferating cells, thus correlating more precisely with observations of primary colon cancer in all stages (UICC I-IV). Moreover, vimentin decreased in SW480 colon cancer cells, indicating a mesenchymal to epithelial transition process, attributed to metastasis formation. Only SW480 cells cocultured with fibroblasts induced the formation of tumor-like aggregates surrounded by fibroblasts, whereas in Caco2 cocultures, a separate Caco2 cell layer was formed separated from the fibroblast compartment beneath. To foster tissue generation, a bioreactor was constructed for dynamic culture approaches. This induced a close tissue-like association of cultured tumor cells with fibroblasts reflecting tumor biopsies. Therapy with 5-fluorouracil (5-FU) was effective only in 3D coculture. In conclusion, our 3D tumor model reflects human tissue-related tumor characteristics, including lower tumor cell proliferation. It is now available for drug testing in metastatic context-especially for substances targeting tumor-stroma interactions.
Over the last 20 years, there has been increasing focus on the development of novel stem cell based therapies for the treatment of disorders and diseases affecting the enteric nervous system (ENS) of the gastrointestinal tract (so-called enteric neuropathies). Here, the idea is that ENS progenitor/stem cells could be transplanted into the gut wall to replace the damaged or absent neurons and glia of the ENS. This White Paper sets out experts' views on the commonly used methods and approaches to identify, isolate, purify, expand and optimize ENS stem cells, transplant them into the bowel, and assess transplant success, including restoration of gut function. We also highlight obstacles that must be overcome in order to progress from successful preclinical studies in animal models to ENS stem cell therapies in the clinic.
To replace the Draize skin irritation assay (OECD guideline 404) several test methods based on reconstructed human epidermis (RHE) have been developed and were adopted in the OECD test guideline 439. However, all validated test methods in the guideline are linked to RHE provided by only three companies. Thus,the availability of these test models is dependent on the commercial interest of the producer. To overcome this limitation and thus to increase the accessibility of in vitro skin irritation testing, an open source reconstructed epidermis (OS-REp) was introduced. To demonstrate the capacity of the OS-REp in regulatory risk assessment, a catch-up-validation study was performed. The participating laboratories used in-house generated OS-REp to assess the set of 20 reference substances according to the performance standards amending the OECD test guideline 439. Testing was performed under blinded conditions. The within-laboratory reproducibility of 87% and the inter-laboratory reproducibility of 85% prove a high reliability of irritancy testing using the OS-REp protocol. In addition, the prediction capacity was with an accuracy of 80% comparable to previous published RHE based test protocols. Taken together the results indicate that the OS-REp test method can be used as a standalone alternative skin irritation test replacing the OECD test guideline 404.
This study aimed to evaluate the tumorigenic potential of functionalising poly(LLA-co-CL) scaffolds. The copolymer scaffolds were functionalised with nanodiamonds (nDP) or with nDP and physisorbed BMP-2 (nDP-PHY) to enhance osteoinductivity. Culturing early neoplastic dysplastic keratinocytes (DOK\(^{Luc}\)) on nDP modified scaffolds reduced significantly their subsequent sphere formation ability and decreased significantly the cells' proliferation in the supra-basal layers of in vitro 3D oral neoplastic mucosa (3D-OT) when compared to DOK\(^{Luc}\) previously cultured on nDP-PHY scaffolds. Using an in vivo non-invasive environmentally-induced oral carcinogenesis model, nDP scaffolds were observed to reduce bioluminescence intensity of tumours formed by DOK\(^{Luc}\) + carcinoma associated fibroblasts (CAF). nDP modification was also found to promote differentiation of DOK\(^{Luc}\) both in vitro in 3D-OT and in vivo in xenografts formed by DOKLuc alone. The nDP-PHY scaffold had the highest number of invasive tumours formed by DOK\(^{Luc}\) + CAF outside the scaffold area compared to the nDP and control scaffolds. In conclusion, in vitro and in vivo results presented here demonstrate that nDP modified copolymer scaffolds are able to decrease the tumorigenic potential of DOK\(^{Luc}\), while confirming concerns for the therapeutic use of BMP-2 for reconstruction of bone defects in oral cancer patients due to its tumour promoting capabilities.
Als die häufigste tödliche Tumorerkrankung weltweit ist das Lungenkarzinom mit einer sehr schlechten Prognose verbunden. Eine Behandlungsoption für Lungenadenokarzinome, die eine aktivierende EGFR-Mutation aufweisen, ist der orale EGFR-TKI Gefitinib (Iressa®, ZD1839). Die Resistenzentwicklung von Tumoren gegen diese Therapie stellt ein großes klinisches Problem dar.
Das Ziel dieser Arbeit war es, die Dosis-Wirkungs-Beziehung von Gefitinib, sowie die Entwicklung von Resistenzen in einem etablierten humanen 3D Lungentumormodell zu untersuchen und dieses Testsystem für eben diese Fragestellungen zu validieren.
Vorliegende Arbeit bestätigt, dass pharmakologische Untersuchungen in Zellkulturen häufig zu einer Überschätzung des Behandlungserfolges führen. Das verwendete Modell entspricht mehr den in vivo Bedingungen. In dieser Arbeit wurden zwei ATP-Zellvitalitätsassays für die statischen 3D Lungentumormodelle etabliert und erfolgreich angewendet. Dabei zeigte sich eine konzentrationsabhängige Wirkung von Gefitinib auf das Wachstum, die Proliferation, die Apoptose, die Markerexpression sowie die Signalwegsaktivierungen. Im statischen 3D Lungentumormodell lag der IC50-Wert zwischen 0,05-0,1 µM Gefitinib welches den Werten aus klinischen Beobachtungen entspricht. Auch der in der Klinik bereits nach wenigen Stunden eintretende zeitliche Effekt von Gefitinib konnte in unserem Modell bestätigt werden. Eine dynamische Kultivierung der Lungentumorzellen, mit von Scherkräften verursachtem schnellerem Zellwachstum, führte zu einer weiteren Annährung an die klinischen Gegebenheiten. Das Netzwerk der Gefitinib-Wirkung auf die EGFR-Signalkaskade wurde in unserem Modell charakterisiert.
Die Betrachtung einer resistenten Zell-Subpopulation zeigte einen Resistenzmechanismus über eine Epitheliale-Mesenchymale-Transition. Zusätzlich wurde versucht, eine neue medikamenten-resistente Zell-Subpopulation zu generieren.
Das beschriebene 3D Lungentumormodell ermöglicht richtungsweisende Untersuchungen zu Dosis-Wirkungs-Beziehung von Gefitinib. Ansätze für eine weitere Optimierung des Modells wurden herausgearbeitet.
Automated real-time monitoring of human pluripotent stem cell aggregation in stirred tank reactors
(2019)
The culture of human induced pluripotent stem cells (hiPSCs) at large scale becomes feasible with the aid of scalable suspension setups in continuously stirred tank reactors (CSTRs). Innovative monitoring options and emerging automated process control strategies allow for the necessary highly defined culture conditions. Next to standard process characteristics such as oxygen consumption, pH, and metabolite turnover, a reproducible and steady formation of hiPSC aggregates is vital for process scalability. In this regard, we developed a hiPSC-specific suspension culture unit consisting of a fully monitored CSTR system integrated into a custom-designed and fully automated incubator. As a step towards cost-effective hiPSC suspension culture and to pave the way for flexibility at a large scale, we constructed and utilized tailored miniature CSTRs that are largely made from three-dimensional (3D) printed polylactic acid (PLA) filament, which is a low-cost material used in fused deposition modelling. Further, the monitoring tool for hiPSC suspension cultures utilizes in situ microscopic imaging to visualize hiPSC aggregation in real-time to a statistically significant degree while omitting the need for time-intensive sampling. Suitability of our culture unit, especially concerning the developed hiPSC-specific CSTR system, was proven by demonstrating pluripotency of CSTR-cultured hiPSCs at RNA (including PluriTest) and protein level.
Spin echo based cardiac diffusion imaging at 7T: An ex vivo study of the porcine heart at 7T and 3T
(2019)
Purpose of this work was to assess feasibility of cardiac diffusion tensor imaging (cDTI) at 7 T in a set of healthy, unfixed, porcine hearts using various parallel imaging acceleration factors and to compare SNR and derived cDTI metrics to a reference measured at 3 T. Magnetic resonance imaging was performed on 7T and 3T whole body systems using a spin echo diffusion encoding sequence with echo planar imaging readout. Five reference (b = 0 s/mm\(^2\)) images and 30 diffusion directions (b = 700 s/mm\(^2\)) were acquired at both 7 T and 3 T using a GRAPPA acceleration factor R = 1. Scans at 7 T were repeated using R = 2, R = 3, and R = 4. SNR evaluation was based on 30 reference (b = 0 s/mm\(^2\)) images of 30 slices of the left ventricle and cardiac DTI metrics were compared within AHA segmentation. The number of hearts scanned at 7 T and 3 T was n = 11. No statistically significant differences were found for evaluated helix angle, secondary eigenvector angle, fractional anisotropy and apparent diffusion coefficient at the different field strengths, given sufficiently high SNR and geometrically undistorted images. R≥3 was needed to reduce susceptibility induced geometric distortions to an acceptable amount. On average SNR in myocardium of the left ventricle was increased from 29±3 to 44±6 in the reference image (b = 0 s/mm\(^2\)) when switching from 3 T to 7 T. Our study demonstrates that high resolution, ex vivo cDTI is feasible at 7 T using commercial hardware.
Transmission of measles virus (MV) from dendritic to airway epithelial cells is considered as crucial to viral spread late in infection. Therefore, pathways and effectors governing this process are promising targets for intervention. To identify these, we established a 3D respiratory tract model where MV transmission by infected dendritic cells (DCs) relied on the presence of nectin-4 on H358 lung epithelial cells. Access to recipient cells is an important prerequisite for transmission, and we therefore analyzed migration of MV-exposed DC cultures within the model. Surprisingly, enhanced motility toward the epithelial layer was observed for MV-infected DCs as compared to their uninfected siblings. This occurred independently of factors released from H358 cells indicating that MV infection triggered cytoskeletal remodeling associated with DC polarization enforced velocity. Accordingly, the latter was also observed for MV-infected DCs in collagen matrices and was particularly sensitive to ROCK inhibition indicating infected DCs preferentially employed the amoeboid migration mode. This was also implicated by loss of podosomes and reduced filopodial activity both of which were retained in MV-exposed uninfected DCs. Evidently, sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) as produced in response to virus-infection in DCs contributed to enhanced velocity because this was abrogated upon inhibition of sphingosine kinase activity. These findings indicate that MV infection promotes a push-and-squeeze fast amoeboid migration mode via the SphK/S1P system characterized by loss of filopodia and podosome dissolution. Consequently, this enables rapid trafficking of virus toward epithelial cells during viral exit.
Objective
To establish individually expandable primary fibroblast and keratinocyte cultures from 3‐mm skin punch biopsies for patient‐derived in vitro skin models to investigate of small fiber pathology.
Methods
We obtained 6‐mm skin punch biopsies from the calf of two patients with small fiber neuropathy (SFN) and two healthy controls. One half (3 mm) was used for diagnostic intraepidermal nerve fiber density (IENFD). From the second half, we isolated and cultured fibroblasts and keratinocytes. Cells were used to generate patient‐derived full‐thickness three‐dimensional (3D) skin models containing a dermal and epidermal component. Cells and skin models were characterized morphologically, immunocyto‐ and ‐histochemically (vimentin, cytokeratin (CK)‐10, CK 14, ki67, collagen1, and procollagen), and by electrical impedance.
Results
Distal IENFD was reduced in the SFN patients (2 fibers/mm each), while IENFD was normal in the controls (8 fibers/mm, 7 fibers/mm). Two‐dimensional (2D) cultured skin cells showed normal morphology, adequate viability, and proliferation, and expressed cell‐specific markers without relevant difference between SFN patient and healthy control. Using 2D cultured fibroblasts and keratinocytes, we obtained subject‐derived 3D skin models. Morphology of the 3D model was analogous to the respective skin biopsy specimens. Both, the dermal and the epidermal layer carried cell‐specific markers and showed a homogenous expression of extracellular matrix proteins.
Interpretation
Our protocol allows the generation of disease‐specific 2D and 3D skin models, which can be used to investigate the cross‐talk between skin cells and sensory neurons in small fiber pathology.
Meningococcal meningitis is a severe central nervous system infection that occurs when Neisseria meningitidis (Nm) penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, in vivo models are greatly limited and pose a significant challenge. In vitro cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific Nm interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study Nm host-pathogen interactions, and provide an overview of host responses to Nm infection. Using iPSC-BECs, we first confirmed that multiple Nm strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, and the secretion of IFN-γ and RANTES. For the first time, we directly observe that Nm disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon Nm challenge. In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of Nm-responsive host genes. Altogether, this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to Nm traversal of BECs.
Bone Morphogenetic Proteins (BMPs) together with the Growth and Differentiation Factors (GDFs) form the largest subgroup of the Transforming Growth Factor (TGF)β family and represent secreted growth factors, which play an essential role in many aspects of cell communication in higher organisms. As morphogens they exert crucial functions during embryonal development, but are also involved in tissue homeostasis and regeneration in the adult organism. Their involvement in maintenance and repair processes of various tissues and organs made these growth factors highly interesting targets for novel pharmaceutical applications in regenerative medicine. A hallmark of the TGFβ protein family is that all of the more than 30 growth factors identified to date signal by binding and hetero-oligomerization of a very limited set of transmembrane serine-threonine kinase receptors, which can be classified into two subgroups termed type I and type II. Only seven type I and five type II receptors exist for all 30plus TGFβ members suggesting a pronounced ligand-receptor promiscuity. Indeed, many TGFβ ligands can bind the same type I or type II receptor and a particular receptor of either subtype can usually interact with and bind various TGFβ ligands. The possible consequence of this ligand-receptor promiscuity is further aggravated by the finding that canonical TGFβ signaling of all family members seemingly results in the activation of just two distinct signaling pathways, that is either SMAD2/3 or SMAD1/5/8 activation. While this would implicate that different ligands can assemble seemingly identical receptor complexes that activate just either one of two distinct pathways, in vitro and in vivo analyses show that the different TGFβ members exert quite distinct biological functions with high specificity. This discrepancy indicates that our current view of TGFβ signaling initiation just by hetero-oligomerization of two receptor subtypes and transduction via two main pathways in an on-off switch manner is too simplified. Hence, the signals generated by the various TGFβ members are either quantitatively interpreted using the subtle differences in their receptor-binding properties leading to ligand-specific modulation of the downstream signaling cascade or additional components participating in the signaling activation complex allow diversification of the encoded signal in a ligand-dependent manner at all cellular levels. In this review we focus on signal specification of TGFβ members, particularly of BMPs and GDFs addressing the role of binding affinities, specificities, and kinetics of individual ligand-receptor interactions for the assembly of specific receptor complexes with potentially distinct signaling properties.
Radioresistance is an important cause of head and neck cancer therapy failure. Zinc oxide nanoparticles (ZnO-NP) mediate tumor-selective toxic effects. The aim of this study was to evaluate the potential for radiosensitization of ZnO-NP. The dose-dependent cytotoxicity of ZnO-NP\(_{20 nm}\) and ZnO-NP\(_{100 nm}\) was investigated in FaDu and primary fibroblasts (FB) by an MTT assay. The clonogenic survival assay was used to evaluate the effects of ZnO-NP alone and in combination with irradiation on FB and FaDu. A formamidopyrimidine-DNA glycosylase (FPG)-modified single-cell microgel electrophoresis (comet) assay was applied to detect oxidative DNA damage in FB as a function of ZnO-NP and irradiation exposure. A significantly increased cytotoxicity after FaDu exposure to ZnO-NP\(_{20 nm}\) or ZnO-NP\(_{100 nm}\) was observed in a concentration of 10 µg/mL or 1 µg/mL respectively in 30 µg/mL of ZnO-NP\(_{20 nm}\) or 20 µg/mL of ZnO-NP\(_{100 nm}\) in FB. The addition of 1, 5, or 10 µg/mL ZnO-NP\(_{20 nm}\) or ZnO-NP\(_{100 nm}\) significantly reduced the clonogenic survival of FaDu after irradiation. The sub-cytotoxic dosage of ZnO-NP\(_{100 nm}\) increased the oxidative DNA damage compared to the irradiated control. This effect was not significant for ZnO-NP\(_{20 nm}\). ZnO-NP showed radiosensitizing properties in the sub-cytotoxic dosage. At least for the ZnO-NP\(_{100 nm}\), an increased level of oxidative stress is a possible mechanism of the radiosensitizing effect.
Background: Culturing of cells is typically performed on standard tissue culture plates generating growth conditions, which in general do not reflect the native three-dimensional cellular environment. Recent investigations provide insights in parameters, which strongly affect the general cellular behavior triggering essential processes such as cell differentiation. The physical properties of the used material, such as stiffness, roughness, or topology, as well as the chemical composition of the cell-surface interface are shown to play a key role in the initiation of particular cellular responses. Methods: We extended our previous research, which identified thin films of metallo-supramolecular coordination polyelectrolytes (MEPEs) as substrate to trigger the differentiation of muscular precursor cells. Results: Here, we show that the same MEPEs similarly stimulate the osteogenic differentiation of pre-osteoblasts. Remarkably, MEPE modified surfaces also trigger the differentiation of primary bone derived mesenchymal stem cells (BMSCs) towards the osteogenic lineage. Conclusion: This result leads to the conclusion that these surfaces individually support the specification of cell differentiation toward lineages that correspond to the natural commitment of the particular cell types. We, therefore, propose that Fe-MEPEs may be used as scaffold for the treatment of defects at least in muscular or bone tissue.