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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.