Lehrstuhl für Tissue Engineering und Regenerative Medizin
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Malignant melanoma (MM) is the most dangerous type of skin cancer with rising incidences worldwide. Melanoma skin models can help to elucidate its causes and formation or to develop new treatment strategies. However, most of the current skin models lack a vasculature, limiting their functionality and applicability. MM relies on the vascular system for its own supply and for its dissemination to distant body sites via lymphatic and blood vessels. Thus, to accurately study MM progression, a functional vasculature is indispensable. To date, there are no vascularized skin models to study melanoma metastasis in vitro, which is why such studies still rely on animal experimentation.
In the present thesis, two different approaches for the vascularization of skin models are employed with the aim to establish a vascularized 3D in vitro full-thickness skin equivalent (FTSE) that can serve as a test system for the investigation of the progression of MM.
Initially, endothelial cells were incorporated in the dermal part of FTSEs. The optimal seeding density, a spheroid conformation of the cells and the cell culture medium were tested. A high cell density resulted in the formation of lumen-forming shapes distributed in the dermal part of the model. These capillary-like structures were proven to be of endothelial origin by staining for the endothelial cell marker CD31. The established vascularized FTSE (vFTSE) was characterized histologically after 4 weeks of culture, revealing an architecture similar to human skin in vivo with a stratified epidermis, separated from the dermal equivalent by a basement membrane indicated by collagen type IV. However, this random capillary-like network is not functional as it cannot be perfused.
Therefore, the second vascularization approach focused on the generation of a perfusable tissue construct. A channel was molded within a collagen hydrogel and seeded with endothelial cells to mimic a central, perfusable vessel. The generation and the perfusion culture of the collagen hydrogel was enabled by the use of two custom-made, 3D printed bioreactors. Histological assessment of the hydrogels revealed the lining of the channel with a monolayer of endothelial cells, expressing the cell specific marker CD31.
For the investigation of MM progression in vitro, a 3D melanoma skin equivalent was established. Melanoma cells were incorporated in the epidermal part of FTSEs, representing the native microenvironment of the tumor. Melanoma nests grew at the dermo-epidermal junction within the well stratified epidermis and were characterized by the expression of common melanoma markers. First experiments were conducted showing the feasibility of combining the melanoma model with the vFTSE, resulting in skin models with tumors at the dermo-epidermal junction and lumen-like structures in the dermis.
Taken together, the models presented in this thesis provide further steps towards the establishment of a vascularized, perfusable melanoma model to study melanoma progression and metastasis.
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.
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.
Für die Verwendung von zellbasierten Therapeutika ist vor allem die korrekt Identifikation
sowohl vom Ausgangsmaterial wie auch dem produziertem Material von
zentraler Wichtigkeit. In dieser Arbeit wurde eine Methodik entwickelt, welche eine
nicht-invasive Klassifizierung von Zellen und zellulärer Entwicklung aufgrund ihrer
zweidimensionalen Magnetresonanz-Korrelationsspektren ermöglichte.
Hierzu wurde ein mobiler MR-Scanner mit einer Feldstärke von 0.5T und einem Isozentrum
von 1 cm3 verwendet. Aufgrund der kompakten und leichten Bauweise war
es möglich, das System in normalen Zellkulturlaboren zu verwenden. Von den Proben
wurde ein zweidimensionales T1/T2 -Korrelationsspektrum aufgenommen, anhand
dessen die Zellen klassifiziert werden sollten. Mithilfe von Agarose-Dotagraf® -Zell-
Phantomen konnte die Stabilität und Reproduzierbarkeit des Messsystems und der
verwendeten Sequenz validiert werden.
Aufgrund der unter Umständen recht langen Messzeiten der MR-Technologie war
auch die Handhabung und Kultur der Zellproben während des Messprozesses von
großer Bedeutung. Um hierfür den Durchsatz an Proben zu erhöhen, wurde eine kostengünstige
und ebenfalls mobile Robotikanlage entwickelt. Diese basierte auf dem
kommerziell erhältlichen Roboterarm Braccio, welcher durch einen Arduino Mega
Mikrocontroller gesteuert wurde. Mit bis zu 24 Proben pro Tag konnte durch die
Automatisierung der Durchsatz an Proben um den Faktor 3 – 4 gesteigert werden.
Durch den entwickelten Prozess war es möglich, eine umfangreiche Datenbank –
bestehend aus 362 unabhängigen Messungen (biologische Replikate) – aufzubauen.
Die Datenbank enthielt Messungen von zehn unterschiedlichen Zelllinien. Zusätzlich
wurden T1/T2 -Korrelationsspektren von mesenchymalen Stromazellen (MSCs)
vor und nach deren Differenzierung zu Adipocyten aufgenommen, um ihre zelluläre
Entwicklung nicht-invasiv charakterisieren zu können.
Die aufgenommenen Daten wurden mithilfe einer geeigneten Support Vector Machine
wie auch angepassten künstlichen neuronalen Netzwerken klassifiziert. Mithilfe
dieser Methoden konnten die Zelllinien und MSCs anhand ihrer aufgenommenen
Korrelationsspektren mit einer Genauigkeit von bis zu 98% klassifiziert werden.
Diese hohe Treffsicherheit legte den Schluss nahe, dass die Kombination aus nichtinvasiver,
zweidimensionaler T1/T2 -MR-Relaxometrie und der Verwendung von geeigneten
Methoden des machine learning und der künstlichen Intelligenz eine effiziente
Methodik für die nicht-invasive Klassifizierung von Zellen sowie zellulärer
Entwicklung darstellt.
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.
Diabetes mellitus is an incurable, metabolic disease, which is associated with severe long-term complications. The in vitro generation of pancreatic β-cells from human induced pluripotent stem cells (hiPSCs) represent a promising strategy for a curative therapy of diabetes mellitus. However, current differentiation strategies largely fail to produce functional β-cells in vitro and require an additional in vivo transplantation to achieve terminal maturation. Previous studies demonstrated a beneficial effect of the extracellular matrix (ECM) on the survival and sustained function of adult, isolated islets of Langerhans. This raises the question whether organ-specific cell-ECM interactions might represent the missing link driving the final stage of β-cell development. In order to address this issue, this study investigated the impact of the pancreas ECM on in vitro β-cell differentiation and its use for the establishment of a pancreatic endocrine organ model.
To this purpose, a pancreas-specific ECM scaffolds (PanMa) was derived from porcine pancreata using whole organ decellularization with Sodium Deoxycholate. In a first step, the generated PanMa was thoroughly characterized using (immuno-) histological stainings, scanning electron microscopy and DNA quantification as well as perfusion and recellularization experiments with endothelial cells. Based on these data, a scoring system (PancScore) for a standardized PanMa generation was developed. Next, the generated PanMa was tested for the presence of tissue-specific ECM features. Therefore, the biophysical and physico-structural characteristics, such as rigidity, porosity and hygroscopy were analyzed using rheological measurements, particle diffusion analyses as well as a water evaporation assay and compared to the properties of ECM scaffolds derived from porcine small intestine (SISser) and lung (LungMa) to examine organ-specific scaffold cues. Following the thorough scaffold characterization, the impact of the PanMa on pluripotency and early development of hiPSC was studied. To this purpose, gene and protein expression of hiPSCs during maintenance culture and spontaneous differentiation on the PanMa were assessed. In a next step, the impact of the PanMa on the pancreatic endocrine differentiation of hiPSCs was tested. Therefore, the PanMa was used as a liquid media supplement or as a solid scaffold during the directed differentiation of hiPSC towards either pancreatic hormone-expressing cells (Rezania et al. 2012; Rezania et al. 2014) or maturing β-cells (Rezania et al. 2014). The impact of the PanMa on the generated cells was examined by gene expression analysis, immunohistochemical staining of important stage markers, as well as glucose stimulated insulin secretion assays. In a last part of this study, the potential of the PanMa for the prolonged culture of hiPSC derived endocrine cells for the establishment of an in vitro organ model of the endocrine pancreas was examined. Therefore, a PanMa-derived hydrogel was generated and used for the encapsulation and culture of hiPSC-derived hormone-expressing cells (HECs). The influence of the PanMa-hydrogel culture was analyzed on gene, protein and functional level by gene expression analysis, immunohistochemical stainings and glucose stimulated insulin secretion.
Whole organ decellularization resulted in the generation of an acellular PanMa scaffold, with low amounts of residual DNA and a preserved ECM micro- and ultrastructure, including important ECM components, such as collagen I, III and IV. Furthermore, the PanMa maintained an intact vessel system and was verified as cytocompatible as demonstrated by the successful recellularization of the arterial system with human endothelial cells. In comparison to SISser and LungMa, the PanMa was characterized as a relative soft, hygroscopic scaffold with a collagen-fiber based structure. Furthermore, the findings indicate that the ECM-specific properties have a relevant effect on the stem cell character and early multi-lineage decisions of hiPSCs. In this regard, maintenance of hiPSCs on the PanMa resulted in a slightly changed expression of pluripotency genes (OCT4, SOX2 and NANOG) and a weak immunohistochemical signal for NANOG protein, indicating a PanMa-dependent impact on hiPSC pluripotency. Strikingly, this presumption was corroborated by the finding that culture on the PanMa promoted an endodermal development of hiPSCs during spontaneous differentiation. In line with that, pancreatic differentiation of hiPSC on both the PanMa and SISser resulted in a significant decrease of glucagon and somatostatin gene expression as well as an unaltered insulin expression, suggesting an ECM-driven suppression of the development of non β-cell endocrine cells. However, this change did not result in an improved glucose stimulated insulin secretion of the generated HECs. Moreover, use of the PanMa as a hydrogel allowed prolonged culture of these cells in a defined culture system. HECs were viable after 21 days of culture, however already showed an altered islet morphology as well as a slightly decreased glucose stimulated insulin secretion.
Altogether, this study demonstrates a relevant biological effect of tissue specific ECM cues on the in vitro differentiation of hiPSCs. More specifically, the data indicate an involvement of the ECM in the endocrine commitment of hiPSC-derived pancreatic cells during directed differentiation highlighting the ECM as an important regulator of pancreatic development. Collectively, these findings emphasize the relevance of the ECM for the fabrication of functional hiPSC-derived cell types and suggest a much stronger consideration of organ specific ECM cues for tissue engineering approaches as well as clinical translation in regenerative medicine.
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.
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.
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.
The small intestine represents a strong barrier separating the lumen from blood circulation thereby playing a major role in the absorption and the transport of pharmacological agents prior to their arrival on the respective target site. In order to gain more knowledge about specialized uptake mechanisms and risk assessment for the patient after oral admission of drugs, intestinal in vitro models demonstrating a close similarity to the in vivo situation are needed.
In the past, cell line-based in vitro models composed of Caco-2 cells cultured on synthetic cell carriers represented the “gold standard” in the field of intestinal tissue engineering. Expressive advantages of these models are a reproducible, cost-efficient and standardized model set up, but cell function can be negatively influenced by the low porosity or unwanted molecular adhesion effects of the artificial scaffold material. Natural extracellular matrices (ECM) such as the porcine decellularized small intestinal submucosa (SIS) are used as alternative to overcome some common drawbacks; however, the fabrication of these scaffolds is time- and cost-intensive, less well standardized and the 3Rs (replacement, reduction, refinement) principle is not entirely fulfilled. Nowadays, biopolymer-based scaffolds such as the bacterial nanocellulose (BNC) suggest an interesting option of novel intestinal tissue engineered models, as the BNC shows comparable features to the native ECM regarding fiber arrangement and hydrophilic properties. Furthermore, the BNC is of non-animal origin and the manufacturing process is faster as well as well standardized at low costs.
In this context, the first part of this thesis analyzed the BNC as alternative scaffold to derive standardized and functional organ models in vitro. Therefore, Caco-2 cells were cultured on two versions of BNC with respect to their surface topography, the unmodified BNC as rather smooth surface and the surface-structured BNC presenting an aligned fiber arrangement. As controls, Caco-2 in vitro models were set up on PET and SIS matrices. In this study, the BNC-based models demonstrated organ-specific properties comprising typical cellular morphologies, a characteristic tight junction protein expression profile, representative ultrastructural features and the formation of a tight epithelial barrier together with a corresponding transport activity. In summary, these results validated the high quality of the BNC-based Caco-2 models under cost-efficient conditions and their suitability for pre-clinical research purposes. However, the full functional diversity of the human intestine cannot be presented by Caco-2 cells due to their tumorigenic background and their exclusive representation of mature enterocytes.
Next to the scaffold used for the setup of in vitro models, the cellular unit mainly drives functional performance, which demonstrates the crucial importance of mimicking the cellular diversity of the small intestine in vitro. In this context, intestinal primary organoids are of high interest, as they show a close similarity to the native epithelium regarding their cellular diversity comprising enterocytes, goblet cells, enteroendocrine cells, paneth cells, transit amplifying cells and stem cells. In general, such primary organoids grow in a 3D Matrigel® based environment and a medium formulation supplemented with a variety of growth factors to maintain stemness, to inhibit differentiation and to stimulate cell migration supporting long term in vitro culture.
Intestinal primary spheroid/organoid cultures were set up as Transwell®-like models on both BNC variants, which resulted in a fragmentary cell layer and thereby unfavorable properties of these scaffold materials under the applied circumstances. As the BNC manufacturing process is highly flexible, surface properties could be adapted in future studies to enable a good cell adherence and barrier formation for primary intestinal cells, too. However, the application of these organoid cultures in pre-clinical research represents an enormous challenge, as the in vitro culture is complex and additionally time- and cost-intensive.
With regard to the high potential of primary intestinal spheroids/organoids and the necessity of a simplified but predictive model in pre-clinical research purposes, the second part of this thesis addressed the establishment of a primary-derived immortalized intestinal cell line, which enables a standardized and cost-efficient culture (including in 2D), while maintaining the cellular diversity of the organoid in vitro cultures. In this study, immortalization of murine and human intestinal primary organoids was induced by ectopic expression of a 10- (murine) or 12 component (human) pool of genes regulating stemness and the cell cycle, which was performed in cooperation with the InSCREENeX GmbH in a 2D- and 3D-based transduction strategy. In first line, the established cell lines (cell clones) were investigated for their cell culture prerequisites to grow under simplified and cost-efficient conditions. While murine cell clones grew on uncoated plastic in a medium formulation supplemented with EGF, Noggin, Y-27632 and 10% FCS, the human cell clones demonstrated the necessity of a Col I pre coating together with the need for a medium composition commonly used for primary human spheroid/organoid cultures. Furthermore, the preceding analyses resulted in only one human cell clone and three murine cell clones for ongoing characterization. Studies regarding the proliferative properties and the specific gene as well as protein expression profile of the remaining cell clones have shown, that it is likely that transient amplifying cells (TACs) were immortalized instead of the differentiated cell types localized in primary organoids, as 2D, 3D or Transwell®-based cultures resulted in slightly different gene expression profiles and in a dramatically reduced mRNA transcript level for the analyzed marker genes representative for the differentiated cell types of the native epithelium. Further, 3D cultures demonstrated the formation of spheroid-like structures; however without forming organoid-like structures due to prolonged culture, indicating that these cell populations have lost their ability to differentiate into specific intestinal cell types. The Transwell®-based models set up of each clone exhibit organ-specific properties comprising an epithelial-like morphology, a characteristic protein expression profile with an apical mucus-layer covering the villin-1 positive cell layer, thereby representing goblet cells and enterocytes, together with representative tight junction complexes indicating an integer epithelial barrier. The proof of a functional as well as tight epithelial barrier in TEER measurements and in vivo-like transport activities qualified the established cell clones as alternative cell sources for tissue engineered models representing the small intestine to some extent. Additionally, the easy handling and cell expansion under more cost-efficient conditions compared to primary organoid cultures favors the use of these newly generated cell clones in bioavailability studies.
Altogether, this work demonstrated new components, structural and cellular, for the establishment of alternative in vitro models of the small intestinal epithelium, which could be used in pre-clinical screenings for reproducible drug delivery studies.