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Purpose:
The biologic relevance of human connective tissue growth factor (hCTGF) for primary human tenon fibroblasts (HTFs) was investigated by RNA expression profiling using affymetrix (TM) oligonucleotide array technology to identify genes that are regulated by hCTGF.
Methods:
Recombinant hCTGF was expressed in HEK293T cells and purified by affinity and gel chromatography. Specificity and biologic activity of hCTGF was confirmed by biosensor interaction analysis and proliferation assays. For RNA expression profiling HTFs were stimulated with hCTGF for 48h and analyzed using affymetrix (TM) oligonucleotide array technology. Results were validated by real time RT-PCR.
Results:
hCTGF induces various groups of genes responsible for a wound healing and inflammatory response in HTFs. A new subset of CTGF inducible inflammatory genes was discovered (e.g., chemokine [C-X-C motif] ligand 1 [CXCL1], chemokine [C-X-C motif] ligand 6 [CXCL6], interleukin 6 [IL6], and interleukin 8 [IL8]). We also identified genes that can transmit the known biologic functions initiated by CTGF such as proliferation and extracellular matrix remodelling. Of special interest is a group of genes, e.g., osteoglycin (OGN) and osteomodulin (OMD), which are known to play a key role in osteoblast biology.
Conclusions:
This study specifies the important role of hCTGF for primary tenon fibroblast function. The RNA expression profile yields new insights into the relevance of hCTGF in influencing biologic processes like wound healing, inflammation, proliferation, and extracellular matrix remodelling in vitro via transcriptional regulation of specific genes. The results suggest that CTGF potentially acts as a modulating factor in inflammatory and wound healing response in fibroblasts of the human eye.
Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy
(2012)
Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 °C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 °C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.
In situ guided tissue regeneration, also addressed as in situ tissue engineering or endogenous regeneration, has a great potential for population-wide “minimal invasive” applications. During the last two decades, tissue engineering has been developed with remarkable in vitro and preclinical success but still the number of applications in clinical routine is extremely small. Moreover, the vision of population-wide applications of ex vivo tissue engineered constructs based on cells, growth and differentiation factors and scaffolds, must probably be deemed unrealistic for economic and regulation-related issues. Hence, the progress made in this respect will be mostly applicable to a fraction of post-traumatic or post-surgery situations such as big tissue defects due to tumor manifestation. Minimally invasive procedures would probably qualify for a broader application and ideally would only require off the shelf standardized products without cells. Such products should mimic the microenvironment of regenerating tissues and make use of the endogenous tissue regeneration capacities. Functionally, the chemotaxis of regenerative cells, their amplification as a transient amplifying pool and their concerted differentiation and remodeling should be addressed. This is especially important because the main target populations for such applications are the elderly and diseased. The quality of regenerative cells is impaired in such organisms and high levels of inhibitors also interfere with regeneration and healing. In metabolic bone diseases like osteoporosis, it is already known that antagonists for inhibitors such as activin and sclerostin enhance bone formation. Implementing such strategies into applications for in situ guided tissue regeneration should greatly enhance the efficacy of tailored procedures in the future.
Stem cell- and growth factor-based regenerative therapies for avascular necrosis of the femoral head
(2012)
Avascular necrosis (AVN) of the femoral head is a debilitating disease of multifactorial genesis, predominately affects young patients, and often leads to the development of secondary osteoarthritis. The evolving field of regenerative medicine offers promising treatment strategies using cells, biomaterial scaffolds, and bioactive factors, which might improve clinical outcome. Early stages of AVN with preserved structural integrity of the subchondral plate are accessible to retrograde surgical procedures, such as core decompression to reduce the intraosseous pressure and to induce bone remodeling. The additive application of concentrated bone marrow aspirates, ex vivo expanded mesenchymal stem cells, and osteogenic or angiogenic growth factors (or both) holds great potential to improve bone regeneration. In contrast, advanced stages of AVN with collapsed subchondral bone require an osteochondral reconstruction to preserve the physiological joint function. Analogously to strategies for osteochondral reconstruction in the knee, anterograde surgical techniques, such as osteochondral transplantation (mosaicplasty), matrix-based autologous chondrocyte implantation, or the use of acellular scaffolds alone, might preserve joint function and reduce the need for hip replacement. This review summarizes recent experimental accomplishments and initial clinical findings in the field of regenerative medicine which apply cells, growth factors, and matrices to address the clinical problem of AVN.
Growth and Differentiation Factor 5 (GDF5) is a secreted growth factor that belongs to the Bone Morphogenetic Protein (BMP) family and plays a pivotal role during limb development. GDF5 is a susceptibility gene for osteoarthritis (OA) and mutations in GDF5 are associated with a wide variety of skeletal malformations ranging from complex syndromes such as acromesomelic chondrodysplasias to isolated forms of brachydactylies or multiple synostoses syndrome 2 (SYNS2). Here, we report on a family with an autosomal dominant inherited combination of SYNS2 and additional brachydactyly type A1 (BDA1) caused by a single point mutation in GDF5 (p.W414R). Functional studies, including chondrogenesis assays with primary mesenchymal cells, luciferase reporter gene assays and Surface Plasmon Resonance analysis, of the GDF5 W-414R variant in comparison to other GDF5 mutations associated with isolated BDA1 (p.R399C) or SYNS2 (p.E491K) revealed a dual pathomechanism characterized by a gain-and loss-of-function at the same time. On the one hand insensitivity to the main GDF5 antagonist NOGGIN (NOG) leads to a GDF5 gain of function and subsequent SYNS2 phenotype. Whereas on the other hand, a reduced signaling activity, specifically via the BMP receptor type IA (BMPR1A), is likely responsible for the BDA1 phenotype. These results demonstrate that one mutation in the overlapping interface of antagonist and receptor binding site in GDF5 can lead to a GDF5 variant with pathophysiological relevance for both, BDA1 and SYNS2 development. Consequently, our study assembles another part of the molecular puzzle of how loss and gain of function mutations in GDF5 affect bone development in hands and feet resulting in specific types of brachydactyly and SYNS2. These novel insights into the biology of GDF5 might also provide further clues on the pathophysiology of OA.
Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.
Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).
Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.
In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.
Background
Fibrosis poses a substantial setback in regenerative medicine. Histopathologically, fibrosis is an excessive accumulation of collagen affected by myofibroblasts and this can occur in any tissue that is exposed to chronic injury or insult. Transforming growth factor (TGF)-β1, a crucial mediator of fibrosis, drives differentiation of fibroblasts into myofibroblasts. These cells exhibit α-smooth muscle actin (α-SMA) and synthesize high amounts of collagen I, the major extracellular matrix (ECM) component of fibrosis. While hormones stimulate cells in a pulsatile manner, little is known about cellular response kinetics upon growth factor impact. We therefore studied the effects of short TGF-β1 pulses in terms of the induction and maintenance of the myofibroblast phenotype.
Results
Twenty-four hours after a single 30 min TGF-β1 pulse, transcription of fibrogenic genes was upregulated, but subsided 7 days later. In parallel, collagen I secretion rate and α-SMA presence were elevated for 7 days. A second pulse 24 h later extended the duration of effects to 14 days. We could not establish epigenetic changes on fibrogenic target genes to explain the long-lasting effects. However, ECM deposited under singly pulsed TGF-β1 was able to induce myofibroblast features in previously untreated fibroblasts. Dependent on the age of the ECM (1 day versus 7 days’ formation time), this property was diminished. Vice versa, myofibroblasts were cultured on fibroblast ECM and cells observed to express reduced (in comparison with myofibroblasts) levels of collagen I.
Conclusions
We demonstrated that short TGF-β1 pulses can exert long-lasting effects on fibroblasts by changing their microenvironment, thus leaving an imprint and creating a reciprocal feed-back loop. Therefore, the ECM might act as mid-term memory for pathobiochemical events. We would expect this microenvironmental memory to be dependent on matrix turnover and, as such, to be erasable. Our findings contribute to the current understanding of fibroblast induction and maintenance, and have bearing on the development of antifibrotic drugs.
The zona pellucida (ZP) domain is present in extracellular proteins such as the zona pellucida proteins and tectorins and participates in the formation of polymeric protein networks. However, the ZP domain also occurs in the cytokine signaling co-receptor transforming growth factor beta (TGF-\(\beta\)) receptor type 3 (TGFR-3, also known as betaglycan) where it contributes to cytokine ligand recognition. Currently it is unclear how the ZP domain architecture enables this dual functionality. Here, we identify a novel major TGF-beta-binding site in the FG loop of the C-terminal subdomain of the murine TGFR-3 ZP domain (ZP-C) using protein crystallography, limited proteolysis experiments, surface plasmon resonance measurements and synthetic peptides. In the murine 2.7 angstrom crystal structure that we are presenting here, the FG-loop is disordered, however, well-ordered in a recently reported homologous rat ZP-C structure. Surprisingly, the adjacent external hydrophobic patch (EHP) segment is registered differently in the rat and murine structures suggesting that this segment only loosely associates with the remaining ZP-C fold. Such a flexible and temporarily-modulated association of the EHP segment with the ZP domain has been proposed to control the polymerization of ZP domain-containing proteins. Our findings suggest that this flexibility also extends to the ZP domain of TGFR-3 and might facilitate co-receptor ligand interaction and presentation via the adjacent FG-loop. This hints that a similar C-terminal region of the ZP domain architecture possibly regulates both the polymerization of extracellular matrix proteins and cytokine ligand recognition of TGFR-3.
Age-related gene expression analysis in enteric ganglia of human colon after laser microdissection
(2014)
The enteric nervous system (ENS) poses the intrinsic innervation of the gastrointestinal tract and plays a critical role for all stages of postnatal life. There is increasing scientific and clinical interest in acquired or age-related gastrointestinal dysfunctions that can be manifested in diseases such as gut constipation or fecal incontinence. In this study, we sought to analyze age-dependent changes in the gene expression profile of the human ENS, particularly in the myenteric plexus. Therefore, we used the laser microdissection technique which has been proven as a feasible tool to analyze distinct cell populations within heterogeneously composed tissues. Full biopsy gut samples were prepared from children (4–12 months), middle aged (48–58 years) and aged donors (70–95 years). Cryosections were histologically stained with H&E, the ganglia of the myenteric plexus identified and RNA isolated using laser microdissection technique. Quantitative PCR was performed for selected neural genes, neurotransmitters and receptors. Data were confirmed on protein level using NADPH-diaphorase staining and immunohistochemistry. As result, we demonstrate age-associated alterations in site-specific gene expression pattern of the ENS. Thus, in the adult and aged distal parts of the colon a marked decrease in relative gene expression of neural key genes like NGFR, RET, NOS1 and a concurrent increase of CHAT were observed. Further, we detected notable regional differences of RET, CHAT, TH, and S100B comparing gene expression in aged proximal and distal colon. Interestingly, markers indicating cellular senescence or oxidative stress (SNCA, CASP3, CAT, SOD2, and TERT) were largely unchanged within the ENS. For the first time, our study also describes the age-dependent expression pattern of all major sodium channels within the ENS. Our results are in line with previous studies showing spatio-temporal differences within the mammalian ENS.
Recent advances in the in vitro characterization of human adult enteric neural progenitor cells have opened new possibilities for cell-based therapies in gastrointestinal motility disorders. However, whether these cells are able to integrate within an in vivo gut environment is still unclear. In this study, we transplanted neural progenitor-containing neurosphere-like bodies (NLBs) in a mouse model of hypoganglionosis and analyzed cellular integration of NLB-derived cell types and functional improvement. NLBs were propagated from postnatal and adult human gut tissues. Cells were characterized by immunohistochemistry, quantitative PCR and subtelomere fluorescence in situ hybridization (FISH). For in vivo evaluation, the plexus of murine colon was damaged by the application of cationic surfactant benzalkonium chloride which was followed by the transplantation of NLBs in a fibrin matrix. After 4 weeks, grafted human cells were visualized by combined in situ hybridization (Alu) and immunohistochemistry (PGP9.5, GFAP, SMA). In addition, we determined nitric oxide synthase (NOS)-positive neurons and measured hypertrophic effects in the ENS and musculature. Contractility of treated guts was assessed in organ bath after electrical field stimulation. NLBs could be reproducibly generated without any signs of chromosomal alterations using subtelomere FISH. NLB-derived cells integrated within the host tissue and showed expected differentiated phenotypes i.e. enteric neurons, glia and smooth muscle-like cells following in vivo transplantation. Our data suggest biological effects of the transplanted NLB cells on tissue contractility, although robust statistical results could not be obtained due to the small sample size. Further, it is unclear, which of the NLB cell types including neural progenitors have direct restoring effects or, alternatively may act via 'bystander' mechanisms in vivo. Our findings provide further evidence that NLB transplantation can be considered as feasible tool to improve ENS function in a variety of gastrointestinal disorders.
Integrated approaches using different in vitro methods in combination with bioinformatics can (i) increase the success rate and speed of drug development; (ii) improve the accuracy of toxicological risk assessment; and (iii) increase our understanding of disease. Three-dimensional (3D) cell culture models are important building blocks of this strategy which has emerged during the last years. The majority of these models are organotypic, i.e., they aim to reproduce major functions of an organ or organ system. This implies in many cases that more than one cell type forms the 3D structure, and often matrix elements play an important role. This review summarizes the state of the art concerning commonalities of the different models. For instance, the theory of mass transport/metabolite exchange in 3D systems and the special analytical requirements for test endpoints in organotypic cultures are discussed in detail. In the next part, 3D model systems for selected organs liver, lung, skin, brain are presented and characterized in dedicated chapters. Also, 3D approaches to the modeling of tumors are presented and discussed. All chapters give a historical background, illustrate the large variety of approaches, and highlight up- and downsides as well as specific requirements. Moreover, they refer to the application in disease modeling, drug discovery and safety assessment. Finally, consensus recommendations indicate a roadmap for the successful implementation of 3D models in routine screening. It is expected that the use of such models will accelerate progress by reducing error rates and wrong predictions from compound testing.
Age-related macular degeneration (AMD) is the leading cause of blindness in the Western world. With an ageing population, it is anticipated that the number of AMD cases will increase dramatically, making a solution to this debilitating disease an urgent requirement for the socioeconomic future of the European Union and worldwide. The present paper reviews the limitations of the current therapies as well as the socioeconomic impact of the AMD. There is currently no cure available for AMD, and even palliative treatments are rare. Treatment options show several side effects, are of high cost, and only treat the consequence, not the cause of the pathology. For that reason, many options involving cell therapy mainly based on retinal and iris pigment epithelium cells as well as stem cells are being tested. Moreover, tissue engineering strategies to design and manufacture scaffolds to mimic Bruch's membrane are very diverse and under investigation. Both alternative therapies are aimed to prevent and/or cure AMD and are reviewed herein.
Hintergrund
Jede Implantation alloplastischer Materialien führt durch Aktivierung der körpereigenen Immunabwehr zu einer Fremdkörperreaktion. An der Synthese der Extrazellulärmatrix und der entstehenden Kollagenkapsel sind insbesondere Makrophagen und Fibroblasten beteiligt. Diese Reaktionen können die Material-Funktionsfähigkeit abschwächen, aufheben oder zu deren operativer Entfernung zwingen.
Fragestellung und Ziele
Spinnenseide ist ein Material mit hoher Biokompatibilität. Nachdem es gelungen ist, Spinnenseide rekombinant herzustellen, soll untersucht werden, wie sich die Verträglichkeit alloplastischer Materialien durch eine Beschichtung mit biotechnologisch hergestellter Spinnenseide beeinflussen lässt.
Eine weitere Möglichkeit ist der TGF-β-Synthese-Inhibitor Halofuginon, der ebenfalls hinsichtlich seiner Potenz, die Ausbildung einer Fibrosekapsel zu vermindern, untersucht werden soll.
Methodik
Anhand von in-vitro-Untersuchungen wurden die bei der Fremdkörperreaktion beteiligten Zelltypen auf ihr Proliferationsverhalten und die Expression unterschiedlicher Genprodukte hinsichtlich bestehender Unterschiede zwischen den jeweiligen Oberflächenbeschichtungen untersucht. Es wurden immunhistochemische Färbungen zum Nachweis spezifischer Oberflächenantigene, Bestimmungen von ATP- und DNA-Gehalt als Maß für die Zellzahl, sowie molekulargenetische Untersuchungen hinsichtlich der Expression relevanter Markergene (rtPCR) durchgeführt.
Ergebnisse
Eine Beschichtung mit rekombinanter Spinnseide führt - im Vergleich zu reinen Silikonimplantaten - zu einer verzögerten und reduzierten Immunreaktion. Die EZM-Synthese und die damit verbundene fremdkörperassoziierte Fibrose werden vermindert und so die Biokompatibilität alloplastischer Materialien gesteigert.
The mono-6-deoxy-6-azides of 2,6-di-O-methyl-beta-cyclodextrin (DIMEB) and randomly methylated-beta-cyclodextrin (RAMEB) were conjugated to propargylated hydroxyethyl starch (HES) by Cu+-catalysed [2 + 3] cycloaddition. The resulting water soluble polymers showed lower critical solution temperatures (LCST) at 52.5 degrees C (DIMEB-HES) and 84.5 degrees C (RAMEB-HES), respectively. LCST phase separations could be completely avoided by the introduction of a small amount of carboxylate groups at the HES backbone. The methylated CDs conjugated to the HES backbone exhibited significantly lower cytotoxicities than the corresponding monomeric CD derivatives. Since the binding potentials of these CD conjugates were very high, they are promising candidates for new oral dosage forms of anaesthetic actives.
Background
Bone morphogenetic protein (BMP)-2 and growth and differentiation factor (GDF)-5 are two related transforming growth factor (TGF)-β family members with important functions in embryonic development and tissue homeostasis. BMP-2 is best known for its osteoinductive properties whereas GDF-5—as evident from its alternative name, cartilage derived morphogenetic protein 1—plays an important role in the formation of cartilage. In spite of these differences both factors signal by binding to the same subset of BMP receptors, raising the question how these different functionalities are generated. The largest difference in receptor binding is observed in the interaction with the type I receptor BMPR-IA. GDF-5, in contrast to BMP-2, shows preferential binding to the isoform BMPR-IB, which is abrogated by a single amino acid (A57R) substitution. The resulting variant, GDF-5 R57A, represents a “BMP-2 mimic” with respect to BMP receptor binding. In this study we thus wanted to analyze whether the two growth factors can induce distinct signals via an identically composed receptor.
Results
Unexpectedly and dependent on the cellular context, GDF-5 R57A showed clear differences in its activity compared to BMP-2. In ATDC-5 cells, both ligands induced alkaline phosphatase (ALP) expression with similar potency. But in C2C12 cells, the BMP-2 mimic GDF-5 R57A (and also wild-type GDF-5) clearly antagonized BMP-2-mediated ALP expression, despite signaling in both cell lines occurring solely via BMPR-IA. The BMP-2- antagonizing properties of GDF-5 and GDF-5 R57A could also be observed in vivo when implanting BMP-2 and either one of the two GDF-5 ligands simultaneously at heterotopic sites.
Conclusions
Although comparison of the crystal structures of the GDF-5 R57A:BMPR-IAEC- and BMP-2:BMPR-IAEC complex revealed small ligand-specific differences, these cannot account for the different signaling characteristics because the complexes seem identical in both differently reacting cell lines. We thus predict an additional component, most likely a not yet identified GDF-5-specific co-receptor, which alters the output of the signaling complexes. Hence the presence or absence of this component then switches GDF-5′s signaling capabilities to act either similar to BMP-2 or as a BMP-2 antagonist. These findings might shed new light on the role of GDF-5, e.g., in cartilage maintenance and/or limb development in that it might act as an inhibitor of signaling events initiated by other BMPs.
In this study we used differentiated adult human upcyte (R) cells for the in vitro generation of liver organoids. Upcyte (R) cells are genetically engineered cell strains derived from primary human cells by lenti-viral transduction of genes or gene combinations inducing transient proliferation capacity (upcyte (R) process). Proliferating upcyte (R) cells undergo a finite number of cell divisions, i.e., 20 to 40 population doublings, but upon withdrawal of proliferation stimulating factors, they regain most of the cell specific characteristics of primary cells. When a defined mixture of differentiated human upcyte (R) cells (hepatocytes, liver sinusoidal endothelial cells (LSECs) and mesenchymal stem cells (MSCs)) was cultured in vitro on a thick layer of Matrigel\(^{TM}\), they self-organized to form liver organoid-like structures within 24 hours. When further cultured for 10 days in a bioreactor, these liver organoids show typical functional characteristics of liver parenchyma including activity of cytochromes P450, CYP3A4, CYP2B6 and CYP2C9 as well as mRNA expression of several marker genes and other enzymes. In summary, we hereby describe that 3D functional hepatic structures composed of primary human cell strains can be generated in vitro. They can be cultured for a prolonged period of time and are potentially useful ex vivo models to study liver functions.
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.
Models of the outer epithelia of the human body namely the skin, the intestine and the lung have found valid applications in both research and industrial settings as attractive alternatives to animal testing. A variety of approaches to model these barriers are currently employed in such fields, ranging from the utilization of ex vivo tissue to reconstructed in vitro models, and further to chip-based technologies, synthetic membrane systems and, of increasing current interest, in silico modeling approaches. An international group of experts in the field of epithelial barriers was convened from academia, industry and regulatory bodies to present both the current state of the art of non-animal models of the skin, intestinal and pulmonary barriers in their various fields of application, and to discuss research-based, industry-driven and regulatory-relevant future directions for both the development of new models and the refinement of existing test methods. Issues of model relevance and preference, validation and standardization, acceptance, and the need for simplicity versus complexity were focal themes of the discussions. The outcomes of workshop presentations and discussions, in relation to both current status and future directions in the utilization and development of epithelial barrier models, are presented by the attending experts in the current report.
Tumors are characterized by a rigid, highly cross-linked extracellular matrix (ECM), which impedes homogeneous drug distribution and potentially protects malignant cells from exposure to therapeutics. Lysyl oxidases are major contributors to tissue stiffness and the elevated expression of these enzymes observed in most cancers might influence drug distribution and efficacy. We examined the effect of lysyl oxidases on drug distribution and efficacy in 3D in vitro assay systems. In our experiments elevated lysyl oxidase activity was responsible for reduced drug diffusion under hypoxic conditions and consequently impaired cytotoxicity of various chemotherapeutics. This effect was only observed in 3D settings but not in 2D-cell culture, confirming that lysyl oxidases affect drug efficacy by modification of the ECM and do not confer a direct desensitizing effect. Both drug diffusion and efficacy were strongly enhanced by inhibition of lysyl oxidases. The results from the in vitro experiments correlated with tumor drug distribution in vivo, and predicted response to therapeutics in murine tumor models. Our results demonstrate that lysyl oxidase activity modulates the physical barrier function of ECM for small molecule drugs influencing their therapeutic efficacy. Targeting this process has the potential to significantly enhance therapeutic efficacy in the treatment of malignant diseases.
Human upcyte\(^{®}\) hepatocytes are proliferating hepatocytes that retain many characteristics of primary human hepatocytes. We conducted a comprehensive evaluation of the application of second-generation upcyte\(^{®}\) hepatocytes from four donors for inhibition and induction assays using a selection of reference inhibitors and inducers. CYP1A2, CYP2B6, CYP2C9, and CYP3A4 were reproducibly inhibited in a concentration-dependent manner and the calculated IC\(_{50}\) values for each compound correctly classified them as potent inhibitors. Upcyte\(^{®}\) hepatocytes were responsive to prototypical CYP1A2, CYP2B6, CYP2C9, and CYP3A4 inducers, confirming that they have functional AhR-, CAR-, and PXR-mediated CYP regulation. A panel of 11 inducers classified as potent, moderate or noninducers of CYP3A4 and CYP2B6 were tested. There was a good fit of data from upcyte\(^{®}\) hepatocytes to three different predictive models for CYP3A4 induction, namely the Relative Induction Score (RIS), AUC\(_{u}\)/F\(_{2}\), and C\(_{max,u}\)/Ind\(_{50}\). In addition, PXR (rifampicin) and CAR-selective (carbamazepine and phenytoin) inducers of CYP3A4 and CYP2B6 induction, respectively, were demonstrated. In conclusion, these data support the use of second-generation upcyte\(^{®}\) hepatocytes for CYP inhibition and induction assays. Under the culture conditions used, these cells expressed CYP activities that were equivalent to or higher than those measured in primary human hepatocyte cultures, which could be inhibited or induced by prototypical CYP inhibitors and inducers, respectively. Moreover, they can be used to predict in vivo CYP3A4 induction potential using three prediction models. Bulk availability of cells from multiple donors makes upcyte\(^{®}\) hepatocytes suitable for DDI screening, as well as more in-depth mechanistic investigations.
Aktueller Goldstandard bei der Rekonstruktion des ACL des Menschen sind au-tologe Transplantate. Diese sind allerdings je nach Entnahmeort mit einer mehr oder weniger hohen Entnahmemorbidität und dem Risiko für Folgeerkrankungen verbunden. Um dies zu umgehen, wurde ein xenogenes Kollagenimplantat aus
Kollagen-I-Fasern von Ratten entwickelt und das native Konstrukt bereits in einer Vorläuferstudie getestet.
Im Rahmen dieser Arbeit wurden diese Kreuzbandkonstrukte mit Hilfe diverser
Crosslinker modifiziert und hinsichtlich ihrer Biomechanik, Biokompatibilität und ihres in-vivo Verhaltens untersucht.
Bewusst wurde dabei auf die Zellbesiedlung dieser Konstrukte verzichtet, da un-ter Berücksichtigung wirtschaftlicher Gesichtspunkte eines späteren humanen
Einsatzes hierfür eine Arzneimittelzulassung notwendig gewesen wäre. Mit Hilfe der Crosslinker wurde versucht, die mechanische Stabilität sowie die Resistenz gegen kollagenabbauende Enzyme der Synovia zu erhöhen, um die Gefahr post-operativer Instabilitäten zu verringern. Dabei sollten Fragen bezüglich Immun-antwort, Biokompatibilität sowie Biodegradierbarkeit genau berücksichtigt wer-den. Als Crosslinker wurden für einen Vergleich in vitro neben 0,5 % Genipin auch 10 % HMDI sowie Glukose und EDC/NHS herangezogen.
Dabei zeigten die Genipin-gecrosslinkten Einzelfasern die größte Reißfestigkeits-zunahme, wohingegen auf Minikonstruktbasis 10 % HMDI zu den höchsten UTS-Werten führte. Ebenso ließen sich bezüglich der Biokompatibilutät in vitro bei den Crosslinkern 0,5 % Genipin und 10 % HMDI Vorteile gegenüber den beiden an-deren erkennen.
Schließlich erfolgte im Rahmen eines Tierversuchs an 16 Minipigs der Einbau von 0,5 % Genipin-gecrosslinkten Konstrukten als Kreuzbandersatz und an-schließend die biomechanische Testung sowie nach Paraffineinbettung auch eine durchlichtmikrokopische deskriptive Auswertung der Transplantate.
Während nach 6 Wochen eine deutliche Reißfestigkeitsabnahme zu verzeichnen war, erreichte diese nach 6 Monaten wieder fast 60 % ihrer ursprünglichen UTS.
Somit konnte ein Remodeling des eingesetzten Implantats angenommen wer-den. Dies bestätigte sich in der durchgeführten histologischen Untersuchung.
Hier war das Implantat deutlich vaskularisiert, von zahlreichen Fibroblasten durchsetzt und wies eine synoviale Deckschicht auf. Allerdings scheint vor allem wegen der Schwäche der Konstrukte nach 6 Wochen sowie den vermutlich auf-grund des Crosslinkers auftretenden Reaktionserscheinungen innerhalb des
Kniegelenks ein Einsatz im humanen Bereich zum gegenwärtigen Zeitpunkt noch nicht ausgereift.
Dennoch lässt sich gerade anhand des stattfindenden Remodelings das große Potential kollagenbasierter Materialien für den Kreuzbandersatz erkennen. Eine weitere Optimierung des bestehenden Konstrukts sollte deshalb forciert werden.
The main function of the small intestine is the absorption of essential nutrients, water and vitamins. Moreover, it constitutes a barrier protecting us from toxic xenobiotics and pathogens. For a better understanding of these processes, the development of intestinal in vitro models is of great interest to the study of pharmacological and pathological issues such as transport mechanisms and barrier function. Depending on the scientific questions, models of different complexity can be applied.
In vitro Transwell® systems based on a porous PET-membrane enable the standardized study of transport mechanisms across the intestinal barrier as well as the investigation of the influence of target substances on barrier integrity. However, this artificial setup reflects only limited aspects of the physiology of the native small intestine and can pose an additional physical barrier. Hence, the applications of this model for tissue engineering are limited.
Previously, tissue models based on a biological decellularized scaffold derived from porcine gut tissue were demonstrated to be a good alternative to the commonly used Transwell® system. This study showed that preserved biological extracellular matrix components like collagen and elastin provide a natural environment for the epithelial cells, promoting cell adhesion and growth. Intestinal epithelial cells such as Caco-2 cultured on such a scaffold showed a confluent, tight monolayer on the apical surface. Additionally, myofibroblasts were able to migrate into the scaffold supporting intestinal barrier formation.
In this thesis, dendritic cells were additionally introduced to this model mimicking an important component of the immune system. This co-culture model was then successfully proven to be suitable for the screening of particle formulations developed as delivery system for cancer antigens in peroral vaccination studies. In particular, nanoparticles based on PLGA, PEG-PAGE-PLGA, Mannose-PEG-PAGE-PLGA and Chitosan were tested. Uptake studies revealed only slight differences in the transcellular transport rate among the different particles. Dendritic cells were shown to phagocytose the particles after they have passed the intestinal barrier. The particles demonstrated to be an effective carrier system to transport peptides across the intestinal barrier and therefore present a useful tool for the development of novel drugs.
Furthermore, to mimic the complex structure and physiology of the gut including the presence of multiple different cell types, the Caco-2 cell line was replaced by primary intestinal cells to set up a de novo tissue model. To that end, intestinal crypts including undifferentiated stem cells and progenitor cells were isolated from human small intestinal tissue samples (jejunum) and expanded in vitro in organoid cultures. Cells were cultured on the decellularized porcine gut matrix in co-culture with intestinal myofibroblasts. These novel tissue models were maintained under either static or dynamic conditions.
Primary intestinal epithelial cells formed a confluent monolayer including the major differentiated cell types positive for mucin (goblet cells), villin (enterocytes), chromogranin A (enteroendocrine cells) and lysozyme (paneth cells). Electron microscopy images depicted essential functional units of an intact epithelium, such as microvilli and tight junctions. FITC-dextran permeability and TEER measurements were used to assess tightness of the cell layer. Models showed characteristic transport activity for several reference substances. Mechanical stimulation of the cells by a dynamic culture system had a great impact on barrier integrity and transporter activity resulting in a tighter barrier and a higher efflux transporter activity.
In Summary, the use of primary human intestinal cells combined with a biological decellularized scaffold offers a new and promising way to setup more physiological intestinal in vitro models. Maintenance of primary intestinal stem cells with their proliferation and differentiation potential together with adjusted culture protocols might help further improve the models. In particular, dynamic culture systems and co culture models proofed to be a first crucial steps towards a more physiological model. Such tissue models might be useful to improve the predictive power of in vitro models and in vitro in vivo correlation (IVIVC) studies. Moreover, these tissue models will be useful tools in preclinical studies to test pharmaceutical substances, probiotic active organisms, human pathogenic germs and could even be used to build up patient-specific tissue model for personalized medicine.
Zusammenfassung
In der Regenerativen Medizin sind polymerbasierte Biomaterialien von großer Bedeutung für
die Entwicklung und Anwendung verbesserter bzw. neuer Therapien. Die Erforschung der
Oberflächeneigenschaften von Biomaterialien, welche als Implantate eingesetzt werden, ist
eine grundlegende Voraussetzung für deren erfolgreichen Einsatz. Die Protein-Oberflächen-
Interaktion geschieht initial, sobald ein Implantat mit Körperflüssigkeiten oder mit Gewebe
in Kontakt kommt, und trägt maßgeblich zur direkten Wechselwirkung von Implantat und
umgebenden Zellen bei. Dieser Prozess wird in der vorliegenden Arbeit an Gelatine untersucht.
Daher bestand ein Ziel darin, stabile, nanometerdünne Gelatineoberflächen herzustellen
und darauf die Adsorption von humanen Plasmaproteinen und bakteriellen Proteinen zu
analysieren.
Die Abscheidung der Gelatinefilme in variabler Schichtdicke auf zuvor mit PPX-Amin modifizierten
Oberflächen wurde unter Verwendung eines Rotationsbeschichters durchgeführt.
Um stabile Hydrogelfilme zu erhalten, wurden die Amingruppen der disaggregierten Gelatinefibrillen
untereinander und mit denen der Amin-Modifizierung durch ein biokompatibles
Diisocyanat quervernetzt. Dieser Prozess lieferte einen reproduzierbaren und chemisch stabilen
Gelatinefilm, welcher durch die substratunabhängige Amin-Modifizierung kovalent auf
unterschiedlichste Oberflächen aufgebracht werden konnte. Die durch den Herstellungsprozess
präzise eingestellte Schichtdicke (Nano- bzw. Mikrometermaßstab) wurde mittels Ellipsometrie
und Rasterkraftmikroskopie ermittelt. Die ebenso bestimmte Rauheit war unabhängig
von der Schichtdicke sehr gering. Gelatinefilme, die auf funktionalisierte und strukturierte
Proben aufgebracht wurden, konnten durch Elektronenmikroskopie dargestellt werden. Mit
Hilfe der Infrarot-Reflexions-Absorptions-Spektroskopie wurden die Gelatinefilme im Hinblick
auf ihre Stabilität chemisch charakterisiert. Zur Quantifizierung der Adsorption humaner
Plasmaproteine (Einzelproteinlösungen) und komplexer Proteingemische aus steril filtrierten
Kulturüberständen des humanpathogenen Bakteriums Pseudomonas aeruginosa wurde die
Quarzkristall-Mikrowaage mit Dissipationsüberwachung eingesetzt. Hiermit konnte nicht
nur die adsorbierte Menge an Proteinen auf dem Gelatinehydrogel bzw. Referenzoberflächen
(Gold, PPX-Amin, Titan), sondern auch die viskoelastischen Eigenschaften des adsorbierten
Proteinfilms bestimmt werden. Allgemein adsorbierte auf dem Gelatinehydrogel eine geringere
Proteinmasse im Vergleich zu den Referenzoberflächen. Circa ein Viertel der adsorbierten
Proteine migrierte in die Poren des gequollenen Gels und veränderte dessen viskoelastische
Eigenschaften. Durch anschließende MALDI-ToF/MS- und MS/MS-Analyse konnten die bakteriellen
Proteine auf den untersuchten Oberflächen identifiziert und untereinander verglichen
werden. Hierbei zeigten sich nur geringfügige Unterschiede in der Proteinzusammensetzung.
Zudem wurde eine Sekundärionenmassenspektrometrie mit Flugzeitanalyse an reinen Gelatinefilmen
und an mit humanen Plasmaproteinen beladenen Gelatinefilmen durchgeführt.
Durch eine anschließende multivariante Datenanalyse konnte zwischen den untersuchten
Proben eindeutig differenziert werden. Dieser Ansatz ermöglicht es, die Adsorption von
unterschiedlichen Proteinen auf proteinbasierten Oberflächen markierungsfrei zu untersuchen
und kann zur Aufklärung der in vivo-Situation beitragen. Darüber hinaus bietet dieser
Untersuchungsansatz neue Perspektiven für die Gestaltung und das schnelle und effiziente
Screening von unterschiedlichen Proteinzusammensetzungen.
Biomaterialien können jedoch nicht nur als Implantate oder Implantatbeschichtungen eingesetzt
werden. Im Bereich des drug delivery und der Depotarzneimittel sind biologisch
abbaubare Polymere, aufgrund ihrer variablen Eigenschaften, von großem Interesse. Die
Behandlung von bakteriellen und fungalen Pneumonien stellt insbesondere bei Menschen mit
Vorerkrankungen wie Cystische Fibrose oder primäre Ziliendyskinesie eine große Herausforderung
dar. Oral oder intravenös applizierte Wirkstoffe erreichen die Erreger aufgrund der
erhöhten Zähigkeit des Bronchialsekretes oft nicht in ausreichender Konzentration. Daher
besteht ein weiteres Ziel der vorliegenden Arbeit darin, mittels electrohydrodynamic cojetting
mikrometergroße, inhalierbare, wirkstoffbeladene Partikel mit zwei Kompartimenten
(Janus-Partikel) herzustellen und deren Eignung für die therapeutische Anwendung bei
Lungeninfektionen zu untersuchen.
Durch das in dieser Arbeit entwickelte Lösungsmittelsystem können Janus-Partikel aus
biologisch abbaubaren Co-Polymeren der Polymilchsäure (Poly(lactid-co-glycolid), PLGA)
hergestellt und mit verschiedenen Wirkstoffen beladen werden. Darunter befinden sich ein
Antibiotikum (Aztreonam, AZT), ein Antimykotikum (Itraconazol, ICZ), ein Mukolytikum
(Acetylcystein, ACC) und ein Antiphlogistikum (Ibuprofen, IBU). Die Freisetzung der eingelagerten
Wirkstoffe, mit Ausnahme von ICZ, konnte unter physiologischen Bedingungen
mittels Dialyse und anschließender Hochleistungsflüssigkeitschromatographie gemessen werden.
Die Freisetzungsrate wird von der Kettenlänge des Polymers beeinflusst, wobei eine
kürzere Kettenlänge zu einer schnelleren Freisetzung führt. Das in die Partikel eingelagerte
Antimykotikum zeigte in vitro eine gute Wirksamkeit gegen Aspergillus nidulans. Durch das
Einlagern von ICZ in die Partikel ist es möglich diesen schlecht wasserlöslichen Wirkstoff in
eine für Patienten zugängliche und wirksame Applikationsform zu bringen. In Interaktion mit
P. aeruginosa erzielten die mit Antibiotikum beladenen Partikel in vitro bessere Ergebnisse
als der Wirkstoff in Lösung, was sich in einem in vivo-Infektionsmodell mit der Wachsmotte
Galleria mellonella bestätigte. AZT-beladene Partikel hatten gegenüber einer identischen
Wirkstoffmenge in Lösung eine 27,5% bessere Überlebensrate der Wachsmotten zur Folge.
Des Weiteren hatten die Partikel keinen messbaren negativen Einfluss auf die Wachsmotten.
Dreidimensionale Atemwegsschleimhautmodelle, hergestellt mit Methoden des Tissue Engineerings,
bildeten die Basis für Untersuchungen der Partikel in Interaktion mit humanen
Atemwegszellen. Die Untersuchung von Apoptose- und Entzündungsmarkern im Überstand
der 3D-Modelle zeigte diesbezüglich keinen negativen Einfluss der Partikel auf die humanen
Zellen. Diese gut charakterisierten und standardisierten in vitro-Testsysteme machen es
möglich, Medikamentenuntersuchungen an menschlichen Zellen durchzuführen. Hinsichtlich
der histologischen Architektur und funktionellen Eigenschaften der 3D-Modelle konnte eine
hohe in vitro-/in vivo-Korrelation zu menschlichem Gewebe festgestellt werden. Humane
Mucine auf den 3D-Modellen dienten zur Untersuchung der schleimlösenden Wirkung von
ACC-beladenen Partikeln. Standen diese in räumlichem Kontakt zu den Mucinen, wurde deren
Zähigkeit durch das freigesetzte ACC herabgesetzt, was qualitativ mittels histologischen
Methoden bestätigt werden konnte.
Die in dieser Arbeit entwickelten Herstellungsprotokolle dienen als Grundlage und können
für die Synthese ähnlicher Systeme, basierend auf anderen Polymeren und Wirkstoffen,
modifiziert werden. Gelatine und PLGA erwiesen sich als vielseitig einsetzbare Werkstoffe
und bieten eine breite Anwendungsvielfalt in der Regenerativen Medizin, was die erzielten
Resultate bekräftigen.
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.
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.
Lung cancer is currently the leading cause of cancer related mortality due to late diagnosis and limited treatment intervention. Non-coding RNAs are not translated into proteins and have emerged as fundamental regulators of gene expression. Recent studies reported that microRNAs and long non-coding RNAs are involved in lung cancer development and progression. Moreover, they appear as new promising non-invasive biomarkers for early lung cancer diagnosis. Here, we highlight their potential as biomarker in lung cancer and present how bioinformatics can contribute to the development of non-invasive diagnostic tools. For this, we discuss several bioinformatics algorithms and software tools for a comprehensive understanding and functional characterization of microRNAs and long non-coding RNAs.
Background
Enteric glial cells (EGCs) are the main constituent of the enteric nervous system and share similarities with astrocytes from the central nervous system including their reactivity to an inflammatory microenvironment. Previous studies on EGC pathophysiology have specifically focused on mucosal glia activation and its contribution to mucosal inflammatory processes observed in the gut of inflammatory bowel disease (IBD) patients. In contrast knowledge is scarce on intestinal inflammation not locally restricted to the mucosa but systemically affecting the intestine and its effect on the overall EGC network.
Methods and Results
In this study, we analyzed the biological effects of a systemic LPS-induced hyperinflammatory insult on overall EGCs in a rat model in vivo, mimicking the clinical situation of systemic inflammation response syndrome (SIRS). Tissues from small and large intestine were removed 4 hours after systemic LPS-injection and analyzed on transcript and protein level. Laser capture microdissection was performed to study plexus-specific gene expression alterations. Upon systemic LPS-injection in vivo we observed a rapid and dramatic activation of Glial Fibrillary Acidic Protein (GFAP)-expressing glia on mRNA level, locally restricted to the myenteric plexus. To study the specific role of the GFAP subpopulation, we established flow cytometry-purified primary glial cell cultures from GFAP promotor-driven EGFP reporter mice. After LPS stimulation, we analyzed cytokine secretion and global gene expression profiles, which were finally implemented in a bioinformatic comparative transcriptome analysis. Enriched GFAP+ glial cells cultured as gliospheres secreted increased levels of prominent inflammatory cytokines upon LPS stimulation. Additionally, a shift in myenteric glial gene expression profile was induced that predominantly affected genes associated with immune response.
Conclusion and Significance
Our findings identify the myenteric GFAP-expressing glial subpopulation as particularly susceptible and responsive to acute systemic inflammation of the gut wall and complement knowledge on glial involvement in mucosal inflammation of the intestine.
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.
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.
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.
Tissue-engineered skin equivalents mimic key aspects of the human skin, and can thus be employed as wound coverage for large skin defects or as in vitro test systems as an alternative to animal models. However, current skin equivalents lack a functional vasculature limiting clinical and research applications. This study demonstrates the generation of a vascularized skin equivalent with a perfused vascular network by combining a biological vascularized scaffold (BioVaSc) based on a decellularized segment of a porcine jejunum and a tailored bioreactor system. Briefly, the BioVaSc was seeded with human fibroblasts, keratinocytes, and human microvascular endothelial cells. After 14 days at the air-liquid interface, hematoxylin & eosin and immunohistological staining revealed a specific histological architecture representative of the human dermis and epidermis including a papillary-like architecture at the dermal-epidermal-junction. The formation of the skin barrier was measured non-destructively using impedance spectroscopy. Additionally, endothelial cells lined the walls of the formed vessels that could be perfused with a physiological volume flow. Due to the presence of a complex in-vivo-like vasculature, the here shown skin equivalent has the potential for skin grafting and represents a sophisticated in vitro model for dermatological research.
In the last decades significant regulatory attempts were made to replace, refine and reduce animal testing to assess the risk of consumer products for the human eye. As the original in vivo Draize eye test is criticized for limited predictivity, costs and ethical issues, several animal-free test methods have been developed to categorize substances according to the global harmonized system (GHS) for eye irritation. This review summarizes the progress of alternative test methods for the assessment of eye irritation. Based on the corneal anatomy and current knowledge of the mechanisms causing eye irritation, different ex vivo and in vitro methods will be presented and discussed with regard to possible limitations and status of regulatory acceptance. In addition to established in vitro models, this review will also highlight emerging, full thickness cornea models that might be suited to predict all GHS categories.
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.
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.
The limited intrinsic self-healing capability of articular cartilage requires treatment of
cartilage defects. Material assisted and cell based therapies are in clinical practice but
tend to result in formation of mechanical inferior fibro-cartilage in long term follow up. If
a lesion has not been properly restored degenerative diseases are diagnosed as late sequela
causing pain and loss in morbidity. Complex three dimensional tissue models mimicking
physiological situation allow investigation of cartilage metabolism and mechanisms involved
in repair. A standardized and reproducible model cultured under controllable conditions
ex vivo to maintain tissue properties is of relevance for comparable studies.
Topic of this thesis was the establishment of an cartilage defect model that allows for
testing novel biomaterials and investigate the effect of defined defect depths on formation
of repair tissue.
In part I an ex vivo osteochondral defect model was established based on isolation of
porcine osteochondral explants (OCE) from medial condyles, 8 mm in diameter and 5 mm
in height. Full thickness cartilage defects with 1 mm to 4 mm in diameter were created
to define ex vivo cartilage critical size after 28 days culture with custom developed static
culture device. In part II of this thesis hydrogel materials, namely collagen I isolated from
rat tail, commercially available fibrin glue, matrix-metalloproteinase clevable poly(ethylene
glycol) polymerized with heparin (starPEGh), methacrylated poly(N-(2-hydroxypropyl)
methacrylamide mono-dilactate-poly(ethylene glycol) triblock copolymer/methacrylated
hyaluronic acid (MP/HA), thiol functionalized HA/allyl functionalized poly(glycidol)
(P(AGE/G)-HA-SH), were tested cell free and chondrocyte loaded (20 mio/ml) as implant
in 4 mm cartilage defects to investigate cartilage regeneration. Reproducible chondral
defects, 8 mm in diameter and 1 mm in height, were generated with an artificial tissue
cutter (ARTcut®) to investigate effect of defect depth on defect regeneration in part III.
In all approaches OCE were analyzed by Safranin-O staining to visualize proteoglycans
in cartilage and/or hydrogels. Immuno-histological and -fluorescent stainings (aggrecan,
collagen II, VI and X, proCollagen I, SOX9, RUNX2), gene expression analysis (aggrecan,
collagen II and X, SOX9, RUNX2) of chondrocyte loaded hydrogels (part II) and proteoglycan
and DNA content (Part I & II) were performed for detailed analysis of cartilage
regeneration.
Part I: The development of custom made static culture device, consisting of inserts in which OCE is fixed and deep well plate, allowed tissue specific media supply without
supplementation of TGF . Critical size diameter was defined to be 4 mm.
Part II: Biomaterials revealed differences in cartilage regeneration. Collagen I and fibrin
glue showed presence of cells migrated from OCE into cell free hydrogels with indication
of fibrous tissue formation by presence of proCollagen I. In chondrocyte loaded study
cartilage matrix proteins aggrecan, collagen II and VI and transcription factor SOX9 were
detected after ex vivo culture throughout the two natural hydrogels collagen I and fibrin
glue whereas markers were localized in pericellular matrix in starPEGh. Weak stainings resulted
for MP/HA and P(AGE/G)-HA-SH in some cell clusters. Gene expression data and
proteoglycan quantification supported histological findings with tendency of hypertrophy
indicated by upregulation of collagen X and RunX2 in MP/HA and P(AGE/G)-HA-SH.
Part III: In life-dead stainings recruitment of cells from OCE into empty or cell free
collagen I treated chondral defects was seen.
Separated and tissue specific media supply is critical to maintain ECM composition in
cartilage. Presence of OCE stimulates cartilage matrix synthesis in chondrocyte loaded
collagen I hydrogel and reduces hypertrophy compared to free swelling conditions and
pellet cultures. Differences in cartilage repair tissue formation resulted in preference of
natural derived polymers compared to synthetic based materials. The ex vivo cartilage
defect model represents a platform for testing novel hydrogels as cartilage materials, but
also to investigate the effect of cell seeding densities, cell gradients, cell co-cultures on
defect regeneration dependent on defect depth. The separated media compartments allow
for systematic analysis of pharmaceutics, media components or inflammatory cytokines on
bone and cartilage metabolism and matrix stability.
In Deutschland erkranken jährlich etwa 500.000 Menschen an Krebs, wovon circa
12.000 die Diagnose „Leukämie“ gestellt bekommen [1]. Unter den Leukämien weist
die akute myeloische Leukämie (AML) die ungünstigste Prognose auf, sodass hier
erheblicher Forschungsbedarf besteht. Zusätzlich schnitten viele potentielle Therapeutika,
die sich in bisherigen präklinischen Testsystemen als vielversprechend erwiesen
haben, in klinischen Studien schlecht ab [8]. Ziel dieser Arbeit war daher die
Etablierung eines 3D in vitro Blutgefäß-/Gewebemodells als verbessertes präklinisches
System zur Testung von Therapeutika, die zur erfolgreichen Behandlung von
Leukämien beitragen sollen.
Das 3D Blutgefäßmodell bestand aus humanen primären Endothelzellen, welche
als Monolayer auf der Serosaseite einer dezellularisierten, porzinen, intestinalen Kollagenmatrix
(SIS-Ser) wuchsen. Nach 14-tägiger Zellkultur wurden dem Versuchsansatz
entsprechend nichtadhärente THP-1 Zellen (AML-M5-Zelllinie) und Tipifarnib
oder entsprechende Kontrolllösungen beziehungsweise bimolekulare Antikörperkonstrukte
mit PBMCs als Effektorzellen hinzupipettiert. Nach 5-tägiger Inkubation
mit Tipifarnib beziehungsweise 24-stündiger Behandlung mit Antikörperkonstrukten
wurde der therapiebedingte Anstieg der Apoptoserate in den malignen THP-1 Zellen
mittels durchflusszytometrischer Analyse der Modellüberstände ermittelt. Zum
Ausschluss verbliebener und durchflusszytometrisch zu analysierender Zellen wurde,
stellvertretend für alle Suspensionszellen, eine Anti-CD13/DAB-Färbung durchgeführt,
welche negativ ausfiel. Mögliche Kollateralschäden am Endothel wurden
mittels histologischen Färbemethoden an Gewebeparaffinschnitten untersucht.
In der Durchflusszytometrie zeigte Tipifarnib sowohl im 2D als auch im 3D Modell
äquivalente, dosisabhängige und antileukämische Auswirkungen auf die THP-1 Zellen.
Bei Applikation der Antikörperkonstrukte ließ lediglich die Kombination beider Hemibodies signifikante Effekte auf die THP-1 Zellen erkennen. Dabei zeigten sich
bei konstanten Konzentrationen der Antikörperkonstrukte im 3D Modell deutlich
höhere Apoptoseraten (58%) als im 2D Modell (38%). Stellt man Vergleiche von
Tipifarnib mit den T-Zell-rekrutierenden Antikörperkonstrukten an, so ließen sich
im 2D Modell ähnliche Apoptoseraten in den THP-1 Zellen erzielen (jeweils 38% bei
Anwendung von 500 nM Tipifarnib). In den 3D Modellen erzielten jedoch die niedriger
konzentrierten Antikörperkonstrukte bei kürzerer Inkubationsdauer eine noch
höhere spezifische Apoptoserate in den THP-1 Zellen (im Mittel 58%) als 500 nM
Tipifarnib (mittlere Apoptoserate 40%). Bezüglich der Nebenwirkungen ließ sich
im 3D Modell nach Applikation von Antikörperkonstrukten kein wesentlicher Einfluss
auf das Endothel erkennen, während Tipifarnib/DMSO als auch die mit DMSO
versetzten Kontrolllösungen zu einer dosisabhänigen Destruktion des ursprünglichen
Endothelzellmonolayers führten. Damit stellt die hier beschriebene, hoch spezifische,
Hemibody-vermittelte Immuntherapie einen vielversprechenden Ansatz für zukünftige
onkologische Therapien dar.
Mithilfe des etablierten humanen 3D in vitro Modells konnte im Vergleich zur
konventionellen Zellkultur eine natürlichere Mikroumgebung für Zellen geschaffen
und die Auswirkungen der Testsubstanzen sowohl auf maligne Zellen, als auch auf
die Gefäßstrukturen untersucht werden.
Multiple myeloma (MM), a malignancy of the bone marrow, is characterized by a pathological increase in antibody-producing plasma cells and an increase in immunoglobulins (plasmacytosis). In recent years, bone morphogenetic proteins (BMPs) have been reported to be activators of apoptotic cell death in neoplastic B cells in MM. Here, we use bone morphogenetic protein 2 (BMP2) to show that the "apoptotic" effect of BMPs on human neoplastic B cells is dominated by anti-proliferative activities and cell cycle arrest and is apoptosis-independent. The anti-proliferative effect of BMP2 was analysed in the human cell lines KMS12-BM and L363 using WST-1 and a Coulter counter and was confirmed using CytoTox assays with established inhibitors of programmed cell death (zVAD-fmk and necrostatin-1). Furthermore, apoptotic activity was compared in both cell lines employing western blot analysis for caspase 3 and 8 in cells treated with BMP2 and FasL. Additionally, expression profiles of marker genes of different cell death pathways were analysed in both cell lines after stimulation with BMP2 for 48h using an RT-PCR-based array. In our experiments we observed that there was rather no reduction in absolute cell number, but cells stopped proliferating following treatment with BMP2 instead. The time frame (48–72 h) after BMP2 treatment at which a reduction in cell number is detectable is too long to indicate a directly BMP2-triggered apoptosis. Moreover, in comparison to robust apoptosis induced by the approved apoptotic factor FasL, BMP2 only marginally induced cell death. Consistently, neither the known inhibitor of apoptotic cell death zVAD-fmk nor the necroptosis inhibitor necrostatin-1 was able to rescue myeloma cell growth in the presence of BMP2.
Multiple myeloma (MM) represents a haematological cancer characterized by the pathological hyper proliferation of antibody-producing B-lymphocytes. Patients typically suffer from kidney malfunction and skeletal disorders. In the context of MM, the transforming growth factor β (TGFβ) member Activin A was recently identified as a promoter of both accompanying symptoms. Because studies have shown that bone morphogenetic protein (BMP)-2-mediated activities are counteracted by Activin A, we analysed whether BMP2, which also binds to the Activin A receptors ActRII and ActRIIB but activates the alternative SMAD-1/5/8 pathway, can be used to antagonize Activin A activities, such as in the context of MM. Therefore three BMP2 derivatives were generated with modified binding activities for the type II (ActRIIB) and/or type I receptor (BMPRIA) showing either increased or decreased BMP2 activity. In the context of MM these BMP2 muteins show two functionalities since they act as a) an anti-proliferative/apoptotic agent against neoplastic B-cells, b) as a bone-formation promoting growth factor. The molecular basis of both activities was shown in two different cellular models to clearly rely on the properties of the investigated BMP2 muteins to compete for the binding of Activin A to the Activin type II receptors. The experimental outcome suggests new therapeutic strategies using BMP2 variants in the treatment of MM-related pathologies.
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.
Site Directed Immobilization of BMP-2: Two Approaches for the Production of Osteoinductive Scaffolds
(2017)
Bone fractures typically heal without surgical intervention. However, pathological situations exist which impede the healing process resulting in so-called non-union fractures. Such fractures are nowadays treated with scaffold material being introduced into the defect area. These scaffolds can be doped with osteogenic factors, such as bone morphogenetic protein (BMP)2. BMP2 belongs to the most osteogenic growth factors known to date. Its medical use, efficiency and safety have been approved by FDA for certain applications. Currently, BMP2 is distributed with a stabilizing scaffold, which is simply soaked with the growth factor. Due to fast release kinetics supraphysiological high doses of BMP2 are required which are causally associated with severe side effects observed in certain applications being most harmful in the area of the cervical spine. These side-effects include inflammation, swelling and breathing problems, leading to disastrous consequences or secondary surgical interventions. Since it could be shown that a retardation of BMP2 release from the scaffold resulted in superior bone forming properties in vivo, it seems obvious to further reduce this release to a minimum. This can be achieved by covalent coupling which in the past was already elaborated using mainly classical EDC/NHS chemistry. Using this technique coupling of the protein occurs non-site-directedly leading mainly to an unpredictable product outcome with variable osteogenic activities. In order to improve the reproducibility of scaffold functionalization by BMP2 we created variants one of which contains a unique unnatural amino acid substitution within the mature polypeptide sequence (BMP2-K3Plk) and another, BMP2-A2C, in which an N-terminal alanine has been substituted by cysteine. These modifications enable site-specific and covalent immobilization of BMP2 e.g. onto polymeric beads. Both proteins were expressed in E. coli, renatured and purified by cation-exchange chromatography. Both variants were extensively analyzed in terms of purity and biological activity which was tested by in vitro interaction analyses as well as in cell based assays. Both proteins could be successfully coupled to polymeric beads. The different BMP2 functionalized beads were shown to interact with the ectodomain of the type I receptor BMPR-IA in vitro indicating that the biological activity of both BMP2 variants retained upon coupling. Both functionalized beads induced osteogenic differentiation C2C12 cells but only of those cells which have been in close contact to the particular beads. This strongly indicates that the BMP2 variant are indeed covalently coupled and not just adsorbed.
We claim that we have developed a system for a site-specific and covalent immobilization of BMP-2 onto solid scaffolds, potentially eliminating the necessity of high-dose scaffold loading. Since immobilized proteins are protected from removal by extracellular fluids, their activities now rely mainly on the half-life of the used scaffold and the rate of proteolytic degradation. Assuming that due to prolonged times much lower loading capacities might be required we propose that the immobilization strategy employed in this work may be further refined and optimized to replace the currently used BMP2-containing medical products.
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.
In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm\(^{2}\) and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies.
Despite medical achievements, the number of patients with end-stage kidney disease keeps steadily raising, thereby entailing a high number of surgical and interventional procedures to establish and maintain arteriovenous vascular access for hemodialysis. Due to vascular disease, aneurysms or infection, the preferred access—an autogenous arteriovenous fistula—is not always available and appropriate. Moreover, when replacing small diameter blood vessels, synthetic vascular grafts possess well-known disadvantages. A continuous multilayered gradient electrospinning was used to produce vascular grafts made of collagen type I nanofibers on luminal and adventitial graft side, and poly-ɛ-caprolactone as medial layer. Therefore, a custom-made electrospinner with robust environmental control was developed. The morphology of electrospun grafts was characterized by scanning electron microscopy and measurement of mechanical properties. Human microvascular endothelial cells were cultured in the graft under static culture conditions and compared to cultures obtained from dynamic continuous flow bioreactors. Immunofluorescent analysis showed that endothelial cells form a continuous luminal layer and functional characteristics were confirmed by uptake of acetylated low-density-lipoprotein. Incorporation of vancomycin and gentamicin to the medial graft layer allowed antimicrobial inhibition without exhibiting an adverse impact on cell viability. Most striking a physiological hemocompatibility was achieved for the multilayered grafts.
New multifunctional nanoparticles (NPs) that can be used as contrast agents (CA) in different imaging techniques, such as photoluminescence (PL) microscopy and magnetic resonance imaging (MRI), open new possibilities for medical imaging, e.g., in the fields of diagnostics or tissue characterization in regenerative medicine. The focus of this study is on the synthesis and characterization of CaF\(_{2}\):(Tb\(^{3+}\),Gd\(^{3+}\)) NPs. Fabricated in a wet-chemical procedure, the spherical NPs with a diameter of 5–10 nm show a crystalline structure. Simultaneous doping of the NPs with different lanthanide ions, leading to paramagnetism and fluorescence, makes them suitable for MR and PL imaging. Owing to the Gd\(^{3+}\) ions on the surface, the NPs reduce the MR T\(_{1}\) relaxation time constant as a function of their concentration. Thus, the NPs can be used as a MRI CA with a mean relaxivity of about r = 0.471 mL·mg\(^{−1}\)·s\(^{−1}\). Repeated MRI examinations of four different batches prove the reproducibility of the NP synthesis and determine the long-term stability of the CAs. No cytotoxicity of NP concentrations between 0.5 and 1 mg·mL\(^{−1}\) was observed after exposure to human dermal fibroblasts over 24 h. Overall this study shows, that the CaF\(_{2}\):(Tb\(^{3+}\),Gd\(^{3+}\)) NPs are suitable for medical imaging.
Despite growing effort to advance materials towards a low fibrotic progression, all implants elicit adverse tissue responses. Pre-clinical biomaterial assessment relies on animals testing, which can be complemented by in vitro tests to address the Russell and Burch’s 3R aspect of reducing animal burden. However, a poor correlation between in vitro and in vivo biomaterial assessments confirms a need for suitable in vitro biomaterial tests. The aim of the study was to identify a test setting, which is predictive and might be time- and cost-efficient. We demonstrated how sensitive in vitro biomaterial assessment based on human primary macrophages depends on test conditions. Moreover, possible clinical scenarios such as lipopolysaccharide contamination, contact to autologous blood plasma, and presence of IL-4 in an immune niche influence the outcome of a biomaterial ranking. Nevertheless, by using glass, titanium, polytetrafluorethylene, silicone, and polyethylene representing a specific material-induced fibrotic response and by comparison to literature data, we were able to identify a test condition that provides a high correlation to state-of-the-art in vivo studies. Most important, biomaterial ranking obtained under native plasma test conditions showed a high predictive accuracy compared to in vivo assessments, strengthening a biomimetic three-dimensional in vitro test platform.
Entwicklungsaspekte eines Medizinproduktes zur Prävention und Überwachung von Hydrierungszuständen
(2018)
Der demografische Wandel und das Populationswachstum stellen eine globale Herausforderung für die Gesundheitssysteme dar. Eine vielversprechende Lösungsstrategie liegt in der digitalen Überwachung, Prävention und Therapie akuter und chronischer Erkrankungen durch die Nutzung von innovativen Technologien aus dem Bereich der personalisierten Medizin.
Die Digitalisierung in der Überwachung von Vitalparametern mittels Sensorik besitzt großes Potential für die längere Gesunderhaltung der Patienten und somit die Entlastung der Gesundheitssyteme im Ganzen.
Da Wassermangel für eine Vielfalt von Krankheiten einen Katalysator darstellt, ist die Hydratation ein wichtiger aber bislang nur invasiv zugänglicher Vitalparameter. Zur Etablierung nicht invasiver Messungen des Wasserhaushaltes am Menschen wurde im Rahmen dieser Arbeit die Eignung der Mikrowellentechnologie untersucht.
Dehydratation resultiert in der Veränderung des Osmolythaushaltes und beeinflusst biochemische Prozesse, was zur Entstehung von Morbidität führen kann. Im Rahmen der Arbeit werden Teilbereiche der Entwicklung eines Medizinproduktes abgebildet. Zu diesem Zweck wird die Machbarkeit der mikrowellenbasierten Analyse des Wasserhaushaltes in einer technischen Machbarkeitsstudie untersucht, um im zweiten Prozessschritt einen technischen Demonstrator in vitro und in vivo am Probanden erproben zu können. Hochfrequente elektromagnetische Wellen interagieren mit Molekülen, speziell Wasser. Enthält eine Probe freie Wassermoleküle, kann dies im reflektierten Signal detektiert werden. Zur Überprüfung des Sensorsystems in vitro dienen humane 3D-Vollhautmodelle mit spezifischer Hydratation und Gewebedichte der Matrixkomponenten als standardisiertes Modell zur Untersuchung definierter Exsikkoseszenarien und des Einflusses verschiedener Modellkomplexitäten. Die Eignungsüberprüfung des Systems mit einem technischen Demonstrator des künftigen Medizinproduktes belegt die Anwendbarkeit des Messsystems zur Erfassung des relativen Wassergehaltes. Die Technologie zeichnet sich durch eine hohe Sensitivität bei der Destinktion von Proben mittels Frequenz- und Signalreflektionsdifferenzen aus. Neben den In-vitro-Testungen wird das entwickelte Sensorsystem aus regulatorischer Sicht zur klinischen Leistungsüberprüfung vorbereitet und im Rahmen eines bewilligten Ethikvotums in vivo erprobt. Die Ergebnisse belegen die Machbarkeit der nichtinvasiven Erfassung des Wasserhaushaltes durch mikrowellenbasierte Messungen. Die Technologie birgt das Potential, in ein körpernahes Sensorsystem integriert zu werden, welches als Medizinprodukt zur persönlichen Gesundheitsüberwachung zugelassen werden kann.
Highly invasive animal based test procedures for risk assessment such as the Draize eye test are under increasing criticism due to poor transferability for the human organism and animal-welfare concerns. However, besides all efforts, the Draize eye test is still not completely replaced by alternative animal-free methods. To develop an in vitro test to identify all categories of eye irritation, we combined organotypic cornea models based on primary human cells with an electrical readout system that measures the impedance of the test models. First, we showed that employing a primary human cornea epithelial cell based model is advantageous in native marker expression to the primary human epidermal keratinocytes derived models. Secondly, by employing a non-destructive measuring system based on impedance spectroscopy, we could increase the sensitivity of the test system. Thereby, all globally harmonized systems categories of eye irritation could be identified by repeated measurements over a period of 7 days. Based on a novel prediction model we achieved an accuracy of 78% with a reproducibility of 88.9% to determine all three categories of eye irritation in one single test. This could pave the way according to the 3R principle to replace the Draize eye test.
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.
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.
In tissue engineering, the generation and functional maintenance of dense voluminous tissues is mainly restricted due to insufficient nutrient supply. Larger three-dimensional constructs, which exceed the nutrient diffusion limit become necrotic and/or apoptotic in long-term culture if not provided with an appropriate vascularization. Here, we established protocols for the generation of a pre-vascularized biological scaffold with intact arterio-venous capillary loops from rat intestine, which is decellularized under preservation of the feeding and draining vascular tree. Vessel integrity was proven by marker expression, media/blood reflow and endothelial LDL uptake. In vitro maintenance persisted up to 7 weeks in a bioreactor system allowing a stepwise reconstruction of fully vascularized human tissues and successful in vivo implantation for up to 4 weeks, although with time-dependent decrease of cell viability. The vascularization of the construct lead to a 1.5× increase in cellular drug release compared to a conventional static culture in vitro. For the first time, we performed proof-of-concept studies demonstrating that 3D tissues can be maintained within a miniaturized vascularized scaffold in vitro and successfully implanted after re-anastomosis to the intrinsic blood circulation in vivo. We hypothesize that this technology could serve as a powerful platform technology in tissue engineering and regenerative medicine.
The pancreas and the small intestine are pivotal organs acting in close synergism to regulate glucose metabolism. After absorption and processing of dietary glucose within the small intestine, insulin and glucagon are released from pancreatic islet cells to maintain blood glucose homeostasis. Malfunctions affecting either individual, organ-specific functions or the sophisticated interplay of both organs can result in massive complications and pathologic conditions. One of the most serious metabolic diseases of our society is diabetes mellitus (DM) that is hallmarked by a disturbance of blood glucose homeostasis. Type 1 (T1DM) and type 2 (T2DM) are the main forms of the disease and both are characterized by chronic hyperglycemia, a condition that evokes severe comorbidities in the long-term. In the past, several standard treatment options allowed a more or less adequate therapy for diabetic patients. Albeit there is much effort to develop new therapeutic interventions to treat diabetic patients in a more efficient way, no cure is available so far. In view of the urgent need for alternative treatment options, a more systemic look on whole organ systems, their biological relation and complex interplay is needed when developing new therapeutic strategies for DM.
T1DM is hallmarked by an autoimmune-mediated destruction of the pancreatic β-cell mass resulting in a complete lack of insulin that is in most patients restored by applying a life-long recombinant insulin therapy. Therefore, novel regenerative medicine-based concepts focus on the derivation of bioartificial β-like cells from diverse stem cell sources in vitro that survive and sustain to secrete insulin after implantation in vivo. In this context, the first part of this thesis analyzed multipotent intestinal stem cells (ISCs) as alternative cell source to derive bioartificial, pancreatic β-like cells in vitro. From a translational perspective, intestinal stem cells pose a particularly attractive cell source since intestinal donor tissues could be obtained via minimal invasive endoscopy in an autologous way. Furthermore, intestinal and pancreatic cells both derive from the same developmental origin, the endodermal gut tube, favoring the differentiation process towards functional β-like cells. In this study, pancreas-specific differentiation of ISCs was induced by the ectopic expression of the pancreatic transcription factor 1 alpha (Ptf1a), a pioneer transcriptional regulator of pancreatic fate. Furthermore, pancreatic lineage-specific culture media were applied to support the differentiation process. In general, ISCs grow in vitro in a 3D Matrigel®-based environment. Therefore, a 2D culture platform for ISCs was established to allow delivery and ectopic expression of Ptf1a with high efficiency. Next, several molecular tools were applied and compared with each other to identify the most suitable technology for Ptf1a delivery and expression within ISCs as well as their survival under the new established 2D conditions. Success of differentiation was investigated by monitoring changes in cellular morphology and induction of pancreatic differentiation-specific gene expression profiles. In summary, the data of this project part suggest that Ptf1a harbors the potential to induce pancreatic differentiation of ISCs when applying an adequate differentiation media. However, gene expression analysis indicated rather an acinar lineage-determination than a pancreatic β-cell-like specification. Nevertheless, this study proved ISCs not only as interesting stem cell source for the generation of pancreatic cell types with a potential use in the treatment of T1DM but alsoPtf1a as pioneer factor for pancreatic differentiation of ISCs in general.
Compared to T1DM, T2DM patients suffer from hyperglycemia due to insulin resistance. In T2DM management, the maintenance of blood glucose homeostasis has highest priority and can be achieved by drugs affecting the stabilization of blood glucose levels. Recent therapeutic concepts are aiming at the inhibition of the intestinal glucose transporter Na+-D-Glucose cotransporter 1 (SGLT1). Pharmacological inhibition of SGLT1 results in reduced postprandial blood glucose levels combined with a sustained and increased Glucagon-like peptide 1 (GLP-1) secretion. So far, systemic side effects of this medication have not been addressed in detail. Of note, besides intestinal localization, SGLT1 is also expressed in various other tissues including the pancreas. In context of having a closer look also on the interplay of organs when developing new therapeutic approaches for DM, the second part of this thesis addressed the effects on pancreatic islet integrity after loss of SGLT1. The analyses comprised the investigation of pancreatic islet size, cytomorphology and function by the use of a global SGLT1 knockout (SGLT1-/-) mouse model. As SGLT1-/- mice develop the glucose-galactose malabsorption syndrome when fed a standard laboratory chow, these animals derived a glucose-deficient, fat-enriched (GDFE) diet. Wildtype mice on either standard chow (WTSC) or GDFE (WTDC) allowed the discrimination between diet- and knockout-dependent effects. Notably, GDFE fed mice showed decreased expression and function of intestinal SGLT1, while pancreatic SGLT1 mRNA levels were unaffected. Further, the findings revealed increased isled sizes, reduced proliferation- and apoptosis rates as well as an increased α-cell and reduced β-cell proportion accompanied by a disturbed cytomorphology in islets when SGLT1 function is lost or impaired. In addition, pancreatic islets were dysfunctional in terms of insulin- and glucagon-secretion. Moreover, the release of intestinal GLP-1, an incretin hormone that stimulates insulin-secretion in the islet, was abnormal after glucose stimulatory conditions. In summary, these data show that intestinal SGLT1 expression and function is nutrient dependent. The data obtained from the islet studies revealed an additional and new role of SGLT1 for maintaining pancreatic islet integrity in the context of structural, cytomorphological and functional aspects. With special emphasis on SGLT1 inhibition in diabetic patients, the data of this project indicate an urgent need for analyzing systemic side effects in other relevant organs to prove pharmacological SGLT1 inhibition as beneficial and safe.
Altogether, the findings of both project parts of this thesis demonstrate that focusing on the molecular and cellular relationship and interplay of the small intestine and the pancreas could be of high importance in context of developing new therapeutic strategies for future applications in DM patients.
Pacemaker systems are an essential tool for the treatment of cardiovascular diseases. However, the immune system’s natural response to a foreign body results in the encapsulation of a pacemaker electrode and an impaired energy efficiency by increasing the excitation threshold. The integration of the electrode into the tissue is affected by implant properties such as size, mechanical flexibility, shape, and dimensionality. Three-dimensional, tissue-like electrode scaffolds render an alternative to currently used planar metal electrodes. Based on a modified electrospinning process and a high temperature treatment, a conductive, porous fiber scaffold was fabricated. The electrical and immunological properties of this 3D electrode were compared to 2D TiN electrodes. An increased surface of the fiber electrode compared to the planar 2D electrode, showed an enhanced electrical performance. Moreover, the migration of cells into the 3D construct was observed and a lower inflammatory response was induced. After early and late in vivo host response evaluation subcutaneously, the 3D fiber scaffold showed no adverse foreign body response. By embedding the 3D fiber scaffold in human cardiomyocytes, a tissue-electrode hybrid was generated that facilitates a high regenerative capacity and a low risk of fibrosis. This hybrid was implanted onto a spontaneously beating, tissue-engineered human cardiac patch to investigate if a seamless electronic-tissue interface is generated. The fusion of this hybrid electrode with a cardiac patch resulted in a mechanical stable and electrical excitable unit. Thereby, the feasibility of a seamless tissue-electrode interface was proven.
Unraveling the connection between fibroblast growth factor and bone morphogenetic protein signaling
(2018)
Ontogeny of higher organisms as well the regulation of tissue homeostasis in adult individuals requires a fine-balanced interplay of regulating factors that individually trigger the fate of particular cells to either stay undifferentiated or to differentiate towards distinct tissue specific lineages. In some cases, these factors act synergistically to promote certain cellular responses, whereas in other tissues the same factors antagonize each other. However, the molecular basis of this obvious dual signaling activity is still only poorly understood. Bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs) are two major signal protein families that have a lot in common: They are both highly preserved between different species, involved in essential cellular functions, and their ligands vastly outnumber their receptors, making extensive signal regulation necessary. In this review we discuss where and how BMP and FGF signaling cross paths. The compiled data reflect that both factors synchronously act in many tissues, and that antagonism and synergism both exist in a context-dependent manner. Therefore, by challenging a generalization of the connection between these two pathways a new chapter in BMP FGF signaling research will be introduced.
Trotz hochmoderner Technologien und ausgefeilter therapeutischer und rekonstruktiver chirurgischer Heilungsmethoden beträgt die 5-Jahres Überlebensrate bei der Diagnose PECA der Mundhöhle im Durchschnitt auch im Jahre 2017 nur 55 % und die Heilungsmethoden haben sich in den letzten drei Jahrzehnten kaum verbessert. Umso wichtiger ist es deshalb, die Forschung voranzutreiben und ein aussagekräftiges Tumormodell zu etablieren, das bei der Entwicklung neuer Therapieansätze schnell und sicher gute Ergebnisse liefert.
In dieser Studie soll mit Hilfe des Tissue Engineering (TE) ein in gesunder Mundschleimhaut (MSH) integriertes 3D-Tumormodell generiert werden, welches bestmöglich die Analyse pathologischer Mechanismen im Tumorzentrum, sowie im Randbereich von gesundem und erkranktem Gewebe, und auch die Analyse der Auswirkungen neuartiger Chemotherapeutika auf gesunde und maligne Zellen in direkter Nachbarschaft ermöglicht – ohne Tierversuche. In der Konsequenz könnte ein erheblicher Fortschritt mit höheren Erfolgsaussichten der Therapieansätze erzielt werden.
Es wird ein Tumormodell generiert, in dem auf Basis eines gesunden MSH-Modells Tumorzellen eingebracht werden, um - genauso, wie die Tumorentstehung in vivo von statten gehen würde – Tumorentstehung und Tumorwachstum in der Umgebung von gesunder MSH analysieren zu können. Das Modell basiert dabei auf einer Matrix aus dezellularisierter, porciner, small-intestinal-submucosa (SIS/MUC), die mit primären Fibroblasten, primären Keratinozyten und Tumorzellen der Tumorzelllinie FaDu besiedelt wird. Eine Besonderheit der FaDu-Zellen ist die vorangegangene Transduktion mit dem Lentivirus RFP – um die eingewanderten Zellen von gesunden Zellen unterscheiden zu können. Der Vorgang der Transduktion war gelungen und es konnte eine Fluoreszenz der noch in Zellkulturschalen kultivierten Zellen erzielt werden. Allerdings waren die fluoreszierenden Zellen in den fixierten Schnitten nicht mehr nachweisbar.
Zur Generierung eines Tumormodelles wurden auf Basis eines OMÄ drei unterschiedliche Applikationsformen zur Integration von Tumorzellen getestet. Die Integration von Tumorzellen fand in Form von Spots, Sphäroiden oder Tumorzellgemischen (prim. Keratinozyten/FaDu-Zellen) in zuvor kultivierte gesunde OMÄ statt. Dabei sollte das Applizieren von Spots oder Sphäroiden das Tumorzellwachstum auch in der Umgebung von gesundem Gewebe initiieren. Dies würde die Möglichkeit schaffen, auch in vitro, gesundes neben pathologischem Gewebe und den Übergang dazu genau analysieren zu können.
Es sollen sowohl die optimale Konzentration der Tumorzellen, welche für die Entstehung von Tumoren nötig ist, als auch die geeignetste Applikationsmethode eruiert werden, um optimale Tumormodelle zuverlässig reproduzierbar ansetzen zu können. Die Modelle wurden histologisch und immunhistochemisch analysiert und die Ergebnisse mit ermittelten TEER-Werte in Korrelation gesetzt.
In dieser Arbeit konnte mit der Applikation von Spots oder Sphäroiden kein suffizientes Tumorwachstum in Umgebung von gesunder MSH erzielt werden. Die Zellen lagen ohne Reaktion des angrenzenden Stratum corneums auf der zu stark ausgeprägten Hornschicht der OMÄ auf und es war keine Einwanderung in das darunterliegende Gewebe möglich.
Allerdings ist es gelungen, durch Applikation eines Zellgemisches variierender Mischungsverhältnisse von primären Keratinozyten und Tumorzellen der Zelllinie FaDu ein 3D-Tumorwachstum unterschiedlicher Malignitätsstufen zu initiieren. Je kleiner das Mischungsverhältnis und je höher in der Konsequenz die Anzahl der FaDu-Tumorzellen, desto ausgeprägter waren die morphologischen Anzeichen einer Tumorbildung. Abhängig vom Mischungsverhältnis war dabei die Ausprägung des Tumors. Auch wenn dadurch keine Kombination von gesundem und pathologischem Gewebe in einem Modell mehr imitiert werden konnte, so konnten zumindest nach histologischen und immunhistochemischen Untersuchungen eindeutige pathologische, maligne Tumormodelle generiert werden. Die Tumormodelle zeigten durchgehend Zell- und Kernpleomorphismen, atypische und vermehrte suprabasale Mitosen, eine Störung der normalen Gewebearchitektur, die Ausbildung von Interzellularbrücken, Einzelzelldyskeratosen und Verhornungsknospen, sowie Stellen der Durchbrechung der Basalmembran und Invasion von Tumorzellen in die darunterliegende Lamina propria. All das sind eindeutige Kennzeichen malignen Wachstums
Auch die Ergebnisse der TEER-Wert Messung stimmten mit den morphologischen Entwicklungen der Modelle überein. So stiegen die TEER-Werte der Kontrollmodelle konsequent an, was für eine deutliche Entwicklung von kontinuierlichem Gewebe spricht und im Gegensatz dazu fielen die TEER-Werte im zeitlichen Verlauf der Tumormodelle, bei denen die Basalmembran und somit die Kontinuität des Gewebes durchbrochen wurde rapide ab, bzw. lagen im konstant niedrigen Bereich.
Der Erfolg der Etablierung dieses zuverlässig rekonstruierbaren 3D, in vitro generierten Tumormodells, das der in vivo Situation eines Plattenepithelkarzinoms sehr nahekommt, bietet der Wissenschaft eine sehr gute Möglichkeit, weitere Studien zum Tumorwachstum durchzuführen. Außerdem kann die Weiterentwicklung und Verbesserung vielversprechender, neuartiger chemotherapeutischer und radiologischer Therapieverfahren erheblich voran¬getrieben und dadurch die Heilungschancen mit geringeren Nebenwirkungen für den Patienten verbessert und eine erhöhte Lebensqualität erzielt werden.
Ziel der vorliegenden Arbeit war es, die Biokompatibilität von Kollagen I-basierten ACL-Konstrukten in-vitro und in-vivo zu überprüfen. Zudem erfolgte eine histologische Charakterisierung der Konstrukte nach sechswöchiger bzw. sechsmonatiger Versuchslaufzeit im Minipig-Tiermodell.
Das Kollagen I wurde durch eine neuartige Methode aus Rattenschwänzen isoliert und zu einem Implantat geknotet und gewickelt. Die Fasern wurden mittels Proliferationsmessung, Proteinbestimmung, Zellzählung und Zellmorphologie auf in-vitro-Biokompatibilität getestet. Hier zeigte sich eine gute Biokompatibilität sowohl für γ-sterilisierte Fasern als auch für nicht sterilisierte Fasern. In der Sterilitätsüberprüfung waren nach Anpassung des Sterilisationsverfahrens weder Bakterien- noch Pilzwachstum nachweisbar. Diese Ergebnisse sind vergleichbar mit vielfältigen Studien zur Biokompatibilität von Kollagen, in denen jeweils gute Zellviabilität und –proliferation im direkten oder indirekten Kontakt mit Kollagen gezeigt werden konnte.
Anschließend wurde das Konstrukt im Tierversuch direkt im Kniegelenk als vorderer Kreuzbandersatz implantiert. Nach Ablauf der Standzeit und Explantation der Kniegelenke wurden Paraffinschnittpräparate der Implantate sowie Paraffinschnittpräparate und Kunststoffschnittpräparate der ossa femora angefertigt und durchlichtmikroskopisch deskriptiv ausgewertet. Zusätzlich wurden die immunhistochemischen Färbungen Kollagen I des Schweins und der Ratte und Faktor VIII angefertigt, wobei in der Faktor VIII-Färbung zusätzlich eine quantitative Auswertung der Gefäßzahl vorgenommen wurde. Es wurde in der Kollagenfärbung ein Ersatz des Rattenkollagens durch das Schweinekollagen einhergehend mit einer hohen Zellzahl gezeigt. Eine synoviale Deckschicht und eine fortschreitende Vaskularisierung, sowie Form und Anordnung der Zellen zeigten Vorgänge des Remodeling. Innerhalb von 6 Monaten nahm die Vaskularisierung zu und neu gebildeter Geflechtknochen verengte die Bohrkanäle. Die Knochen-Implantat-Heilung war im Bohrkanal durch Sharpey´sche Fasern gekennzeichnet. Am Tunnelausgang fanden sich von sechs Wochen zu sechs Monaten Hinweise auf die fortschreitende Entwicklung einer direkten Bandinsertion.
Diese Ergebnisse entsprechen weitgehend den in der Literatur beschriebenen Remodelingvorgängen bei Studien zum Thema Kreuzbandersatz. Die beginnende direkte Bandinsertion spricht für eine gute Fixation und die Einheilung begünstigende Eigenschaften des Implantates. Dies ist ein geeigneter Ansatz für weitere Untersuchungen. Von Seiten der Biokompatibilität und der Integration des Gewebes ist das Implantat zum Kreuzbandersatz geeignet. Es bleibt abzuwarten, inwieweit die erforderlichen mechanischen Eigenschaften erreicht werden können.
Development and proof of concept of a biological vascularized cell‐based drug delivery system
(2019)
A major therapeutic challenge is the increasing incidence of chronic disorders.
The persistent impairment or loss of tissue function requires constitutive on‐demand
drug availability optimally achieved by a drug delivery system ideally directly connected
to the blood circulation of the patient. However, despite the efforts and achievements in
cell‐based therapies and the generation of complex and customized cell‐specific
microenvironments, the generation of functional tissue is still unaccomplished.
This study demonstrates the capability to generate a vascularized platform technology to
potentially overcome the supply restraints for graft development and clinical application
with immediate anastomosis to the blood circulation.
The ability to decellularize segments of the rat intestine while preserving the ECM for
subsequent reendothelialization was proven. The reestablishment of a functional
arteriovenous perfusion circuit enabled the supply of co‐cultured cells capable to replace
the function of damaged tissue or to serve as a drug delivery system. During in vitro
studies, the applicability of the developed miniaturized biological vascularized scaffold
(mBioVaSc‐TERM®) was demonstrated. While indicating promising results in short term
in vivo studies, long term implantations revealed current limitations for the translation
into clinical application. The gained insights will impact further improvements of quality
and performance of this promising platform technology for future regenerative therapies.
The skeletal system forms the mechanical structure of the body and consists of bone, which is hard connective tissue. The tasks the skeleton and bones take over are of mechanical, metabolic and synthetic nature. Lastly, bones enable the production of blood cells by housing the bone marrow. Bone has a scarless self-healing capacity to a certain degree. Injuries exceeding this capacity caused by trauma, surgical removal of infected or tumoral bone or as a result from treatment-related osteonecrosis, will not heal. Critical size bone defects that will not heal by themselves are still object of comprehensive clinical investigation. The conventional treatments often result in therapies including burdening methods as for example the harvesting of autologous bone material. The aim of this thesis was the creation of a prevascularized bone implant employing minimally invasive methods in order to minimize inconvenience for patients and surgical site morbidity. The basis for the implant was a decellularized, naturally derived vascular scaffold (BioVaSc-TERM®) providing functional vessel structures after reseeding with autologous endothelial cells. The bone compartment was built by the combination of the aforementioned scaffold with synthetic β-tricalcium phosphate. In vitro culture for tissue maturation was performed using bioreactor technology before the testing of the regenerative potential of the implant in large animal experiments in sheep. A tibia defect was treated without the anastomosis of the implant’s innate vasculature to the host’s circulatory system and in a second study, with anastomosis of the vessel system in a mandibular defect. While the non-anastomosed implant revealed a mostly osteoconductive effect, the implants that were anastomosed achieved formation of bony islands evenly distributed over the defect.
In order to prepare preconditions for a rapid approval of an implant making use of this vascularization strategy, the manufacturing of the BioVaSc-TERM® as vascularizing scaffold was adjusted to GMP requirements.
Der myokardiale Ischämie-Reperfusions-Schaden (IR) hat eine hohe Relevanz in der Kardiologie und Herzchirurgie. Trotz intensiver Forschung ist es bislang nicht gelungen, eine
effektive Therapie des IR in den klinischen Alltag zu implementieren.
Mitochondrien spielen im IR eine wichtige Rolle. Die Raman-Spektroskopie mit Laserquellen von 785 nm Wellenlänge erlaubt die nicht-invasive Analyse pathophysiologischer Prozesse in vitro in Echtzeit. Daher eignet sich die Raman-spektroskopische Analyse von Mitochondrien möglicherweise dazu, notwendige neue Einblicke in die Pathophysiologie des myokardialen IR zu gewinnen.
Die vorliegende Arbeit analysierte die mitochondriale Funktion von subsarkolemmalen Mitochondrien im IR mit Hilfe bekannter Methoden. Anschließend erfolgte ein Vergleich der etablierten Methode „Clark-Elektrode“ mit der neu etablierten Raman-Spektroskopie zur Analyse der mitochondrialen Funktion im IR.
Cancer remains after cardiovascular diseases the leading cause of death worldwide and an estimated 8.2 million people died of it in 2012. By 2030, 13 million cancer deaths are expected due to the growth and ageing of the population. Hereof, colorectal cancer (CRC) is the third most common cancer in men and the second in women with a wide geographical variation across the world. Usually, CRC begins as a non-cancerous growth leading to an adenomatous polyp, or adenoma, arising from glandular cells. Since research has brought about better understanding of the mechanisms of cancer development, novel treatments such as targeted therapy have emerged in the past decades. Despite that, up to 95% of anticancer drugs tested in clinical phase I trials do not attain a market authorisation and hence these high attrition rates remain a key challenge for the pharmaceutical industry, making drug development processes enormously costly and inefficient. Therefore, new preclinical in vitro models which can predict drug responses in vivo more precisely are urgently needed. Tissue engineering not only provides the possibility of creating artificial three-dimensional (3D) in vitro tissues, such as functional organs, but also enables the investigation of drug responses in pathological tissue models, that is, in 3D cancer models which are superior to conventional two-dimensional (2D) cell cultures on petri dishes and can overcome the limitations of animal models, thereby reducing the need for preclinical in vivo models. In this thesis, novel 3D CRC models on the basis of a decellularised intestinal matrix were established. In the first part, it could be shown that the cell line SW480 exhibited different characteristics when grown in a 3D environment from those in conventional 2D culture. While the cells showed a mesenchymal phenotype in 2D culture, they displayed a more pronounced epithelial character in the 3D model. By adding stromal cells (fibroblasts), the cancer cells changed their growth pattern and built tumour-like structures together with the fibroblasts, thereby remodelling the natural mucosal structures of the scaffold. Additionally, the established 3D tumour model was used as a test system for treatment with standard chemotherapeutic 5-fluorouracil (5-FU). The second part of the thesis focused on the establishment of a 3D in vitro test system for targeted therapy. The US Food and Drug Administration has already approved of a number of drugs for targeted therapy of specific types of cancer. For instance, the small molecule vemurafenib (PLX4032, Zelboraf™) which demonstrated impressive response rates of 50–80% in melanoma patients with a mutation of the rapidly accelerated fibrosarcoma oncogene type B (BRAF) kinase which belongs to the mitogen active protein kinase (MAPK) signalling pathway. However, only 5% of CRC patients harbouring the same BRAF mutation respond to treatment with vemurafenib. An explanation for this unresponsiveness could be a feedback activation of the upstream EGFR, reactivating the MAPK pathway which sustains a proliferative signalling. To test this hypothesis, the two early passage cell lines HROC24 and HROC87, both presenting the mutation BRAF V600E but differing in other mutations, were used and their drug response to vemurafenib and/or gefitinib was assessed in conventional 2D cell culture and compared to the more advanced 3D model. Under 3D culture conditions, both cell lines showed a reduction of the proliferation rate only in the combination therapy approach. Furthermore, no significant differences between the various treatment approaches and the untreated control regarding apoptosis rate and viability for both cell lines could be found in the 3D tumour model which conferred an enhanced chemoresistance to the cancer cells. Because of the observed unresponsiveness to BRAF inhibition by vemurafenib as can be seen in the clinic for patients with BRAF mutations in CRC, the cell line HROC87 was used for further xenografting experiments and analysis of activation changes in the MAPK signalling pathway. It could be shown that the cells presented a reactivation of Akt in the 3D model when treated with both inhibitors, suggesting an escape mechanism for apoptosis which was not present in cells cultured under conventional 2D conditions. Moreover, the cells exhibited an activation of the hepatocyte growth factor receptor (HGFR, c-Met) in 2D and 3D culture, but this was not detectable in the xenograft model. This shows the limitations of in vivo models. The results suggest another feedback activation loop than that to the EGFR which might not primarily be involved in the resistance mechanism. This reflects the before mentioned high attrition rates in the preclinical drug testing.
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.
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.
Die Risikobewertung von Chemikalien ist für die öffentliche Gesundheit von entschei-dender Bedeutung, weshalb strenge Testverfahren zu deren toxikologischer Begutach-tung angewandt werden. Die ursprünglich tierbasierten Testverfahren werden aufgrund von neuen wissenschaftlichen Erkenntnissen und wegen ökonomischer Ineffizienz sowie ethischer Fragwürdigkeit immer mehr durch alternative Methoden ohne Tiermodelle ersetzt. Für den toxikologischen Endpunkt der Augenreizung wurden bereits die ersten alternativen Testsysteme auf der Basis von ex vivo- oder in vitro-Modellen entwickelt. Jedoch ist bis dato kein alternatives Testsystem in der Lage, das gesamte Spektrum der verschiedenen Kategorien der Augenreizungen nach dem global harmonisierten System zur Einstufung und Kennzeichnung von Chemikalien (GHS) vorherzusagen und damit den tierbasierten Draize-Augenreizungstest vollends zu ersetzen. Gründe hierfür sind fehlende physiologische Merkmale im Modell sowie eine destruktive Analysemethode.
Aufgrund dessen wurden in dieser Studie die Hypothesen getestet, ob ein verbessertes In-vitro-Modell oder eine zerstörungsfreie, hochsensitive Analysemethode die Vorher-sagekraft des Augenreizungstests verbessern können. Dafür wurden zunächst neue Mo-delle aus humanen Hornhaut- und Hautepithelzellen entwickelt. Die Modelle aus pri-mären cornealen Zellen zeigten eine gewebespezifische Expression der Marker Zytokera-tin 3 und 12 sowie Loricrin. In beiden Modellen konnte durch die Verkürzung der Kul-turdauer die Ausbildung einer Hornschicht verhindert werden. Die Modelle wiesen dadurch eine sensiblere Barriere vergleichbar der nativen Cornea auf. Darüber hinaus konnte durch die chemische Quervernetzung mit Polyethylenglykolsuccinimidylglutara-tester ein transparentes, nicht kontrahierendes Stroma-Äquivalent etabliert werden. Der Stroma-Ersatz konnte zur Generierung von Hemi- und Voll-Cornea-Äquivalenten einge-setzt werden und lieferte somit erste Ansatzpunkte für die Rekonstruktion der nativen Hornhaut.
Parallel dazu konnte ein zerstörungsfreies Analyseverfahren basierend auf der Impe-danzspektroskopie entwickelt werden, das wiederholte Messungen der Gewebeintegri-tät zulässt. Zur verbesserten Messung der Barriere in dreidimensionalen Modelle wurde hierfür ein neuer Parameter, der transepitheliale elektrische Widerstand (TEER) bei der Frequenz von 1000 Hz, der TEER1000 Hz definiert, der eine genauere Aussage über die Integrität der Modelle zulässt. Durch die Kombination der entwickelten cornealen Epithelzellmodelle mit der TEER1000 Hz-Messung konnte die Prädikitivität des Augenrei-zungstests auf 78 - 100 % erhöht werden. Von besonderer Bedeutung ist dabei, dass die nicht destruktive Messung des TEER1000 Hz zum ersten Mal erlaubte, die Persistenz von Irritationen durch wiederholte Messungen in einem in vitro-Modell zu erkennen und somit die GHS-Kategorie 1 von GHS-Kategorie 2 zu unterscheiden. Der wissenschaftli-che Gewinn dieser Forschungsarbeit ist ein neues Testverfahren, das alle GHS-Kategorien in einem einzigen in vitro-Test nachweisen und den Draize-Augenreizungstest gänzlich ersetzen kann.
Gonorrhea is the second most common sexually transmitted infection in the world and is caused by Gram-negative diplococcus Neisseria gonorrhoeae. Since N. gonorrhoeae is a human-specific pathogen, animal infection models are only of limited use. Therefore, a suitable in vitro cell culture model for studying the complete infection including adhesion, transmigration and transport to deeper tissue layers is required. In the present study, we generated three independent 3D tissue models based on porcine small intestinal submucosa (SIS) scaffold by co-culturing human dermal fibroblasts with human colorectal carcinoma, endometrial epithelial, and male uroepithelial cells. Functional analyses such as transepithelial electrical resistance (TEER) and FITC-dextran assay indicated the high barrier integrity of the created monolayer. The histological, immunohistochemical, and ultra-structural analyses showed that the 3D SIS scaffold-based models closely mimic the main characteristics of the site of gonococcal infection in human host including the epithelial monolayer, the underlying connective tissue, mucus production, tight junction, and microvilli formation. We infected the established 3D tissue models with different N. gonorrhoeae strains and derivatives presenting various phenotypes regarding adhesion and invasion. The results indicated that the disruption of tight junctions and increase in interleukin production in response to the infection is strain and cell type-dependent. In addition, the models supported bacterial survival and proved to be better suitable for studying infection over the course of several days in comparison to commonly used Transwell® models. This was primarily due to increased resilience of the SIS scaffold models to infection in terms of changes in permeability, cell destruction and bacterial transmigration. In summary, the SIS scaffold-based 3D tissue models of human mucosal tissues represent promising tools for investigating N. gonorrhoeae infections under close-to-natural conditions.
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.
Die Implantation eines Medizinprodukts in den menschlichen Körper ruft eine Immunreaktion hervor, die zur fibrösen Einkapselung führen kann. Makrophagen in direktem Kontakt mit der Oberfläche des Implantats erfassen sensorisch den Fremdkörper und übersetzten das Signal in die Freisetzung zahlreicher löslicher Mediatoren. Das generierte Entzündungsmilieu moduliert die Heilungsreaktion und kann zur Anreicherung von Fibroblasten sowie zur Erhöhung der Matrixsyntheserate in der Wundumgebung führen. Eine dichte fibröse Kapsel um ein Medizinprodukt beeinträchtigt den Ersatz von Körperstrukturen, das Unterstützen physiologischer Körperfunktionen sowie die Effizienz einer medizinischen Therapie. Zur Identifizierung potenzieller Biomaterialkandidaten mit optimalen Eigenschaften ist jedoch eine evidenzbasierte Entscheidungsfindung notwendig und diese wiederum muss durch geeignete Testmethoden unterstützt werden.
Zur Erfassung lokaler Effekte nach Implantation eines Biomaterials begründet die Komplexi-tät der ablaufenden Fremdkörperreaktion die Anwendung von Tiermodellen als Goldstandard. Die Eingliederung von in vitro Modellsystemen in standardisierte Testverfahren scheitert oft an der Verfügbarkeit validierter, verlässlicher und reproduzierbarer Methoden. Demnach ist kein standardisiertes in vitro Testverfahren beschrieben, das die komplexen dreidimensionalen Gewebsstrukturen während einer Fremdkörperreaktion abbildet und sich zur Testung über längere Kontaktphasen zwischen Blutkomponenten und Biomaterialien eignet. Jedoch können in vitro Testungen kosten- und zeiteffizienter sein und durch die Anwendung humaner Zellen eine höhere Übertragbarkeit auf den Menschen aufweisen. Zusätzlich adressiert die Präferenz zu in vitro Testmethoden den Aspekt „Reduzierung“ der 3R-Prinzipien „Replacement, Reduction, Refinement“ (Ersatz, Reduzierung, Verbesserung) von Russel und Burch (1959) zu einer bewussten und begründeten Anwendung von Tiermodellen in der Wissenschaft. Ziel von diesem Forschungsvorhaben war die Entwicklung von humanen in vitro Modellsystemen, die den Kontakt zu Blutkomponenten sowie die Reaktion des umliegenden Bindegewebes bei lokaler Implantation eines Biomaterials abbilden. Referenzmaterialien, deren Gewebsantwort nach Implantation in Tiere oder den Menschen bekannt ist, dienten als Validierungskriterium für die entwickelten Modellsysteme. Die Anreicherung von Zellen sowie die Bildung extrazellulärer Matrix in der Wundumgebung stellen wichtige Teilprozesse während einer Fremdkörperreaktion dar. Für beide Teilprozesse konnte in einem indirekten zellbasierten Modellsystem der Einfluss einer zellvermittelten Konditionierung wie die Freisetzung von löslichen Mediatoren durch materialadhärente Makrophagen auf die gerichtete Wanderung von Fibroblasten sowie den Umbau eines dreidimensionalen Bindegewebsmodells aufgezeigt werden.
Des Weiteren ließ sich das Freisetzungsprofil von Zytokinen durch materialständige Makrophagen unter verschiedenen Testbedingungen wie der Kontamination mit LPS, der Oberflächenbehandlung mit humanem Blutplasma und der Gegenwart von IL-4 bestimmen. Die anschließende vergleichende statistische Modellierung der generierten komplexen multifaktoriellen Datenmatrix ermöglichte die Übersetzung in eine Biomaterialbewertung. Dieses entwickelte Testverfahren eignete sich einerseits zur Validierung von in vitro Testbedingungen sowie andererseits zur Bewertung von Biomaterialien. Darüber hinaus konnte in einem dreidimensionalen Fremdkörpermodell die komplexe dreidimensionale Struktur der extrazellulären Matrix in einer Wunde durch die Kombination unterschiedlicher Zell- und Matrixkomponenten biomimetisch nachgebaut werden. Diese neuartigen dreidimensionalen Fremdkörpermodelle ermöglichten die Testung von Biomaterialien über längere Testphasen und können in anschließenden Studien angewandt werden, um dynamische Prozesse zu untersuchen. Zusammenfassend konnten in dieser Arbeit drei unterschiedliche Teststrategien entwickelt werden, die (I) die Bewertung von Teilprozessen ermöglichen, (II) die Identifizierung verlässlicher Testbedingungen unterstützen und (III) biomimetisch ein Wundgewebe abbilden. Wesentlich ist, dass biomimetisch ein dreidimensionales Gewebemodell entwickelt werden konnte, das eine verlässliche Unterscheidungskapazität zwischen Biomaterialien aufweist.
Herzschrittmachersysteme sind eine weitverbreitete Möglichkeit Herz-Kreislauf-Erkrankungen zu behandeln. Wegen der natürlichen Reaktion des Immunsystems auf Fremdkörper, erfolgt aber eine fortschreitende Verkapselung der Herzschrittmacherelektrode. Die Folge ist eine ansteigende Verminderung der Stimulationseffizienz durch Erhöhung der Anregungsschwelle. Die Integration der Elektrode in das Gewebe ist dabei mangelhaft und wird bestimmt durch Implantateigenschaften wie Größe, Flexibilität und Dimensionalität. Um die Integration zu verbessern, stellen dreidimensionale (3D) bzw. gewebeartige Elektroden eine Alternative zu den derzeit verwendeten planaren Metallelektroden dar. Zur Entwicklung einer leitfähigen, 3D und faserförmigen Elektrode wurden in dieser Arbeit Kohlenstoff-Nanofaser-Scaffolds über Elektrospinnen hergestellt. Durch die Modifikation des Fasergerüstes mit Natriumchlorid (NaCl) während der Scaffoldherstellung, konnte das Fasernetzwerk aufgelockert und Poren generiert werden. Die Kohlenstofffaser-Elektroden zeigten einen effizienten Energieübertrag, welcher vergleichbar mit heutigen Titannitrid (TiN) -Elektroden ist. Die Auflockerung des Fasergewebes hatte eine verbesserte Flexibilität des Faserscaffolds zu Folge. Neben der Flexibilität, konnte auch die Infiltration von Zellen in das poröse Faserscaffold erheblich verbessert werden. Dabei konnten Fibroblasten durch das gesamte Scaffold migrieren. Die Kompatibilität mit kardialen Zellen, die Grundvoraussetzung von Herzschrittmacherelektroden, wurde in vitro nachgewiesen. Durch die Kombination aus dem 3D-Elektrodengerüst mit einer Co-Kultur aus humanen Kardiomyozyten, mesenchymalen Stammzellen und Fibroblasten, erfolgte eine Einbettung der Elektrode in funktionelles kardiales Gewebe. Dadurch konnte ein lebender Gewebe-Elektroden-Hybrid generiert werden, welcher möglicherweise die Elektrode vor Immunzellen in vivo abschirmen kann. Eine Zusammenführung der hybriden Elektrode mit einen Tissue-Engineerten humanen kardialen Patch in vitro, führte zu Bildung einer nahtlosen Elektronik-Gewebe-Schnittstelle. Die fusionierte Einheit wurde abschließend auf ihre mechanische Belastbarkeit getestet und konnte über einen Elektroden-Anschluss elektrisch stimuliert werden.
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.
Cardiovascular diseases are considered the leading cause of death worldwide according to the World Health Organization. Heart failure is the last stage of most of these diseases, where loss of myocardium leads to architectural and functional decline.
The definitive treatment option for patients with CVDs is organ or tissue transplantation, which relies on donor availability. Therefore, generating an autologous bioengineered myocardium or heart could overcome this limitation. In addition, generating cardiac patches will provide ventricular wall support and enable reparative stem cells delivery to damaged areas. Although many hurdles still exist, a good number of researches have attempted to create an engineered cardiac tissue which can induce endogenous cardiac repair by replacing damaged myocardium.
The present study provided cardiac patches in two models, one by a detergent coronary perfusion decellularization protocol that was optimized, and the other that resulted in a 3D cell-free extracellular matrix with intact architecture and preserved s-glycosaminoglycan and vasculature conduits. Perfusion with 1% Sodium dodecyle sulfate (SDS) under constant pressure resulted in cell-free porcine scaffold within two and cell-free rat scaffold in 7 days, whereas scaffold perfused with 4% sodium deoxycholate (SDO) was not able to remove cells completely. Re-reendothelialization of tissue vasculature was obtained by injecting human microvascular endothelial cell and human fibroblast in 2:1 ratio in a dynamic culture. One-week later, CD31 positive cells and endothelium markers were observed, indicating new blood lining. Moreover, functionality test of re-endothelialized tissue revealed improvement in clotting seen in decellularized tissues. When the tissue was ready to be repopulated, porcine induced pluripotent stem cells (PiPSc) were generated by transfected reprogramming of porcine skin fibroblast and then differentiated to cardiac cells following a robust protocol, for an autologous cardiac tissue model. However, due to the limitation in the PiPSc cell number, alternatively, human induced pluripotent stem cells generated cardiac cells were used.
For reseeding a coculture of human iPSc generated cardiac cells, human mesenchymal stem cells and human fibroblast in 2:1:1 ratio respectively were used in a dynamic culture for 6-8 weeks. Contractions at different areas of the tissue were recorded at an average beating rate of 67 beats/min. In addition, positive cardiac markers (Troponin T), Fibroblast (vemintin), and mesenchymal stem cells (CD90) were detected. Not only that, but by week 3, MSC started differentiating to cardiac cells progressively until few CD90 positive cells were very few by week 6 with increasing troponin t positive cells in parallel. Electrophysiological and drug studies were difficult to obtain due to tissue thickness and limited assessment sources. However, the same construct was established using small intestine submucosa (SISer) scaffold, which recorded a spontaneous beating rate between 0.88 and 1.2 Hz, a conduction velocity of 23.9 ± 0.74 cm s−1, and a maximal contraction force of 0.453 ± 0.015 mN. Moreover, electrophysiological studies demonstrated a drug-dependent response on beating rate; a higher adrenalin frequency was revealed in comparison to the untreated tissue and isoproterenol administration, whereas a decrease in beating rate was observed with propranolol and untreated tissue.
The present study demonstrated the establishment of vascularized cardiac tissue, which can be used for human clinical application.
Breast cancer is the most common cancer among women worldwide and the second most common cause of cancer death in the developed countries. As the current state of the art in first-line drug screenings is highly ineffective, there is an urgent need for novel test systems that allow for reliable predictions of drug sensitivity.
In this study, a tissue engineering approach was used to successfully establish and standardize a 3-dimensional (3D) mamma carcinoma test system that was optimized for the testing of anti-tumour therapies as well as for the investigation of tumour biological issues. This 3D test system is based on the decellularised scaffold of a porcine small intestinal segment and represents the three molecular subsets of oestrogen receptor-positive, HER2/Neu-overexpressing and triple negative breast cancer (TNBC). The characterization of the test system with respect to morphology as well as the expression of markers for epithelial-mesenchymal transition (EMT) and differentiation indicate that the 3D tumour models cultured under static and dynamic conditions reflect tumour relevant features and have a good correlation with in vivo tumour tissue from the corresponding xenograft models. In this respect, the dynamic culture in a flow bioreactor resulted in the generation of tumour models that exhibited best reflection of the morphology of the xenograft material. Furthermore, the proliferation indices of 3D models were significantly reduced compared to 2-dimensional (2D) cell culture and therefore better reflect the in vivo situation. As this more physiological proliferation index prevents an overestimation of the therapeutic effect of cytostatic compounds, this is a crucial advantage of the test system compared to 2D culture. Moreover, it could be shown that the 3D models can recapitulate different tumour stages with respect to tumour cell invasion. The scaffold SISmuc with the preserved basement membrane structure allowed the investigation of invasion over this barrier which tumour cells of epithelial origin have to cross in in vivo conditions during the process of metastasis formation. Additionally, the data obtained from ultrastructural analysis and in situ zymography indicate that the invasion observed is connected to a tumour cell-associated change in the basement membrane in which matrix metalloproteinases (MMPs) are also involved. This features of the model in combination with the mentioned methods of analysis could be used in the future to mechanistically investigate invasive processes and to test anti-metastatic therapy strategies.
The validation of the 3D models as a test system with respect to the predictability of therapeutic effects was achieved by the clinically relevant targeted therapy with the monoclonal antibody trastuzumab which induces therapeutic response only in patients with HER2/Neu-overexpressing mamma carcinomas due to its specificity for HER2. While neither in 2D nor in 3D models of all molecular subsets a clear reduction of cell viability or an increase in apoptosis could be observed, a distinct increase in antibody-dependent cell-mediated cytotoxicity (ADCC) was detected only in the HER2/NEU-overexpressing 3D model with the help of an ADCC reporter gene assay that had been adapted for the application in the 3D model in the here presented work. This correlates with the clinical observations and underlines the relevance of ADCC as a mechanism of action (MOA) of trastuzumab. In order to measure the effects of ADCC on the tumour cells in a direct way without the indirect measurement via a reporter gene, the introduction of an immunological component into the models was required. This was achieved by the integration of peripheral blood mononuclear cells (PBMCs), thereby allowing the measurement of the induction of tumour cell apoptosis in the HER2/Neu-overexpressing model. Hence, in this study an immunocompetent model could be established that holds the potential for further testing of therapies from the emergent field of cancer immunotherapies.
Subsequently, the established test system was used for the investigation of scientific issues from different areas of application. By the comparison of the sensitivity of the 2D and 3D model of TNBC towards the water-insoluble compound curcumin that was applied in a novel nanoformulation or in a DMSO-based formulation, the 3D test system was successfully applied for the evaluation of an innovative formulation strategy for poorly soluble drugs in order to achieve cancer therapy-relevant concentrations. Moreover, due to the lack of targeted therapies for TNBC, the TNBC model was applied for testing novel treatment strategies. On the one hand, therapy with the WEE1 kinase inhibitor MK 1775 was evaluated as a single agent as well as in combination with the chemotherapeutic agent doxorubicin. This therapy approach did not reveal any distinct benefits in the 3D test system in contrast to testing in 2D culture. On the other hand, a novel therapy approach from the field of cellular immunotherapies was successfully applied in the TNBC 3D model. The treatment with T cells that express a chimeric antigen receptor (CAR) against ROR1 revealed in the static as well as in the dynamic model a migration of T cells into the tumour tissue, an enhanced proliferation of T cells as well as an efficient lysis of the tumour cells via apoptosis and therefore a specific anti-cancer effect of CAR-transduced T cells compared to control T cells. These results illustrate that the therapeutic application of CAR T cells is a promising strategy for the treatment of solid tumours like TNBC and that the here presented 3D models are suitable for the evaluation and optimization of cellular immunotherapies.
In the last part of this work, the 3D models were expanded by components of the tumour stroma for future applications. By coculture with fibroblasts, the natural structures of the intestinal scaffold comprising crypts and villi were remodelled and the tumour cells formed tumour-like structures together with the fibroblasts. This tissue model displayed a strong correlation with xenograft models with respect to morphology, marker expression as well as the activation of dermal fibroblasts towards a cancer-associated fibroblast (CAF) phenotype. For the integration of adipocytes which are an essential component of the breast stroma, a coculture with human adipose-derived stromal/stem cells (hASCs) which could be successfully differentiated along the adipose lineage in 3D static as well as dynamic models was established. These models are suitable especially for the mechanistic analysis of the reciprocal interaction between tumour cells and adipocytes due to the complex differentiation process.
Taken together, in this study a human 3D mamma carcinoma test system for application in the preclinical development and testing of anti-tumour therapies as well as in basic research in the field of tumour biology was successfully established. With the help of this modular test system, relevant data can be obtained concerning the efficacy of therapies in tumours of different molecular subsets and different tumour stages as well as for the optimization of novel therapy strategies like immunotherapies. In the future this can contribute to improve the preclinical screening and thereby to reduce the high attrition rates in pharmaceutical industry as well as the amount of animal experiments.
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.
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.
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.
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.
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.
To improve and focus preclinical testing, we combine tumor models based on a decellularized tissue matrix with bioinformatics to stratify tumors according to stage-specific mutations that are linked to central cancer pathways. We generated tissue models with BRAF-mutant colorectal cancer (CRC) cells (HROC24 and HROC87) and compared treatment responses to two-dimensional (2D) cultures and xenografts. As the BRAF inhibitor vemurafenib is—in contrast to melanoma—not effective in CRC, we combined it with the EGFR inhibitor gefitinib. In general, our 3D models showed higher chemoresistance and in contrast to 2D a more active HGFR after gefitinib and combination-therapy. In xenograft models murine HGF could not activate the human HGFR, stressing the importance of the human microenvironment. In order to stratify patient groups for targeted treatment options in CRC, an in silico topology with different stages including mutations and changes in common signaling pathways was developed. We applied the established topology for in silico simulations to predict new therapeutic options for BRAF-mutated CRC patients in advanced stages. Our in silico tool connects genome information with a deeper understanding of tumor engines in clinically relevant signaling networks which goes beyond the consideration of single drivers to improve CRC patient stratification.
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.
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.
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.
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.
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.
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.
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.
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.
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.