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Despite advances in cartilage repair strategies, treatment of focal chondral lesions remains an important challenge to prevent osteoarthritis. Articular cartilage is organized into several layers and lack of zonal organization of current grafts is held responsible for insufficient biomechanical and biochemical quality of repair-tissue. The aim was to develop a zonal approach for cartilage regeneration to determine whether the outcome can be improved compared to a non-zonal strategy. Hydrogel-filled polycaprolactone (PCL)-constructs with a chondrocyte-seeded upper-layer deemed to induce hyaline cartilage and a mesenchymal stromal cell (MSC)-containing bottom-layer deemed to induce calcified cartilage were compared to chondrocyte-based non-zonal grafts in a minipig model. Grafts showed comparable hardness at implantation and did not cause visible signs of inflammation. After 6 months, X-ray microtomography (µCT)-analysis revealed significant bone-loss in both treatment groups compared to empty controls. PCL-enforcement and some hydrogel-remnants were retained in all defects, but most implants were pressed into the subchondral bone. Despite important heterogeneities, both treatments reached a significantly lower modified O’Driscoll-score compared to empty controls. Thus, PCL may have induced bone-erosion during joint loading and misplacement of grafts in vivo precluding adequate permanent orientation of zones compared to surrounding native cartilage.
Tissue-engineered anterior segment eye cultures demonstrate hallmarks of conventional organ culture
(2023)
Background
Glaucoma is a blinding disease largely caused by dysregulation of outflow through the trabecular meshwork (TM), resulting in elevated intraocular pressure (IOP). We hypothesized that transplanting TM cells into a decellularized, tissue-engineered anterior segment eye culture could restore the outflow structure and function.
Methods
Porcine eyes were decellularized with freeze–thaw cycles and perfusion of surfactant. We seeded control scaffolds with CrFK cells transduced with lentiviral vectors to stably express eGFP and compared them to scaffolds seeded with primary TM cells as well as to normal, unaltered eyes. We tracked the repopulation behavior, performed IOP maintenance challenges, and analyzed the histology.
Results
Transplanted cells localized to the TM and progressively infiltrated the extracellular matrix, reaching a distribution comparable to normal, unaltered eyes. After a perfusion rate challenge to mimic a glaucomatous pressure elevation, transplanted and normal eyes reestablished a normal intraocular pressure (transplanted = 16.5 ± 0.9 mmHg, normal = 16.9 ± 0.9). However, eyes reseeded with eGFP-expressing CrFK cells could not regulate IOP, remaining high and unstable (27.0 ± 6.2 mmHg) instead.
Conclusion
Tissue-engineered anterior segment scaffolds can serve as readily available, scalable ocular perfusion cultures. This could reduce dependency on scarce donor globes in outflow research and may allow engineering perfusion cultures with specific geno- and phenotypes.
Die Erzeugung von klinisch in der plastischen und rekonstruktiven Chirurgie nutzbarem Fettgewebe stellt einen sehr wichtigen Aspekt in aktuellen Arbeiten des Tissue Engineerings, also der Erzeugung von spezifischem Gewebe aus Spenderzellen dar. Sollte es gelingen, aus patienteneigenen Zellen wieder neues Gewebe zu züchten, so würden daraus eine Fülle neuer Behandlungsmöglichkeiten für Gewebedefekte resultieren. In einer Vorgängerarbeit zu der vorliegenden Arbeit konnte gezeigt werden, dass die Adipogenese in vivo von Fettgewebe aus Vorläuferzellen, den Präadipozyten, durch geeignete Methoden der Vorkultivierung in vitro beeinflusst werden kann.
Die Unterschiede in der Vorbehandlung lagen in einer Induktion der Differenzierung der Präadipozyten bei gleichzeitigem Stopp der Proliferation und einer anschließenden verschieden langen Ausdifferenzierungsphase der Zellen in vitro im Brutschrank. Die resultierenden Konstrukte wurden in jeweils drei Mäuse in vier Gruppen implantiert und nach 1, 5, 12 und 24 Wochen entnommen und untersucht. Während die Präadipozyten von Gruppe 1 keine Induktion erfuhren, erfolgte diese bei den anderen drei Gruppen. Die Konstrukte der Gruppe 2 wurden dann bereits nach 2 Tagen der Induktion der Präadipozyten implantiert, die Konstrukte der Gruppe 3 blieben zur Differenzierung noch 7 Tage, die der Gruppe 4 noch 33 Tage im Brutschrank, bevor sie in die Versuchstiere eingebracht wurden.
Ziel der vorliegenden Arbeit war es zunächst, an den Gewebekonstrukten der Vorgängerarbeit eine histomorphometrische Analyse der resultierenden Adipozyten in vivo über die Zeit durchzuführen, um eine detaillierte Beurteilung des Verlaufs der Fettgewebeentwicklung anhand resultierender Zellzahlen darzustellen. Hierfür wurden die Gewebedünnschnitte der Mäuse nach einer HE-Anfärbung mikroskopisch untersucht und die Zellzahlen resultierend jeweils aus unreifen und reifen Adipozyten histomorphometrisch quantifiziert. Die Unterscheidung erfolgte mittels einer Größenzuordnung, wobei Zellen kleiner 20 µm Durchmesser den unreifen und Zellen größer 20 µm Durchmesser den reifen Adipozyten zugeordnet wurden.
Aus der quantitativen Analyse mittels Histomorphometrie ergab sich, dass in allen Konstrukten die Zahlen an Zellen der den unreifen Adipozyten zugeordneten Größenordnung von kleiner als 20µm tendenziell während der gesamten Zeit in vivo klein bleibt. Die Zellzahlen resultierend aus großen Zellen mit einem Durchmesser mehr als 20µm, die den reifen Adipozyten zugeordnet wurden, steigen dagegen in allen Proben leicht an, wobei die Konstrukte der Gruppe 4 den absolut höchsten Wert aufwiesen. In der HE-Anfärbung ist demgemäß in Gruppe 4 eine Vielzahl reifer Adipozyten zu erkennen.
Das zweite Ziel dieser Arbeit war es, durch Anfärbung charakteristischer Proteine der extrazellulären Matrix mittels markierter Antikörper und einer anschließenden immunohistochemischen Analyse des Verlaufs der Signalintensität dieser markierten Komponenten in der EZM die Adipogenese mittels Analyse der entstehenden Gerüstproteine zu verfolgen. Hierfür wurde durch eine umfangreiche immunohistochemische Analyse die Bildung der Kollagene I, IV und VI sowie von Laminin als Bestandteile der EZM analysiert und damit die Art und der Umfang der entstandenen extrazellulären Matrix während der Adipogenese qualitativ beurteilt. Die Fluoreszenz-Bilder der Proben nach den jeweiligen Gruppen und Wochen in vivo zeigen einen deutlichen Hinweis im Sinne der Bildung von Fettgewebe in den Gewebe-Konstrukten der Gruppe 4. Während in den Gruppen 1 und 2 fast durchweg faserartige Bindegewebsstrukturen, verbunden mit den entsprechenden eher fibrillärem Aussehen der Signale für die untersuchten Kollagene I, IV, VI und für Laminin gefunden werden konnten, zeigen die Konstrukte der Gruppe 3 und insbesondere von Gruppe 4 in den Fluoreszenz-Abbildungen deutlich ausgeprägtere, netzartig ausgebildete Strukturen.
Aus den Resultaten der vorliegenden Arbeit kann demnach geschlossen werden, dass die Art der Vorkultivierung eine spätere Adipogenese eindeutig beeinflussen kann. Eine längere Inkubationszeit nach erfolgter Induktion der Präadipozyten zur Förderung der Reifung zu Adipozyten vor der Implantation fördert die Bildung einer höheren Anzahl von Adipozyten und die Ausbildung einer charakteristischen EZM. Diese Erkenntnisse eröffnen für zukünftige Arbeiten die Möglichkeit, durch die weitere Optimierung der Vorkultivierung, verbunden mit einer eventuell noch besseren Überlebensrate der ursprünglich eingebrachten Zellen, die Herstellung von klinisch geeigneten Konstrukten aus Fettgewebe weiter voranzutreiben.
The knee joint is a complex composite joint containing the C-shaped wedge-like menisci composed of fibrocartilage. Due to their complex composition and structure, they provide mechanical resilience to the knee joint protecting the articular cartilage. Because of the limited repair potential, meniscal injuries do not only affect the meniscus itself but also lead to altered joint homeostasis and inevitably to secondary osteoarthritis.
The meniscus was characterized focusing on its anatomy, structure and meniscal markers such as aggrecan, collagen type I (Col I) and Col II. The components relevant for meniscus tissue engineering, namely cells, Col I scaffolds, biochemical and biomechanical stimuli were studied. Meniscal cells (MCs) were isolated from meniscus, mesenchymal stem cells (MSCs) from bone marrow and dermal microvascular endothelial cells (d-mvECs) from foreskin biopsies. For the human (h) meniscus model, wedge-shape compression of a hMSC-laden Col I gel was successfully established. During three weeks of static culture, the biochemical stimulus transforming growth factor beta-3 (TGF beta-3) led to a compact collagen structure. On day 21, this meniscus model showed high metabolic activity and matrix remodeling as confirmed by matrix metalloproteinases detection. The fibrochondrogenic properties were illustrated by immunohistochemical detection of meniscal markers, significant GAG/DNA increase and increased compressive properties. For further improvement, biomechanical stimulation systems by compression and hydrostatic pressure were designed. As one vascularization approach, direct stimulation with ciclopirox olamine (CPX) significantly increased sprouting of hd-mvEC spheroids even in absence of auxiliary cells such as MSCs. Second, a cell sheet composed of hMSCs and hd-mvECs was fabricated by temperature triggered cell sheet engineering and transferred onto the wedge-shaped meniscus model. Third, a biological vascularized scaffold (BioVaSc-TERM) was re-endothelialized with hd-mvECs providing a viable vascularized network. The vascularized BioVaSc-TERM was suggested as wrapping scaffold of the meniscus model by using two suture techniques, the all-inside-repair (AIR) for the posterior horn, and the outside-in-refixation (OIR) for the anterior horn and the middle part.
This meniscus model for replacing torn menisci is a promising approach to be further optimized regarding vascularization, biochemical and biomechanical stimuli.
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.
Zusammenfassung
Einleitung: Die Inzidenz von Narbenhernien (operativ erworbene Schwachstellen der Bauchwand) ist abhängig von der Art der vorhergegangen Operation, nach Laparaskopien ist sie um einiges niedriger als nach Laparotomien, wird aber mit 2-20% in der Literatur angegeben. Aufgrund der möglichen Komplikationen (Platzbauch, Darminkarzeration, Schmerzen, Funktionseinschränkung, …) stellen Narbenhernien oftmals große Belastungen für die Patienten dar. Die operative Sanierung, in Abhängigkeit von Größe und Lage, wird zumeist durch einbringen eines Netzgewebes erreicht. Dieser Fremdkörper kann seinerseits wieder Komplikationen hervorrufen (Infektionen, Funktionsverlust, Schmerzen, Fisteln), die bis zur Explantation des Netzgewebes führen können. Das Risiko für das Auftreten von Narbenhernien bzw. deren Rezidiven hängt von vielen Faktoren ab, als Risikofaktoren wurden unter anderem Rauchen, männliches Geschlecht, Alter >45 Jahre und ein BMI >25 kg/cm² ausgemacht. Ein Teilbereich des Tissue Engineerings ist die Entwicklung von Modellen, anhand derer in vitro Prozesse des menschlichen Körpers nachvollzogen werden können. Mit dieser Arbeit soll ein Modell etabliert werden Anhand dessen die Untersuchung der Kollagenproduktion und der Netzinkorporation bzw. die Auswirkungen verschiedener Risikofaktoren auf diese Prozesse in vitro ermöglicht werden soll. Weiterhin wurden Studienfragen formuliert, die sich sowohl mit der Durchführbarkeit dieser Methode abzielten, als auch gezielt nach der Stützung der These der „guten und schlechten Heiler“ durch diese Arbeit abzielten. Sowie nach der Vergleichbarkeit der Ergebnisse mit bekannten Kollagenmustern die aus Netzexplantaten bekannt sind. Material und Methode: Für die vorliegende Arbeit wurden Biopsien von Faszien bzw. Narbenhernien im Rahmen einer Operation gewonnen, aus diesen wurden die Fibroblasten isoliert und anschliessend entweder eingefroren bzw. expandiert, um sie in einer Rattenkollagenmatrix mit und ohne synthetischem Netz im dynamisch mechanischen Bioreaktor zu kultivieren. Die Biopsien wurden Anhand der Kollagen I/III Ratio in „gute und schlechte Heiler“ eingruppiert. Anschließend wurden die so gezüchteten Neofaszien HE und Pikrosiriusrot gefärbt um zum einen einen Eindruck von der Verteilung der Fibroblasten innerhalb der Neofaszie zu gewinnen, als auch Aussagen zum Kollagenmuster, der Kollagen I/III Ratio und zur Kollagendensität treffen zu können. Die Dicke der kultivierten Neofaszien wurde sowohl in Sirius als auch in HE Färbung untersucht. Weiterhin wurden RT-PCR und Gene Arrays von Nativgeweben und von Neofaszien mit unterschiedlichen Netztypen durchgeführt. Ergebnisse: Bei gesunden Probanden konnten oftmals nicht genügend Zellen aus den Faszienbiopsaten gewonnen werden, deshalb wurde im Verlauf der Arbeit auf die Gewinnung von gesundem Fasziengewebe als Vergleichsgruppe verzichtet. Fibroblasten von als „schlechten Heilern“ klassifizierten Patienten zeigten meist ein langsameres Wachstum in der Expansionsphase. Der Bioreaktor bereitete kaum Probleme (ein paar Faszien trockneten anfänglich aus, dieses Problem lies sich durch bei Bedarf verkürzten Medienwechselintervallen in den Griff bekommen. Probleme mit Kontaminationen traten nicht auf. Bei den Histologischen Untersuchungen der Neofaszien waren Fibroblasten über den gesamten Bereich der Neofaszie zu sehen, auch in unmittelbarer Umgebung der Netzstrukturen. Die Kollagenmuster stimmten in Ansätzen mit den aus klinischen Netzexplantaten bekannten Mustern überein (Polydirektional bei Polyesternetz, Konzentrisch um die Netzstrukturen bei Polypropylen). Weiterhin war eine verstärkte Kollagenbildung quer zur Druckrichtung des Bioreaktors zu erkennen. Bei der Betrachtung der Dicke der Neofaszien zeigte sich (unter Vorbehalt, aufgrund der geringen Probenanzahl) eine Tendenz zu meist dünneren Faszien bei „schlechten Heilern“ während die Neofaszien von „guten Heilern“ meist eine kleinere Streuung um den Mittelwert zeigten (einheitlicher waren). Die Kollagendensität und auch die Kollagen I/III Ratio lieferten Ergebnisse Anhand derer Gesagt werden kann, dass je höher die Ausgangswerte im Nativgewebe waren, diese mit höherer Wahrscheinlichkeit von den Neofaszien nicht erreicht werden konnten. qRT-PCR und Gene Array zeigten in der Rangkorrelation nach Spearman große Übereinstimmungen. Beantwortung der Studienfragen: Es konnte gezeigt werden, dass es möglich ist Neofaszien mit synthetischen Netzen zu züchten, die über den gesamten Bereich mit Fibroblasten besiedelt waren. Die Ergebnisse der Kollagenmorphologie zeigten in Ansätzen die aus Netzexplantaten bekannten Muster. Bei Kollagen I/III Ratio und Densität war lediglich erkennbar, dass je höher die Ausgangswerte waren, diese mit zunehmender Wahrscheinlichkeit nicht reproduziert werden konnten. Es ließ sich keine Verbindung zwischen der Kollagen I/III Ratio der Histologischen Gewebeproben und den Molekularbiologischen Ergebnissen feststellen. Weiterhin konnte die Theorie der „guten und schlechten Heiler“ molekularbiologisch nicht gestützt werden, da die Proben der als „schlechte Heiler“ Klassifizierten Biopsien stärkere Gemeinsamkeiten mit als „gute Heiler“ Klassifizierten Biopsien aufwiesen als untereinander. Es konnte gezeigt werden dass die Kultur auf die MMP-8 und Elastinproduktion keinen Einfluss zu haben scheint. Diskussion: Im Verlauf der Diskussion wurde darauf hingewiesen, dass die Kollagensynthese, und Sekretion ein komplexes und höchst aktives System darstellt, welches im Rahmen der Wundheilung durch Co-Signalling, und der Interaktion zwischen Fibroblasten und Immunzellen (Makrophagen…) nochmals verändert wird, auch dadurch bedingt, dass Fibroblasten im Verlauf der Wundheilung selbst als immunmodulierende Zellen in Erscheinung treten können. So können weiterhin die Kollagen kodierenden Gene (Col1A1, Col1A2, Col3A1) als Marker für die Kollagenaktivität herangezogen werden, da aber zwischen Synthese und Sekretion des Kollagens ein nicht zu vernachlässigender Teil bereits intrazellulär wieder abgebaut wird kann nur durch Betrachtung dieser Gene die Theorie der „guten und schlechten Heiler“ nicht gestützt werden. Durch die hohe Korrelation der Ergebnisse aus gene-Array und qRT-PCR könnte für die Zukunft vorläufig auf die Durchführung von qRT-PCR verzichtet werden, um eventuell unterschiedliche Pathways mit dem Gene-Array zu identifizieren. Offene Fragen Ausblick und Perspektiven: Da das System der Wundheilung und Kollagensynthese und –Sekretion sehr komplex ist sollte für die Zukunft durch eine Kokultur mit Makrophagen bzw. durch die Zugabe von TNF-α, IL-6, PDGF, G-CSF, GM-CSF, Vitamin C oder Lysyloxidase zum Kulturmedium, geprüft werden ob sich eine Aktivitätsveränderung der Fibroblasten und damit eine andere Neofaszienstruktur erreichen lässt. Weiterhin sollte um einer Verfälschung der Ergebnisse durch das für die Gele verwendete Rattenkollagen vorzubeugen, entweder die Kulturdauer verlängert werden (mit dem Gedanken dass dann das gesamte Rattenkollagen durch humanes ersetzt wurde) bzw. ein Kollagenfreies Gel als Trägerstruktur entwickelt und verwendet werden. Um eine bessere Vergleichbarkeit der Ergebnisse des Gene-Arrays aus Spenderbiopsie und Neofaszie zu erreichen sollten die zur RNA-Gewinnung verwendeten Anteile der Biopsie noch innerhalb des OP in RNA-later bzw. in flüssigen Stickstoff gegeben werden, um einer verstärkten Degradation vorzubeugen.
Objectives
The long head of the biceps (LHB) is often resected in shoulder surgery and could therefore serve as a cell source for tissue engineering approaches in the shoulder. However, whether it represents a suitable cell source for regenerative approaches, both in the inflamed and non-inflamed states, remains unclear. In the present study, inflamed and native human LHBs were comparatively characterized for features of regeneration.
Methods
In total, 22 resected LHB tendons were classified into inflamed samples (n = 11) and non-inflamed samples (n = 11). Proliferation potential and specific marker gene expression of primary LHB-derived cell cultures were analyzed. Multipotentiality, including osteogenic, adipogenic, chondrogenic, and tenogenic differentiation potential of both groups were compared under respective lineage-specific culture conditions.
Results
Inflammation does not seem to affect the proliferation rate of the isolated tendon-derived stem cells (TDSCs) and the tenogenic marker gene expression. Cells from both groups showed an equivalent osteogenic, adipogenic, chondrogenic and tenogenic differentiation potential in histology and real-time polymerase chain reaction (RT-PCR) analysis.
Conclusion
These results suggest that the LHB tendon might be a suitable cell source for regenerative approaches, both in inflamed and non-inflamed states. The LHB with and without tendinitis has been characterized as a novel source of TDSCs, which might facilitate treatment of degeneration and induction of regeneration in shoulder surgery.
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.
In vitro co-cultures of different primary human cell types are pivotal for the testing and evaluation of biomaterials under conditions that are closer to the human in vivo situation. Especially co-cultures of macrophages and mesenchymal stem cells (MSCs) are of interest, as they are both present and involved in tissue regeneration and inflammatory reactions and play crucial roles in the immediate inflammatory reactions and the onset of regenerative processes, thus reflecting the decisive early phase of biomaterial contact with the host. A co-culture system of these cell types might thus allow for the assessment of the biocompatibility of biomaterials. The establishment of such a co-culture is challenging due to the different in vitro cell culture conditions. For human macrophages, medium is usually supplemented with human serum (hS), whereas hMSC culture is mostly performed using fetal calf serum (FCS), and these conditions are disadvantageous for the respective other cell type. We demonstrate that human platelet lysate (hPL) can replace hS in macrophage cultivation and appears to be the best option for co-cultivation of human macrophages with hMSCs. In contrast to FCS and hS, hPL maintained the phenotype of both cell types, comparable to that of their respective standard culture serum, as well as the percentage of each cell population. Moreover, the expression profile and phagocytosis activity of macrophages was similar to hS.
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.
Bone metastasis is a frequent and life-threatening complication of breast cancer. The molecular mechanisms supporting the establishment of breast cancer cells in the skeleton are still not fully understood, which may be attributed to the lack of suitable models that interrogate interactions between human breast cancer cells and the bone microenvironment. Although it is well-known that integrins mediate adhesion of malignant cells to bone extracellular matrix, their role during bone colonization remains unclear. Here, the role of β1 integrins in bone colonization was investigated using tissue-engineered humanized in vitro and in vivo bone models. In vitro, bone-metastatic breast cancer cells with suppressed integrin β1 expression showed reduced attachment, spreading, and migration within human bone matrix compared to control cells. Cell proliferation in vitro was not affected by β1 integrin knockdown, yet tumor growth in vivo within humanized bone microenvironments was significantly inhibited upon β1 integrin suppression, as revealed by quantitative in/ex vivo fluorescence imaging and histological analysis. Tumor cells invaded bone marrow spaces in the humanized bone and formed osteolytic lesions; osteoclastic bone resorption was, however, not reduced by β1 integrin knockdown. Taken together, we demonstrate that β1 integrins have a pivotal role in bone colonization using unique tissue-engineered humanized bone models.
(1) Background: The mesenchymal stromal cells (MSCs) of different tissue origins are applied in cell-based chondrogenic regeneration. However, there is a lack of comparability determining the most suitable cell source for the tissue engineering (TE) of cartilage. The purpose of this study was to compare the in vitro chondrogenic potential of MSC-like cells from different tissue sources (bone marrow, meniscus, anterior cruciate ligament, synovial membrane, and the infrapatellar fat pad removed during total knee arthroplasty (TKA)) and define which cell source is best suited for cartilage regeneration. (2) Methods: MSC-like cells were isolated from five donors and expanded using adherent monolayer cultures. Differentiation was induced by culture media containing specific growth factors. Transforming growth factor (TGF)-ß1 was used as the growth factor for chondrogenic differentiation. Osteogenesis and adipogenesis were induced in monolayer cultures for 27 days, while pellet cell cultures were used for chondrogenesis for 21 days. Control cultures were maintained under the same conditions. After, the differentiation period samples were analyzed, using histological and immunohistochemical staining, as well as molecularbiological analysis by RT-PCR, to assess the expression of specific marker genes. (3) Results: Plastic-adherent growth and in vitro trilineage differentiation capacity of all isolated cells were proven. Flow cytometry revealed the clear co-expression of surface markers CD44, CD73, CD90, and CD105 on all isolated cells. Adipogenesis was validated through the formation of lipid droplets, while osteogenesis was proven by the formation of calcium deposits within differentiated cell cultures. The formation of proteoglycans was observed during chondrogenesis in pellet cultures, with immunohistochemical staining revealing an increased relative gene expression of collagen type II. RT-PCR proved an elevated expression of specific marker genes after successful differentiation, with no significant differences regarding different cell source of native tissue. (4) Conclusions: Irrespective of the cell source of native tissue, all MSC-like cells showed multipotent differentiation potential in vitro. The multipotent differentiation capacity did not differ significantly, and chondrogenic differentiation was proven in all pellet cultures. Therefore, cell suitability for cell-based cartilage therapies and tissue engineering is given for various tissue origins that are routinely removed during total knee arthroplasty (TKA). This study might provide essential information for the clinical tool of cell harvesting, leading to more flexibility in cell availability.
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.
No abstract available
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.
In dieser Arbeit konnte erstmals gezeigt werden, dass plastik-adhärent wachsende, multipotente Vorläuferzellen, die eine für MSCs charakteristische Kombination von Oberflächenantigenen tragen, aus allen vier untersuchten Geweben des arthrotischen Hüftgelenks isoliert werden konnten. MSC-ähnliche Zellen können somit nicht nur in der Spongiosa und im Gelenkknorpel, sondern auch in der anterioren Gelenkkapsel und dem Ligamentum capitis femoris (LCF) des arthrotisch veränderten menschlichen Hüftgelenks nachgewiesen werden.
Die FACS Analyse der Oberflächenantigene auf Zellen, die aus den vier unterschiedlichen Geweben eines beispielhaft gewählten Spenders isoliert wurden, zeigte eine deutliche Expression der Antigene CD44, CD73, CD90 und CD105. Unabhängig vom Nativgewebe zeigten somit alle untersuchten Zellen ein für MSCs charakteristisches, aber nicht spezifisches Profil an Antigenen auf ihrer Oberfläche. Eine Übereinstimmung mit den ISCT Kriterien für MSCs war aufgrund der fehlenden Kontrolle hämatopoetischer Marker nicht möglich.
Die multipotente Differenzierung der isolierten Zellen erfolgte mithilfe spezifischer Differenzierungsmedien in Monolayer-Kulturen oder für die chondrogene Differenzierung in dreidimensionalen Pellet-Kulturen. Nach 21 Tagen konnten in allen differenzierten Kulturen histologisch und immunhistochemisch klare Zeichen der Osteo- und Adipogenese detektiert werden, während die Auswertung spezifischer Markergene eine klare Steigerung der Expression dieser im Vergleich zu den Negativkontrollen zeigte.
Histologische und immunhistochemische Auswertungen bestätigten auch eine erfolgreiche chondrogene Differenzierung der Zell-Pellets aus Spongiosa, Knorpel und Kapsel. Lediglich in den chondrogen differenzierten Zell-Pellets aus dem LCF konnte immunhistochemisch keine Bildung des knorpelspezifischen Matrixproteins Col II nachgewiesen werden. Mikroskopisch zeigten vor allem die differenzierten MSC-Pellets aus Spongiosa und Knorpel morphologisch eine starke Ähnlichkeit zu hyalinem Knorpelgewebe. Trotz dieser Abstufungen zeigten sich für die relative Expression der chondrogenen Markergene AGG, Col II und Sox-9 keine signifikanten Unterschiede zwischen den differenzierten MSC-Kulturen der vier unterschiedlichen Nativgewebe. Ein positiver Nachweis des Markers Col X wies nach 27 Tagen sowohl in differenzierten als auch in undifferenzierten Pellet-Kulturen auf eine leichte chondrogene Hypertrophie hin. Zusammenfassend zeigten sich keine signifikanten Unterschiede im Hinblick auf das osteogene und adipogene Differenzierungspotential aller untersuchten Zellen. Während das chondrogene Differenzierungspotential der Zellen aus Spongiosa, Knorpel und Kapsel sich aus histologischer und immunhistochemischer Sicht ähnelte, zeigten Pellets aus dem LCF ein schwächeres chondrogenes Differenzierungspotential in vitro.
Obwohl somit erstmals MSC-ähnliche Zellen aus dem LCF und Gewebsproben, die neben dem Stratum synoviale auch das Stratum fibrosum der Hüftgelenkskapsel beinhalteten, charakterisiert wurden, sind weitere wissenschaftliche Arbeiten notwendig, um das multipotente Differenzierungspotential dieser Zellen zu optimieren.
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.
In this report we describe a human pluripotent stem cell-derived vascular progenitor (MesoT) cell of the mesothelium lineage. MesoT cells are multipotent and generate smooth muscle cells, endothelial cells, and pericytes and self-assemble into vessel-like networks in vitro. MesoT cells transplanted into mechanically damaged neonatal mouse heart migrate into the injured tissue and contribute to nascent coronary vessels in the repair zone. When seeded onto decellularized vascular scaffolds, MesoT cells differentiate into the major vascular lineages and self-assemble into vasculature capable of supporting peripheral blood flow following transplantation. These findings demonstrate in vivo functionality and the potential utility of MesoT cells in vascular engineering applications.
This thesis concerned the design and examination of a scaffold for tissue engineering applications. The template for the presented scaffold came from nature itself: the intercellular space in tissues that provides structure and support to the cells of the respective tissue, known as extracellular matrix (ECM). Fibres are a predominant characteristic feature of ECM, providing adhesion sites for cell-matrix interactions. In this dissertation a fibrous mesh was generated using the electrospinning technique to mimic the fibrous structure of the ECM. Two base polymers were explored: a biodegradable polyester, poly(D,L-lactide-co-glycolide); and a functional PEG-based star polymer, NCO-sP(EO-stat-PO). This topic was described in three major parts: the first part was materials based, concerning the chemical design and characterisation of the polymer scaffolds; the focus was then shifted to the cellular response to this fibrous scaffold; and finally the in vivo performance of the material was preliminarily assessed. The first steps towards an electrospun mesh started with adjusting the spinning parameters for the generation of homogeneous fibres. As reported in Chapter 3 a suitable setup configuration was on the one hand comprised of a spinning solution that consisted of 28.5 w/v% PLGA RG 504 and 6 w/v% NCO-sP(EO-stat-PO) in 450 µL acetone, 50 µL DMSO and 10 µL of an aqueous trifluoroacetic acid solution. On the other hand an ideal spinning behaviour was achieved at process parameters such as a flow rate of 0.5 mL/h, spinneret to collector distance of 12-16 cm and a voltage of 13 kV. The NCO-sP(EO-stat-PO) containing fibres proved to be highly hydrophilic as the functional additive was present on the fibre surface. Furthermore, the fibres featured a bulk degradation pattern as a consequence of the proportion of PLGA. Besides the morphologic similarity to ECM fibres, the functionality of the electrospun fibres is also decisive for a successful ECM mimicry. In Chapter 4, the passive as well as active functionality of the fibres was investigated. The fibres were required to be protein repellent to prevent an unspecific cell adhesion. This was proven as even 6.5 % sP(EO-stat-PO) in the PLGA fibres reduced any unspecific protein adsorption of bovine serum albumin and foetal calf serum to less than 1 %. However, avidin based proteins attached to the fibres. This adhesion process was avoided by an additional fibre surface treatment with glycidol. The active functionalisation of NCO-sP(EO-stat-PO)/PLGA fibres was investigated with two fluorescent dyes and biocytin. A threefold, chemically orthogonal, fibre modification was achieved with these dyes. The chapters about the chemical and mechanical properties laid the basis for the in vitro chapters where a specific fibre functionalisation with peptides was conducted to analyse the cell adhesion and biochemical expressions. Beginning with fibroblasts in Chapter 5 the focus was on the specific cell adhesion on the electrospun fibres. While NCO-sP(EO-stat-PO)/PLGA fibres without peptides did not allow any adhesion of fibroblasts, a fibre modification with GRGDS (an adhesion mediating peptide sequence) induced the adhesion and spreading of human dermal fibroblasts on the fibrous scaffolds. The control sequence GRGES that has no adhesion mediating qualities did not lead to any cell adhesion as observed on fibres without modifications. While the experiments of Chapter 5 were a proof-of-concept, in Chapter 6 a possible application in cartilage tissue engineering was examined. Therefore, primary human chondrocytes were seeded on fibrous scaffolds with various peptide sequences. Though the chondrocytes exhibited high viability on all scaffolds, an active interaction of cells and fibres was only found for the decorin derived sequence CGKLER. Live-cell-imaging revealed both cell attachment and migration within CGKLER-modified meshes. As chondrocytes undergo a de-differentiation towards a fibroblast-like phenotype, the chondrogenic re-differentiation on these scaffolds was investigated in a long term cell culture experiment of 28 days. Therefore, the glycosaminoglycan production was analysed as well as the mRNA expression of genes coding for collagen I and II, aggrecan and proteoglycan 4. In general only low amounts of the chondrogenic markers were measured, suggesting no chondrogenic differentiation. For conclusive evidence follow-up experiments are required that support or reject the findings. The success of an implant for tissue engineering relies not only on the response of the targeted cell type but also on the immune reaction caused by leukocytes. Hence, Chapter 7 dealt with primary human macrophages and their behaviour and phenotype on two-dimensional (2D) surfaces compared to three-dimensional (3D) fibrous substrates. It was found that the general non-adhesiveness of NCO-sP(EO-stat-PO) surfaces and fibres does not apply to macrophages. The cells aligned along the fibres on surfaces or resided in the pores of the meshes. On flat surfaces without 3D structure the macrophages showed a retarded adhesion kinetic accompanied with a high migratory activity indicating their search for a topographical feature to adhere to. Moreover, a detailed investigation of cell surface markers and chemokine signalling revealed that macrophages on 2D surfaces exhibited surface markers indicating a healing phenotype while the chemokine release suggested a pro-inflammatory phenotype. Interestingly, the opposite situation was found on 3D fibrous substrates with pro-inflammatory surface markers and pro-angiogenic cytokine release. As the immune response largely depends on cellular communication, it was concluded that the NCO-sP(EO-stat-PO)/PLGA fibres induce an adequate immune response with promising prospects to be used in a scaffold for tissue engineering. The final chapter of this thesis reports on a first in vivo study conducted with the presented electrospun fibres. Here, the fibres were combined with a polypropylene mesh for the treatment of diaphragmatic hernias in a rabbit model. Two scaffold series were described that differed in the overall surface morphology: while the fibres of Series A were incorporated into a thick gel of NCO-sP(EO-stat-PO), the scaffolds of Series B featured only a thin hydrogel layer so that the overall fibrous structure could be retained. After four months in vivo the treated defects of the diaphragm were significantly smaller and filled mainly with scar tissue. Thick granulomas occurred on scaffolds of Series A while the implants of Series B did not induce any granuloma formation. As a consequence of the generally positive outcome of this study, the constructs were enhanced with a drug release system in a follow-up project. The incorporated drug was the MMP-inhibitor Ilomastat which is intended to reduce the formation of scar tissue. In conclusion, the simple and straight forward fabrication, the threefold functionalisation possibility and general versatile applicability makes the meshes of NCO-sP(EO-stat-PO)/PLGA fibres a promising candidate to be applied in tissue engineering scaffolds in the future.
Advanced Therapy Medicinal Products (ATMP) provide promising treatment options particularly for unmet clinical needs, such as progressive and chronic diseases where currently no satisfying treatment exists. Especially from the ATMP subclass of Tissue Engineered Products (TEPs), only a few have yet been translated from an academic setting to clinic and beyond. A reason for low numbers of TEPs in current clinical trials and one main key hurdle for TEPs is the cost and labor-intensive manufacturing process. Manual production steps require experienced personnel, are challenging to standardize and to scale up. Automated manufacturing has the potential to overcome these challenges, toward an increasing cost-effectiveness. One major obstacle for automation is the control and risk prevention of cross contaminations, especially when handling parallel production lines of different patient material. These critical steps necessitate validated effective and efficient cleaning procedures in an automated system. In this perspective, possible technologies, concepts and solutions to existing ATMP manufacturing hurdles are discussed on the example of a late clinical phase II trial TEP. In compliance to Good Manufacturing Practice (GMP) guidelines, we propose a dual arm robot based isolator approach. Our novel concept enables complete process automation for adherent cell culture, and the translation of all manual process steps with standard laboratory equipment. Moreover, we discuss novel solutions for automated cleaning, without the need for human intervention. Consequently, our automation concept offers the unique chance to scale up production while becoming more cost-effective, which will ultimately increase TEP availability to a broader number of patients.
Der Ersatz von Knochengewebe durch die Methode des Tissue Engineerings stellt eine viel versprechende Alternative zu konventionellen Therapieformen dar. Jedoch müssen die bisherigen Kulturbedingungen verbessert werden, um das Differenzierungsverhalten von Zellen optimal steuern zu können. Dabei spielt nicht nur die Wahl eines geeigneten Scaffolds und der zu verwendenden Zellen, sondern auch die des Kultursystems eine entscheidende Rolle. In einem dynamischen Kultursystem zirkuliert Medium und bietet gegenüber einem statischen Kultursystem veränderte Bedingungen bezüglich Nährstoffversorgung und Stimulation durch Flüssigkeitsscherstress. Um die Einflüsse der veränderten Bedingungen zu analysieren, wird in dieser Arbeit ein dynamisches Kultursystem etabliert. Dazu werden Calciumphosphat(CaP)-Scaffolds mit dem 3D Powder Printing System gedruckt und mit Zellen der Osteosarkomzelllinie MG63 oder der Fibroblastenzelllinie L-929 besiedelt. In 17 Versuchsreihen werden die zellbesiedelten Scaffolds bei unterschiedlichen Fließgeschwindigkeiten und über unterschiedliche Kultivierungszeiträume kontinuierlich perfundiert. Anhand der Wachstumsparameter Zellzahl und Zellviabiltät, sowie der Morphologie und räumlichen Verteilung der Zellen werden die Qualitäten der Kultursysteme untersucht und mit statischen Kultursystemen verglichen. Die mit dem 3D Powder Printing System gedruckten Scaffolds erweisen sich als geeignet: Nach 6-tägiger Kultur können unter dem Rasterelektronenmikroskop auf den CaP-Scaffolds eine reichliche Zellbesiedelung mit morphologisch gesunden Zellen, die in das Porensystem hineinwachsen, beobachtet werden. Bei beiden Zelllinien nehmen in beiden Kultursystemen die Wachstumsparameter über einen 6-tägigen Kultivierungszeitraum stetig zu und eine Langzeitkultur über 30 Tage kann in beiden Kultursystemen am Leben erhalten werden. Die kontinuierliche Perfusion in einem dynamischen Kultursystem wirkt sich auf das Zellwachstum günstig aus. Im Vergleich von dynamischen zu statischem Kultursystem über einen 6-tägigen Kultivierungszeitraum wachsen beide Zelllinien im dynamischen Kultursystem besser. Dabei spielt die Fließgeschwindigkeit im dynamischen Kultursystem auf die verbesserte Nährstoffversorgung und Stimulation durch Flüssigkeitsscherstress eine Rolle. Außerdem ist zu beachten, dass der Einfluss der Fließgeschwindigkeit des Mediums auf die einzelnen Scaffolds innerhalb des Kulturcontainers unterschiedlich ist. Dies hängt vom Strömungsprofil im Container ab und macht sich durch eine erhöhte Standardabweichung der Messwerte gegenüber der statischen Kultur bemerkbar.
Als Therapie des Diabetes mellitus Typ II stellt die Xenotransplantation von porzinen Langerhans-Inseln in mikroverkapselter Form eine attraktive Alternative zu den täglichen Insulininjektionen dar. Kultivierung und Funktionsdiagnostik der isolierten porzinen Langerhans-Inseln sind technisch anspruchsvoll und bieten noch immer Potential für Verbesserungen. Werden die Zellen in Zellkulturflaschen über mehrere Tage kultiviert, sinkt die Vitalität unter ein akzeptables Niveau. Bei der Beurteilung der Vitalität und Funktion der Inseln gehen wertvolle Zellen für eine spätere Transplantation verloren. Dies schränkt eine ausgiebige Diagnostik vor der Transplantation ein. Ziel der Arbeit ist es, eine Möglichkeit zur Verbesserung der Kulturbedingungen zu finden und exakte Ergebnisse bei der Funktions- und Vitalitätsdiagnostik ohne Verlust von Zellen zu erreichen. Vorversuche konnten beweisen, dass eine Verbesserung der Vitalität von Langerhans-Inseln in Kultur an der Methode der Kultivierung in Zellkulturflaschen scheitert. Stattdessen wurde das Prinzip der Perfusionskultur in spezialisierten Behältnissen für die systematische Verbesserung der Kulturbedingungen und zur genaueren Diagnostik mittels Mikroskopie und Funktionsdiagnostik gewählt. Mit einem solchen System ist sowohl Kultivierung als auch Funktions- und Vitalitätsdiagnostik in einem Behälter möglich. Beim Prinzip der Perfusionskultur befindet sich das Medium stets in Bewegung um die Zellen und Gewebe und sorgt so für einen kontinuierlichen Zustrom exakt definierten Mediums und permanenten Abtransport der Stoffwechselprodukte. Im Rahmen dieser Arbeit wurde für die Anforderungen im eigenen Labor ein maßgeschneidertes System mit mehreren Versionen von Behältnissen für Perfusionskulturen entwickelt, deren jeweils neuere Version auf den Erfahrungen mit den Vorversionen aufbaut. In die Entwicklung fließen ebenso umfangreiche theoretische Überlegungen ein, sowie systematische Tests zu den physikalischen Eigenschaften der Behältnisse. Die zuletzt entwickelte ist die Version V6SMTE, die in der Arbeit „Würzburger Kammer“ genannt wird. Dieser Behälter ist aus Edelstahl, mit einem Deckglas zur makro- und mikroskopischen Begutachtung, Zu- und Abläufen und einem Anschluss zum Entfernen von Gasblasen. Im Inneren befindet sich ein Einsatz, der eine stufenlose Regulierung des Volumens um die Zellen ermöglicht, so dass für Kultur und Funktionsprüfung bzw. Mikroskopie jeweils optimale Bedingungen erreicht werden. Weiterhin kann über einen Temperatursensor die Temperatur im Inneren des Behälters gemessen und über Heizelemente an der Außenwand computergesteuert reguliert werden. Die Zellen und Gewebe können auf unterschiedlichen Trägermaterialien eingesetzt werden. Während der Kultur kann ein Deckel geöffnet und die Zellen manipuliert werden. Das System ist unabhängig vom Brutschrank, ist sterilisierbar und wieder verwendbar. Kultiviert wurde endokrines Gewebe (isolierte Langerhans-Inseln, humanes Nebenschilddrüsengewebe). Dieses wurde zur Funktionsprüfung mit verschiedenen Mediatoren stimuliert, das Medium fraktioniert aufgefangen und sein Hormongehalt mit ELISA oder RIA bestimmt. Die Zellen wurden nativ und mit Fluoreszenzfarbstoffen (FDA, PI) gefärbt mit bis zu 400facher Vergrößerung unter dem Auflichtmikroskop beurteilt. Im Zuge der Auswertung der anfallenden Proben auf ihren Insulingehalt wurde für diese Arbeit ein Insulin-ELISA entwickelt, der bei vergleichbarer Genauigkeit deutlich günstiger ist, als der bisher verwendete kommerzielle ELISA. Mit der Würzburger Kammer kultivierte Langerhans-Inseln zeigten eine vergleichbare Vitalität im Vergleich zur Zellkulturflasche, die mit der Würzburger Kammer gewonnenen Perifusionskurven sind in hohem Maß reproduzierbar, Zusammenhänge von Höhe der Glukoseexposition und Kultivierungsdauer mit der Insulinausschüttungskurve konnten eindrucksvoll beschrieben werden. Erstmals wurde auch im eigenen Labor die aus der Literatur bekannte paradoxe Insulinausschüttung beschrieben. Beispielhaft für andere endokrine Gewebe wurde humanes Nebenschilddrüsengewebe erfolgreich in der Würzburger Kammer kultiviert und Vitalitäts- und Funktionsdiagnostik unterzogen. Das Kultursystem ermöglicht die Kultivierung und eine komplette Analyse von Funktion, Vitalität und Morphologie von endokrinen Zellen. Es kann somit in idealer Weise zur Verbesserung der Kulturbedingungen und zur Beurteilung von endokrinen Zellen vor der Transplantation herangezogen werden.
Despite promising clinical results in osteochondral defect repair, a recently developed bi-layered collagen/collagen-magnesium-hydroxyapatite scaffold has demonstrated less optimal subchondral bone repair. This study aimed to improve the bone repair potential of this scaffold by adsorbing bone morphogenetic protein 2 (BMP-2) and/or platelet-derived growth factor-BB (PDGF-BB) onto said scaffold. The in vitro release kinetics of BMP-2/PDGF-BB demonstrated that PDGF-BB was burst released from the collagen-only layer, whereas BMP-2 was largely retained in both layers. Cell ingrowth was enhanced by BMP-2/PDFG-BB in a bovine osteochondral defect ex vivo model. In an in vivo semi-orthotopic athymic mouse model, adding BMP-2 or PDGF-BB increased tissue repair after four weeks. After eight weeks, most defects were filled with bone tissue. To further investigate the promising effect of BMP-2, a caprine bilateral stifle osteochondral defect model was used where defects were created in weight-bearing femoral condyle and non-weight-bearing trochlear groove locations. After six months, the adsorption of BMP-2 resulted in significantly less bone repair compared with scaffold-only in the femoral condyle defects and a trend to more bone repair in the trochlear groove. Overall, the adsorption of BMP-2 onto a Col/Col-Mg-HAp scaffold reduced bone formation in weight-bearing osteochondral defects, but not in non-weight-bearing osteochondral defects.
Objectives
Micro-computed tomography (μ-CT) and histology, the current gold standard methods for assessing the formation of new bone and blood vessels, are invasive and/or destructive. With that in mind, a more conservative tool, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), was tested for its accuracy and reproducibility in monitoring neovascularization during bone regeneration. Additionally, the suitability of blood perfusion as a surrogate of the efficacy of osteoplastic materials was evaluated.
Materials and methods
Sixteen rabbits were used and equally divided into four groups, according to the time of euthanasia (2, 3, 4, and 6 weeks after surgery). The animals were submitted to two 8-mm craniotomies that were filled with blood or autogenous bone. Neovascularization was assessed in vivo through DCE-MRI, and bone regeneration, ex vivo, through μ-CT and histology.
Results
The defects could be consistently identified, and their blood perfusion measured through DCE-MRI, there being statistically significant differences within the blood clot group between 3 and 6 weeks (p = 0.029), and between the former and autogenous bone at six weeks (p = 0.017). Nonetheless, no significant correlations between DCE-MRI findings on neovascularization and μ-CT (r =−0.101, 95% CI [−0.445; 0.268]) or histology (r = 0.305, 95% CI [−0.133; 0.644]) findings on bone regeneration were observed.
Conclusions
These results support the hypothesis that DCE-MRI can be used to monitor neovascularization but contradict the premise that it could predict bone regeneration as well.
The electrohydrodynamic stabilization of direct-written fluid jets is explored to design and manufacture tissue engineering scaffolds based on their desired fiber dimensions. It is demonstrated that melt electrowriting can fabricate a full spectrum of various fibers with discrete diameters (2–50 µm) using a single nozzle. This change in fiber diameter is digitally controlled by combining the mass flow rate to the nozzle with collector speed variations without changing the applied voltage. The greatest spectrum of fiber diameters was achieved by the simultaneous alteration of those parameters during printing. The highest placement accuracy could be achieved when maintaining the collector speed slightly above the critical translation speed. This permits the fabrication of medical-grade poly(ε-caprolactone) into complex multimodal and multiphasic scaffolds, using a single nozzle in a single print. This ability to control fiber diameter during printing opens new design opportunities for accurate scaffold fabrication for biomedical applications.
Identification of articular cartilage progenitor cells (ACPCs) has opened up new opportunities for cartilage repair. These cells may be used as alternatives for or in combination with mesenchymal stromal cells (MSCs) in cartilage engineering. However, their potential needs to be further investigated, since only a few studies have compared ACPCs and MSCs when cultured in hydrogels. Therefore, in this study, we compared chondrogenic differentiation of equine ACPCs and MSCs in agarose constructs as monocultures and as zonally layered co-cultures under both normoxic and hypoxic conditions. ACPCs and MSCs exhibited distinctly differential production of the cartilaginous extracellular matrix (ECM). For ACPC constructs, markedly higher glycosaminoglycan (GAG) contents were determined by histological and quantitative biochemical evaluation, both in normoxia and hypoxia. Differential GAG production was also reflected in layered co-culture constructs. For both cell types, similar staining for type II collagen was detected. However, distinctly weaker staining for undesired type I collagen was observed in the ACPC constructs. For ACPCs, only very low alkaline phosphatase (ALP) activity, a marker of terminal differentiation, was determined, in stark contrast to what was found for MSCs. This study underscores the potential of ACPCs as a promising cell source for cartilage engineering.
In this study, well-defined, 3D arrays of air-suspended melt electrowritten fibers are made from medical grade poly(ɛ-caprolactone) (PCL). Low processing temperatures, lower voltages, lower ambient temperature, increased collector distance, and high collector speeds all aid to direct-write suspended fibers that can span gaps of several millimeters between support structures. Such processing parameters are quantitatively determined using a “wedge-design” melt electrowritten test frame to identify the conditions that increase the suspension probability of long-distance fibers. All the measured parameters impact the probability that a fiber is suspended over multimillimeter distances. The height of the suspended fibers can be controlled by a concurrently fabricated fiber wall and the 3D suspended PCL fiber arrays investigated with early post-natal mouse dorsal root ganglion explants. The resulting Schwann cell and neurite outgrowth extends substantial distances by 21 d, following the orientation of the suspended fibers and the supporting walls, often generating circular whorls of high density Schwann cells between the suspended fibers. This research provides a design perspective and the fundamental parametric basis for suspending individual melt electrowritten fibers into a form that facilitates cell culture.
Compared to cell therapy, where cells are injected into a defect region, the treatment of heart infarction with cells seeded in a vascularized scaffold bears advantages, such as an immediate nutrient supply or a controllable and persistent localization of cells. For this purpose, decellularized native tissues are a preferable choice as they provide an in vivo-like microenvironment. However, the quality of such scaffolds strongly depends on the decellularization process. Therefore, two protocols based on sodium dodecyl sulfate or sodium deoxycholate were tailored and optimized for the decellularization of a porcine heart. The obtained scaffolds were tested for their applicability to generate vascularized cardiac patches. Decellularization with sodium dodecyl sulfate was found to be more suitable and resulted in scaffolds with a low amount of DNA, a highly preserved extracellular matrix composition, and structure shown by GAG quantification and immunohistochemistry. After seeding human endothelial cells into the vasculature, a coagulation assay demonstrated the functionality of the endothelial cells to minimize the clotting of blood. Human-induced pluripotent-stem-cell-derived cardiomyocytes in co-culture with fibroblasts and mesenchymal stem cells transferred the scaffold into a vascularized cardiac patch spontaneously contracting with a frequency of 25.61 ± 5.99 beats/min for over 16 weeks. The customized decellularization protocol based on sodium dodecyl sulfate renders a step towards a preclinical evaluation of the scaffolds.
Cartilage offers limited regenerative capacity. Cell-based approaches have emerged as a promising alternative in the treatment of cartilage defects and osteoarthritis. Due to their easy accessibility, abundancy, and chondrogenic potential mesenchymal stromal cells (MSCs) offer an attractive cell source. MSCs are often combined with natural or synthetic hydrogels providing tunable biocompatibility, biodegradability, and enhanced cell functionality. In this review, we focused on the different advantages and disadvantages of various natural, synthetic, and modified hydrogels. We examined the different combinations of MSC-subpopulations and hydrogels used for cartilage engineering in preclinical and clinical studies and reviewed the effects of added growth factors or gene transfer on chondrogenesis in MSC-laden hydrogels. The aim of this review is to add to the understanding of the disadvantages and advantages of various combinations of MSC-subpopulations, growth factors, gene transfers, and hydrogels in cartilage engineering.
Das Arbeitsgebiet Tissue Engineering befasst sich mit der Klärung der Mechanismen, die der Funktionen verschiedener Gewebearten zu Grunde liegen sowie mit der Entwicklung alternativer Strategien zur Behandlung von Organversagen bzw. Organverlusten. Einer der kritischsten Punkte im Tissue Engineering ist die ausreichende Versorgung der Zellen mit Nährstoffen und Sauerstoff. Bioartifizielle Gewebe mit einer Dicke von bis zu 200 µm können mittels Diffusion ausreichend versorgt werden. Für dickere Transplantate ist die Versorgung der Zellen alleine durch Diffusion jedoch nicht gegeben. Hierfür müssen Mechanismen und Strategien zur Prävaskularisierung der artifiziellen Gewebekonstrukte entwickelt werden, damit die Nährstoff- und Sauerstoffversorgung aller Zellen, auch im Inneren des Transplantates, von Anfang an gewährleistet ist. Eine wichtige Rolle bei der Prävaskularisierung spielt die Angiogenese. Dabei ist die Wahl einer geeigneten Zellquelle entscheidend, da die Zellen die Basis für die Angiogenese darstellen. Mikrovaskuläre Endothelzellen (mvEZ) sind maßgeblich an der Angiogenese beteiligt. Das Problem bei der Verwendung von humanen primären mvEZ ist ihre geringe Verfügbarkeit, ihre limitierte Proliferationskapazität und der schnelle Verlust ihrer typischen Endothelzellmarker in-vitro. Der Aufbau standardisierter in-vitro Testsysteme ist durch die geringe Zellausbeute auch nicht möglich. Die upcyte® Technologie bietet hierfür einen Lösungsansatz. In der vorliegenden Arbeit konnten upcyte® mvEZ als Alternative zu primären mvEZ generiert werden. Es konnte gezeigt werden, dass die Zellen eine erweiterte Proliferationsfähigkeit aufweisen und im Vergleich zu primären mvEZ durchschnittlich 15 zusätzliche Populationsverdopplungen leisten können. Dadurch ist es möglich 3x104-fach mehr upcyte® mvEZ eines Spenders zu generieren verglichen mit den korrespondierenden Primärzellen. Die gute und ausreichende Verfügbarkeit der Zellen macht sie interessant für die Standardisierung von in-vitro Testsystemen, ebenso können die Zellen zur Prävaskularisierung von Transplantaten eingesetzt werden. Upcyte® mvEZ zeigen zahlreiche Primärzellmerkmale, die in der Literatur beschrieben sind. Im konfluenten Zustand zeigen sie die für primäre mvEZ spezifische pflastersteinartige Morphologie. Darüber hinaus exprimieren upcyte® mvEZ typische Endothelzellmarker wie CD31, vWF, eNOS, CD105, CD146 und VEGFR-2 vergleichbar zu primären mvEZ. Eine weitere endothelzellspezifische Eigenschaft ist die Bindung von Ulex europaeus agglutinin I Lektin an die alpha-L-Fucose enthaltene Kohlenhydratstrukturen von mvEZs. Auch hier wurden upcyte® Zellen mit primären mvEZ verglichen und zeigten die hierfür charkteristischen Strukturen. Zusätzlich zu Morphologie, Proliferationskapazität und endothelzellspezifischen Markern, zeigen upcyte® mvEZ auch mehrere funktionelle Eigenschaften, welche in primären mvEZ beobachtet werden können, wie beispielsweise die Aufnahme von Dil-markiertem acetyliertem Low Density Lipoprotein (Dil-Ac-LDL) oder die Fähigkeit den Prozess der Angiognese zu unterstützen. Zusätzlich bilden Sphäroide aus upcyte® mvEZ dreidimensionale luminäre Zellformationen in einer Kollagenmatrix aus. Diese Charakteristika zeigen den quasi-primären Phänotyp der upcyte® mvEZs. Upcyte® mvEZ stellen darüber hinaus eine neuartige mögliche Zellquelle für die Generierung prävaskularisierter Trägermaterialien im Tissue Engineering dar. In der vorliegenden Arbeit konnte die Wiederbesiedlung der biologisch vaskularisierte Matrix (BioVaSc) mit upcyte® mvEZ vergleichbar zu primären mvEZ gezeigt werden. Der Einsatz von upcyte® mvEZ in der BioVaSc stellt einen neuen, vielversprechenden Ansatz zur Herstellung eines vaskularisierten Modells für Gewebekonstrukte dar, wie beispielsweise einem Leberkonstrukt. Zusammenfassend konnte in der vorliegenden Arbeit gezeigt werden, dass upcyte® mvEZ vergleichbar zu primären mvEZs sind und somit eine geeignete Alternative für die Generierung prävaskulierter Trägermaterialien und Aufbau von in-vitro Testsystemen darstellen. Darüber hinaus wurde ein neues, innovatives System für die Generierung einer perfundierten, mit Endothelzellen wiederbesiedelten Matrix für künstliches Gewebe in-vitro entwickelt.
Approaches to mimic the complexity of the skeletal mesenchymal stem/stromal cell niche in vitro
(2019)
Mesenchymal stem/stromal cells (MSCs) are an essential element of most modern tissue engineering and regenerative medicine approaches due to their multipotency and immunoregulatory functions. Despite the prospective value of MSCs for the clinics, the stem cells community is questioning their developmental origin, in vivo localization, identification, and regenerative potential after several years of far-reaching research in the field. Although several major progresses have been made in mimicking the complexity of the MSC niche in vitro, there is need for comprehensive studies of fundamental mechanisms triggered by microenvironmental cues before moving to regenerative medicine cell therapy applications. The present comprehensive review extensively discusses the microenvironmental cues that influence MSC phenotype and function in health and disease – including cellular, chemical and physical interactions. The most recent and relevant illustrative examples of novel bioengineering approaches to mimic biological, chemical, and mechanical microenvironmental signals present in the native MSC niche are summarized, with special emphasis on the forefront techniques to achieve bio-chemical complexity and dynamic cultures. In particular, the skeletal MSC niche and applications focusing on the bone regenerative potential of MSC are addressed. The aim of the review was to recognize the limitations of the current MSC niche in vitro models and to identify potential opportunities to fill the bridge between fundamental science and clinical application of MSCs.
In order to mimic the extracellular matrix for tissue engineering, recent research approaches often involve 3D printing or electrospinning of fibres to scaffolds as cell carrier material. Within this thesis, a micron fibre printing process, called melt electrospinning writing (MEW), combining both additive manufacturing and electrospinning, has been investigated and improved. Thus, a unique device was developed for accurate process control and manufacturing of high quality constructs. Thereby, different studies could be conducted in order to understand the electrohydrodynamic printing behaviour of different medically relevant thermoplastics as well as to characterise the influence of MEW on the resulting scaffold performance.
For reproducible scaffold printing, a commonly occurring processing instability was investigated and defined as pulsing, or in extreme cases as long beading. Here, processing analysis could be performed with the aim to overcome those instabilities and prevent the resulting manufacturing issues. Two different biocompatible polymers were utilised for this study: poly(ε-caprolactone) (PCL) as the only material available for MEW until then and poly(2-ethyl-2-oxazoline) for the first time. A hypothesis including the dependency of pulsing regarding involved mass flows regulated by the feeding pressure and the electrical field strength could be presented. Further, a guide via fibre diameter quantification was established to assess and accomplish high quality printing of scaffolds for subsequent research tasks.
By following a combined approach including small sized spinnerets, small flow rates and high field strengths, PCL fibres with submicron-sized fibre diameters (fØ = 817 ± 165 nm) were deposited to defined scaffolds. The resulting material characteristics could be investigated regarding molecular orientation and morphological aspects. Thereby, an alignment and isotropic crystallinity was observed that can be attributed to the distinct acceleration of the solidifying jet in the electrical field and by the collector uptake. Resulting submicron fibres formed accurate but mechanically sensitive structures requiring further preparation for a suitable use in cell biology. To overcome this handling issue, a coating procedure, by using hydrophilic and cross-linkable star-shaped molecules for preparing fibre adhesive but cell repellent collector surfaces, was used.
Printing PCL fibre patterns below the critical translation speed (CTS) revealed the opportunity to manufacture sinusoidal shaped fibres analogously to those observed using purely viscous fluids falling on a moving belt. No significant influence of the high voltage field during MEW processing could be observed on the buckling phenomenon. A study on the sinusoidal geometry revealed increasing peak-to-peak values and decreasing wavelengths as a function of decreasing collector speeds sc between CTS > sc ≥ 2/3 CTS independent of feeding pressures. Resulting scaffolds printed at 100 %, 90 %, 80 % and 70 % of CTS exhibited significantly different tensile properties, foremost regarding Young’s moduli (E = 42 ± 7 MPa to 173 ± 22 MPa at 1 – 3 % strain). As known from literature, a changed morphology and mechanical environment can impact cell performance substantially leading to a new opportunity of tailoring TE scaffolds.
Further, poly(L-lactide-co-ε-caprolactone-co-acryloyl carbonate) as well as poly(ε-caprolactone-co-acryloyl carbonate) (PCLAC) copolymers could be used for MEW printing. Those exhibit the opportunity for UV-initiated radical cross-linking in a post-processing step leading to significantly increased mechanical characteristics. Here, single fibres of the polymer composed of 90 mol.% CL and 10 mol.% AC showed a considerable maximum tensile strength of σmax = 53 ± 16 MPa. Furthermore, sinusoidal meanders made of PCLAC yielded a specific tensile stress-strain characteristic mimicking the qualitative behaviour of tendons or ligaments. Cell viability by L929 murine fibroblasts and live/dead staining with human mesenchymal stem cells revealed a promising biomaterial behaviour pointing out MEW printed PCLAC scaffolds as promising choice for medical repair of load-bearing soft tissue.
Indeed, one apparent drawback, the small throughput similar to other AM methods, may still prevent MEW’s industrial application yet. However, ongoing research focusses on enlargement of manufacturing speed with the clear perspective of relevant improvement. Thereby, the utilisation of large spinneret sizes may enable printing of high volume rates, while downsizing the resulting fibre diameter via electrical field and mechanical stretching by the collector uptake. Using this approach, limitations of FDM by small nozzle sizes could be overcome. Thinking visionary, such printing devices could be placed in hospitals for patient-specific printing-on-demand therapies one day. Taking the evolved high deposition precision combined with the unique small fibre diameter sizes into account, technical processing of high performance membranes, filters or functional surface finishes also stands to reason.
The skeleton is a preferred homing site for breast cancer metastasis. To date, treatment options for patients with bone metastases are mostly palliative and the disease is still incurable. Indeed, key mechanisms involved in breast cancer osteotropism are still only partially understood due to the lack of suitable animal models to mimic metastasis of human tumor cells to a human bone microenvironment. In the presented study, we investigate the use of a human tissue-engineered bone construct to develop a humanized xenograft model of breast cancer-induced bone metastasis in a murine host. Primary human osteoblastic cell-seeded melt electrospun scaffolds in combination with recombinant human bone morphogenetic protein 7 were implanted subcutaneously in non-obese diabetic/severe combined immunodeficient mice. The tissue-engineered constructs led to the formation of a morphologically intact 'organ' bone incorporating a high amount of mineralized tissue, live osteocytes and bone marrow spaces. The newly formed bone was largely humanized, as indicated by the incorporation of human bone cells and human-derived matrix proteins. After intracardiac injection, the dissemination of luciferase-expressing human breast cancer cell lines to the humanized bone ossicles was detected by bioluminescent imaging. Histological analysis revealed the presence of metastases with clear osteolysis in the newly formed bone. Thus, human tissue-engineered bone constructs can be applied efficiently as a target tissue for human breast cancer cells injected into the blood circulation and replicate the osteolytic phenotype associated with breast cancer-induced bone lesions. In conclusion, we have developed an appropriate model for investigation of species-specific mechanisms of human breast cancer-related bone metastasis in vivo.
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.