TY  - THES
A1  - Mathew-Schmitt, Sanjana
T1  - Development of blood-brain barrier spheroid models based on human induced pluripotent stem cells (hiPSCs) and investigation of shear stress on hiPSC-derived brain capillary endothelial-like cells
T1  - Entwicklung von Sphäroid-Modellen der Blut-Hirn-Schranke basierend auf 
menschlichen induzierten pluripotenten Stammzellen (hiPSCs) und Untersuchung der Scherbeanspruchung von hiPSC-abgeleiteten Hirnkapillarendothel-ähnlichen Zellen
N2  - A highly regulated microenvironment is essential in maintaining normal functioning of the central nervous system (CNS). The existence of a biological barrier, termed as the blood-brain barrier (BBB), at the blood to brain interface effectively allows for selective passage of substances and pathogens into the brain (Kadry, Noorani et al. 2020). The BBB chiefly serves in protecting the brain from extrinsic toxin entry and pathogen invasions. The BBB is formed mainly by brain capillary endothelial cells (BCECs) which are responsible for excluding ∼ 100% of large-molecule neurotherapeutics and more than 98% of all small-molecule drugs from entry into the brain. Minimal BBB transport of major potential CNS drugs allows for attenuated effective treatments for majority of CNS disorders (Appelt-Menzel, Oerter et al. 2020). Animals are generally used as model systems to study neurotherapeutic delivery into the brain, however due to species based disparity, experimental animal models lead to several false positive or false negative drug efficacy predictions thereby being unable to fully predict effects in humans (Ruck, Bittner et al. 2015). An example being that over the last two decades, much of the studies involving animals lead to high failure rates in drug development with ~ 97% failure in cancers and ~ 99% failure for Alzheimer´s disease (Pound 2020). Widespead failures in clinical trials associated with neurological disorders have resulted in questions on whether existing preclinical animal models are genuinely reflective of the human condition (Bhalerao, Sivandzade et al. 2020). Apart from high failure rates in humans, the costs for animal testings is extremely high. According to the Organisation for Economic Co-operation and Development (OECD), responsible for determining animal testing guidelines and methodology for government, industry, and independent laboratories the average cost of a single two-generation reproductive animal toxicity study worldwide is 318,295 € and for Europe alone is ~ 285,842 € (Van Norman 2019). Due to these reasons two separate movements exist within the scientific world, one being to improve animal research and the other to promote new approach methodologies with the European government setting 2025 - 2035 as a deadline for gradually disposing the use of animals in pharmaceutical testing (Pound 2020).
The discovery of human induced pluripotent stem cell (hiPSC) technology in 2006 (Takahashi and Yamanaka 2006, Takahashi, Tanabe et al. 2007) revolutionized the field of drug discovery in-vitro. HiPSCs can be differentiated into various tissue types that mimic disease phenotypes, thereby offering the possibility to deliver humanized in-vitro test systems. With respect to the BBB, several strategies to differentiate hiPSCs to BCECs (iBCECs) are reported over the years (Appelt-Menzel, Oerter et al. 2020). However, iBCECs are said to possess an epithelial or undifferentiated phenotype causing incongruity in BBB lineage specifications (Lippmann,
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Azarin et al. 2020). Therefore, in order to identify a reliable differentiation strategy in deriving iBCECs possessing hallmark BBB characteristics, which can be used for downstream applications, the work in this thesis compared two methods, namely the co-differentiation (CD) and the directed differentiation (DD). Briefly, CD mimics a brain like niche environment for iBCEC specification (Lippmann, Al-Ahmad et al. 2014), while DD focuses on induction of the mesoderm followed by iBCEC specification (Qian, Maguire et al. 2017). The results obtained verified that while iBCECs derived via CD, in comparison to human BCEC cell line hCMEC/D3 showed the presence of epithelial transcripts such as E-Cadherin (CDH1), and gene level downregulation of endothelial specific platelet endothelial cell adhesion molecule-1 (PECAM-1) and VE-cadherin (CDH5) but demonstrated higher barrier integrity. The CD strategy essentially presented iBCECs with a mean trans-endothelial electrical resistance (TEER) of ~ 2000 – 2500 Ω*cm2 and low permeability coefficients (PC) of < 0.50 μm/min for small molecule transport of sodium fluorescein (NaF) and characteristic BCEC tight junction (TJ) protein expression of claudin-5 and occludin. Additionally, iBCECs derived via CD did not form tubes in response to angiogenic stimuli. DD on the other hand resulted in iBCECs with similar down regulations in PECAM-1 and CDH5 gene expression. They were additionally characterized by lower barrier integrity, measured by mean TEER of only ~ 250 – 450 Ω*cm2 and high PC of > 5 μm/min in small molecule transport of NaF. Although iBCECs derived via DD formed tubes in response to angiogenic stimuli, they did not show positive protein expression of characteristic BCEC TJs such as claudin-5 and occludin. These results led to the hypothesis that maturity and lineage specification of iBCECs could be improved by incorporating in-vivo like characteristics in-vitro, such as direct co-culture with neurovascular unit (NVU) cell types via spheroid formation and by induction of shear stress and fluid flow. In comparison to standard iBCEC transwell mono-cultures, BBB spheroids showed enhanced transcript expression of PECAM-1 and reduced expression of epithelial markers such as CDH1 and claudin-6 (CLDN6). BBB spheroids showed classical BCEC-like ultrastructure that was identified by TJ particles on the protoplasmic face (P-face) and exoplasmic face (E-face) of the plasma membrane. TJ strands were organized as particles and particle-free grooves on the E-face, while on the P-face, partly beaded particles and partly continuous strands were identified. BBB spheroids also showed positive protein expression of claudin-5, VE-cadherin, PECAM-1, glucose transporter-1 (GLUT-1), P-glycoprotein (P-gp) and transferrin receptor-1 (Tfr-1). BBB spheroids demonstrated higher relative impedance percentages in comparison to spheroids without an iBCEC barrier. Barrier integrity assessments additionally corresponded with lower permeability to small molecule tracer NaF, with spheroids containing iBCECs showing higher relative fluorescence unit percentages (RFU%) of ~ 90% in apical compartments, compared to ~ 80% in spheroids without iBCECs. In summary, direct cellular contacts in the complex spheroid model resulted in enhanced maturation of iBCECs.
8
A bioreactor system was used to further assess the effect of shear stress. This system enabled inclusion of fluidic flow and shear stress conditions in addition to non-invasive barrier integrity measurements (Choi, Mathew et al. 2022). iBCECs were cultured for a total of seven days post differentiation (d17) within the bioreactor and barrier integrity was non-invasively monitored. Until d17 of long-term culture, TEER values of iBCECs steadily dropped from ~ 1800 Ω*cm2 ~ 400 Ω*cm2 under static conditions and from ~ 2500 Ω*cm2 to ~ 250 Ω*cm2 under dynamic conditions. Transcriptomic analyses, morphometric analyses and protein marker expression showed enhanced maturation of iBECs under long-term culture and dynamic flow. Importantly, on d10 claudin-5 was expressed mostly in the cytoplasm with only ~ 5% iBCECs showing continuous staining at the cell borders. With increase in culture duration, iBCECs at d17 of static culture showed ~ 18% of cells having continuous cell border expression, while dynamic conditions showed upto ~ 30% of cells with continuous cell-cell border expression patterns. Similarly, ~ 33% of cells showed cell-cell border expression of occludin on d10 with increases to ~ 55% under d17 static and up to ~ 65% under d17 dynamic conditions, thereby indicating iBCEC maturation.
In conclusion, the data presented within this thesis demonstrates the maturation of iBCECs in BBB spheroids, obtained via direct cellular contacts and by the application of flow and shear stress. Both established novel models need to be further validated for pharmaceutical drug applications together with in-vitro-in-vivo correlations in order to exploit their full potential.
N2  - Eine hochregulierte Mikroumgebung ist für die Aufrechterhaltung der normalen Funktion des Zentralen Nervensystems (ZNS) unerlässlich. Das Vorhandensein einer biologischen Barriere, der so genannten Blut-Hirn-Schranke (BHS), als Schnittstelle zwischen Blutkreislauf und Gehirn ermöglicht den selektiven Durchgang von Substanzen und Pathogenen in das Gehirn (Kadry, Noorani et al. 2020). Die BHS dient hauptsächlich dazu, das Gehirn vor dem Eindringen von Toxinen von außen und dem Eindringen von Krankheitserregern zu schützen. Die BHS wird hauptsächlich von Hirnkapillarendothelzellen (engl. brain capillary endothelial cells, BCECs) gebildet, die dafür verantwortlich sind, dass ∼ 100% der großmolekularen Neurotherapeutika und mehr als 98% aller kleinmolekularen Medikamente nicht in das Gehirn gelangen können. Ein eingeschränkter BHS-Transport wichtiger potenzieller Wirkstoffe führt zu einer abgeschwächten Wirksamkeit der Behandlung der meisten ZNS-Erkrankungen (Pardridge 2005). Mäuse, Ratten, Schweine und Rinder werden in der Regel als Modellsysteme verwendet, um die Verabreichung von Neurotherapeutika in das Gehirn zu untersuchen. Aufgrund der Unterschiede zwischen den Spezies führen experimentelle Tiermodelle jedoch vermehrt zu falsch positiven oder falsch negativen Vorhersagen über die Wirksamkeit von Medikamenten, so dass sie nicht in der Lage sind, die Wirkungen beim Menschen vollständig vorherzusagen (Ruck, Bittner et al. 2015). Ein Beispiel dafür ist, dass in den letzten zwei Jahrzehnten ein Großteil der Studien an Tieren zu Misserfolgsraten in der Wirkstoffzulassung geführt hat. Bei einer Fehlerrate von 97% im Zusammenhang mit Krebs und ~99% bei Alzheimer führt dies zum Therapieversagen (Pound 2020). Die weit verbreiteten Misserfolge bei klinischen Versuchen im Zusammenhang mit neurologischen Erkrankungen haben zu der Frage geführt, ob die bestehenden präklinischen Tiermodelle wirklich die Physiologie des Menschen widerspiegeln (Bhalerao, Sivandzade et al. 2020). Abgesehen von den hohen Ausfallraten sind die Kosten für Tierversuche extrem hoch. Nach Angaben der Organisation für wirtschaftliche Zusammenarbeit und Entwicklung (engl. Organisation for Economic Co-operation and Development, OECD), welche für die Festlegung von Tierversuchsrichtlinien und -methoden für die Regierung, die Industrie und unabhängige Labore zuständig ist, belaufen sich die durchschnittlichen Kosten für eine einzige Zwei-Generationen-Studie zur Reproduktionstoxizität an Tieren weltweit auf 318.295 € und allein für Europa auf ~ 285.842 € (Van Norman 2019). Aus diesen Gründen gibt es zwei unterschiedliche Bemühungen unter den Wissenschaftlern. Zum einen zur Verbesserung der Tierforschung und zum anderen zur Förderung neuer Methoden gemäß den 3R (eng. replace, reduce, refine), wobei die europäische Regierung die Jahre 2025 bis 2035 als Frist für den schrittweisen Verzicht auf Tierversuche in der Forschung festgelegt hat (Pound 2020). Die
10
Entdeckung der humanen induziert pluripotenten Stammzell (hiPSC)-Technologie im Jahr 2006 (Takahashi und Yamanaka 2006, Takahashi, Tanabe et al. 2007) hat den Bereich der Arzneimittelforschung revolutioniert, da hiPSCs in verschiedene Gewebetypen differenziert werden können und damit die Möglichkeit bieten, humanisierte in-vitro-Testsysteme bereitzustellen, die zur Untersuchung verschiedener Krankheiten verwendet werden können.
In Bezug auf die BHS wurde im Laufe der Jahre mehrere Strategien zur Differenzierung von hiPSCs zu BCECs (iBCECs) etabliert (Appelt-Menzel, Oerter et al. 2020), allerdings wird ihnen ein epithelialer Phänotyp nachgesagt, was zu Fragen in der Spezifikation der BBB führt (Lippmann, Azarin et al. 2020). Um eine verlässliche Differenzierungsstrategie für die Gewinnung von iBCECs mit charakteristischen BHS-Merkmalen zu finden, welche für Downstream-Anwendungengenutztwerden können, wurden in dieser Arbeit zwei Methoden verglichen. Die Ko-Differenzierung (engl. co-differentiation, CD) und die gerichtete Differenzierung (engl. directed differentiation, DD). Zusammengefasst simuliert die CD eine ZNS-ähnliche Mikroumgebung für die Spezifikation der iBCECs nach (Lippmann, Al-Ahmad et al. 2014), während sich die DD auf die Induktion des Mesoderms und die anschließende iBCEC-Spezifikation konzentriert (Qian, Maguire et al. 2017). Die erzielten Ergebnisse bestätigten, dass iBCECs, welche mittels CD abgeleitet wurden, im Vergleich zur humanen BCEC-Zelllinie (hCMEC/D3) zwar epitheliale Transkripte wie E-Cadherin (CDH1) besitzen, aber eine Herabregulierung von Thrombozyten-Endothelzell-Adhäsionsmolekül-1 (PECAM-1) und VE-Cadherin (CDH5) aufweisen. Die von CD abgeleiteten iBCECs hatten zudem eine höhere Barriere-Integrität und Funktionalität gezeigt. Im Wesentlichen führte die CD-Strategie zu iBCECs mit einem hohen transendothelialen elektrischen Widerstand (engl. Transendothelialelectrical resistance, TEER) von 2000 - 2500Ω*cm2, einem niedrigen Permeabilitätskoeffizienten (eng. Permeability co-efficient, PC) von < 0,50 μm/min für den Transport kleiner Moleküle wie Natriumfluorescein (NaF) und einer charakteristischen Expression von BCEC-spezifischen Tight Junction (TJ)-Proteinen wie Claudin-5 und Occludin. Außerdem bildeten iBCECs, welche über CD gewonnen wurden, keine Gefäßstrukturen als Reaktion auf angiogene Stimuli. DD hingegen führte zu iBCECs mit einer ähnlichen Herabregulierung der PECAM-1- und CDH5-Genexpression, die zusätzlich durch eine geringere Barriereintegrität, gemessen an einem niedrigen TEER von nur ~ 250 - 450 Ω*cm2 und einem hohen PC von > 5 μm/min, bei dem Transport von NaF gekennzeichnet war. Obwohl die über DD gewonnenen iBCECs in der Lage waren Gefäßnetze auszubilden, zeigten sie keine Expression der charakteristischen BCEC-TJs wie Claudin-5 und Occludin.
Diese Ergebnisse führten zu der Hypothese, die in-vitro Differenzierung und Reifung von iBCECs zu verbessern, indem in-vivo-ähnliche Stimuli in-vitro angewandt werden, wie z. B. die direkte Kokultur mit Zelltypen der neurovaskulären Einheit (NVE) durch Sphäroidbildung und die Induktion von Scherstress in einem dynamischen Flussmodell. Im Vergleich zu iBCEC-
11
basierten Transwellmodellen, die zumeist in Monokulturen aufgebaut werden, zeigten die BHS-Sphäroide eine erhöhte Expression von PECAM-1 und eine reduzierte Expression von Epithelmarkern wie E-Cadherin (CDH1) und Claudin-6 (CLDN6). BHS-Sphäroide zeigten eine klassische BCEC-ähnliche Ultrastruktur, die durch TJ-Partikel auf der protoplasmatischen Phase (P-Phase) und der exoplasmatischen Phase (E-Phase) der Plasmamembran gekennzeichnet war. Die TJ-Stränge waren als Partikel und partikelfreie Rillen auf der E-Phase organisiert, während auf der P-Phase teils Partikel und teils kontinuierliche Stränge zu erkennen waren. BHS-Sphäroide zeigten auch eine positive Proteinexpression von Claudin-5, VE-Cadherin, PECAM-1, Glukose-Transporter-1 (GLUT-1), P-Glykoprotein (P-gp) und Transferrin-Rezeptor-1 (Tfr-1). Die BHS-Sphäroide wiesen ebenfalls höhere relative Impedanzwerte im Vergleich zu Sphäroiden ohne iBCEC-Barriere auf. Die Bewertung der Integrität der Barriere korrespondierte zudem mit einer geringeren Permeabilität für den niedermolekularen Tracer NaF, wobei Sphäroide mit iBCECs einen höheren Prozentsatz an relativen Fluoreszenzeinheiten (RFU%) von etwa 90% in den apikalen Kompartimenten aufwiesen, verglichen mit etwa 80% in Sphäroiden ohne iBCECs. Zusammenfassend lässt sich sagen, dass direkte zelluläre Kontakte im komplexen Sphäroidmodell zu einer verstärkten Reifung von iBCECs führten.
In-vivo ist die BHS Scherbelastungen ausgesetzt, die einen wichtigen und oft vernachlässigten physiologischen Stimulus darstellen (Cucullo, Hossain et al. 2011). Um die Auswirkung von Scherstress auf die Eigenschaften und die Reifung von iBCECs zu messen, wurden diese in einem Bioreaktorsystem kultiviert.(Choi, Mathew et al. 2022). Hier war es möglich, iBCECs für insgesamt sieben Tage nach der Differenzierung (d17) erfolgreich in diesem System zu kultivieren und zusätzlich die Barriereintegrität nicht-invasiv zu überwachen. Bis d17 der Langzeitkultur fielen die TEER-Werte von iBCECs stetig von ~ 1800 Ω*cm2 ~ 400 Ω*cm2 unter statischen Bedingungen bzw. von ~ 2500 Ω*cm2 auf ~ 250 Ω*cm2 unter dynamischen Bedingungen. Zusätzliche Untersuchungen und Vergleiche von iBCECs unter diesen Kulturbedingungen mittels transkriptioneller und morphometrischer Analysen, sowie Expression von Proteinmarkern zeigten, dass iBCECs aufgrund der Langzeitkultur und des dynamischen Flusses eine verstärkte Reifung vorweisen. Wichtig ist, dass Claudin-5 bei d10 hauptsächlich im Zytoplasma exprimiert wurde und nur etwa 5% der iBCECs eine kontinuierliche Färbung an den Zellgrenzen aufwiesen. Mit zunehmender Kulturdauer zeigten iBCECs bei d17 in statischer Kultur ~ 18% der Zellen mit kontinuierlicher Zellrandexpression, während unter dynamischen Bedingungen bis zu ~ 30% der Zellen kontinuierliche Zellrandexpressionsmuster aufwiesen. In ähnlicher Weise zeigten ~ 33% der Zellen eine Zell-Zell-Grenzexpression von Occludin an d10 mit einem Anstieg auf ~ 55% unter d17 statischen und bis zu ~ 65% unter d17 dynamischen Bedingungen, was auf eine iBCEC-Reifung hinweist. Zusammenfassend zeigen die in dieser Arbeit vorgestellten Daten die Reifung von iBCECs in
12
BHS Sphäroiden, die durch direkte Zell - Zell Kontakte und in dynamischen Strömungsmodellen durch die Anwendung von Scherspannungen erreicht wird. Beide etablierten neuen Modelle müssen für pharmazeutische Anwendungen zusammen mit In-vitro-in-vivo-Korrelationen weiter validiert werden, um ihr volles Potential zu beweisen.
KW  - Blut-Hirn-Schranke
KW  - Blood-brain barrier
Y1  - 2024
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-322475
ER  - 
TY  - JOUR
A1  - Calderon, Dayana
A1  - Peña, Luis
A1  - Suarez, Angélica
A1  - Villamil, Carolina
A1  - Ramirez-Rojas, Adan
A1  - Anzola, Juan M.
A1  - García-Betancur, Juan C.
A1  - Cepeda, Martha L.
A1  - Uribe, Daniel
A1  - Del Portillo, Patricia
A1  - Mongui, Alvaro
T1  - Recovery and functional validation of hidden soil enzymes in metagenomic libraries
JF  - MicrobiologyOpen
N2  - The vast microbial diversity on the planet represents an invaluable source for identifying novel activities with potential industrial and therapeutic application. In this regard, metagenomics has emerged as a group of strategies that have significantly facilitated the analysis of DNA from multiple environments and has expanded the limits of known microbial diversity. However, the functional characterization of enzymes, metabolites, and products encoded by diverse microbial genomes is limited by the inefficient heterologous expression of foreign genes. We have implemented a pipeline that combines NGS and Sanger sequencing as a way to identify fosmids within metagenomic libraries. This strategy facilitated the identification of putative proteins, subcloning of targeted genes and preliminary characterization of selected proteins. Overall, the in silico approach followed by the experimental validation allowed us to efficiently recover the activity of previously hidden enzymes derived from agricultural soil samples. Therefore, the methodology workflow described herein can be applied to recover activities encoded by environmental DNA from multiple sources.
KW  - environmental microbiology
KW  - functional genomics
KW  - metagenomics
KW  - microbial genomics
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-222016
VL  - 8
ER  - 
TY  - JOUR
A1  - Germain, Dominique P.
A1  - Brand, Eva
A1  - Burlina, Alessandro
A1  - Cecchi, Franco
A1  - Garman, Scott C.
A1  - Kempf, Judy
A1  - Laney, Dawn A.
A1  - Linhart, Aleš
A1  - Maródi, László
A1  - Nicholls, Kathy
A1  - Ortiz, Alberto
A1  - Pieruzzi, Federico
A1  - Shankar, Suma P.
A1  - Waldek, Stephen
A1  - Wanner, Christoph
A1  - Jovanovic, Ana
T1  - Phenotypic characteristics of the p.Asn215Ser (p.N215S) GLA mutation in male and female patients with Fabry disease: A multicenter Fabry Registry study
JF  - Molecular Genetics & Genomic Medicine
N2  - Background
The p.Asn215Ser or p.N215S GLA variant has been associated with late-onset cardiac variant of Fabry disease.

Methods
To expand on the scarce phenotype data, we analyzed natural history data from 125 p.N215S patients (66 females, 59 males) enrolled in the Fabry Registry (NCT00196742) and compared it with data from 401 patients (237 females, 164 males) harboring mutations associated with classic Fabry disease. We evaluated interventricular septum thickness (IVST), left ventricular posterior wall thickness (LVPWT), estimated glomerular filtration rate and severe clinical events.

Results
In p.N215S males, mildly abnormal mean IVST and LVPWT values were observed in patients aged 25–34 years, and values gradually increased with advancing age. Mean values were similar to those of classic males. In p.N215S females, these abnormalities occurred primarily in patients aged 55–64 years. Severe clinical events in p.N215S patients were mainly cardiac (males 31%, females 8%) while renal and cerebrovascular events were rare. Renal impairment occurred in 17% of p.N215S males (mostly in patients aged 65–74 years), and rarely in females (3%).

Conclusion
p.N215S is a disease-causing mutation with severe clinical manifestations found primarily in the heart. Cardiac involvement may become as severe as in classic Fabry patients, especially in males.
KW  - cardiac variant
KW  - Fabry disease
KW  - GLA
KW  - p.Asn215Ser
KW  - p.N215S
KW  - phenotype
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-232976
VL  - 6
ER  - 
TY  - JOUR
A1  - Mirza, Myriam
A1  - Vainshtein, Anna
A1  - DiRonza, Alberto
A1  - Chandrachud, Uma
A1  - Haslett, Luke J.
A1  - Palmieri, Michela
A1  - Storch, Stephan
A1  - Groh, Janos
A1  - Dobzinski, Niv
A1  - Napolitano, Gennaro
A1  - Schmidtke, Carolin
A1  - Kerkovich, Danielle M.
T1  - The CLN3 gene and protein: What we know
JF  - Molecular Genetics & Genomic Medicine
N2  - Background
One of the most important steps taken by Beyond Batten Disease Foundation in our quest to cure juvenile Batten (CLN3) disease is to understand the State of the Science. We believe that a strong understanding of where we are in our experimental understanding of the CLN3 gene, its regulation, gene product, protein structure, tissue distribution, biomarker use, and pathological responses to its deficiency, lays the groundwork for determining therapeutic action plans.

Objectives
To present an unbiased comprehensive reference tool of the experimental understanding of the CLN3 gene and gene product of the same name.

Methods
BBDF compiled all of the available CLN3 gene and protein data from biological databases, repositories of federally and privately funded projects, patent and trademark offices, science and technology journals, industrial drug and pipeline reports as well as clinical trial reports and with painstaking precision, validated the information together with experts in Batten disease, lysosomal storage disease, lysosome/endosome biology.

Results
The finished product is an indexed review of the CLN3 gene and protein which is not limited in page size or number of references, references all available primary experiments, and does not draw conclusions for the reader.

Conclusions
Revisiting the experimental history of a target gene and its product ensures that inaccuracies and contradictions come to light, long-held beliefs and assumptions continue to be challenged, and information that was previously deemed inconsequential gets a second look. Compiling the information into one manuscript with all appropriate primary references provides quick clues to which studies have been completed under which conditions and what information has been reported. This compendium does not seek to replace original articles or subtopic reviews but provides an historical roadmap to completed works.
KW  - Batten
KW  - CLN3
KW  - JNCL
KW  - juvenile Batten
KW  - neuronal ceroid lipofuscinosis
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-224138
VL  - 7
ER  - 
TY  - JOUR
A1  - Jandke, Solveig
A1  - Garz, Cornelia
A1  - Schwanke, Daniel
A1  - Sendtner, Michael
A1  - Heinze, Hans-Jochen
A1  - Carare, Roxana O.
A1  - Schreiber, Stefanie
T1  - The association between hypertensive arteriopathy and cerebral amyloid angiopathy in spontaneously hypertensive stroke-prone rats
JF  - Brain Pathology
N2  - We aimed to test the hypothesis that in spontaneously hypertensive stroke-prone rats (SHRSP), non-amyloid cerebral small vessel disease/hypertensive arteriopathy (HA) results in vessel wall injury that may promote cerebral amyloid angiopathy (CAA). Our study comprised 21 male SHRSP (age 17–44 weeks) and 10 age- and sex-matched Wistar control rats, that underwent two-photon (2PM) imaging of the arterioles in the parietal cortex using Methoxy-X04, Dextran and cerebral blood flow (CBF) measurements. Our data suggest that HA in SHRSP progresses in a temporal and age-dependent manner, starting from small vessel wall damage (stage 1A), proceeding to CBF reduction (stage 1B), non-occlusive (stage 2), and finally, occlusive thrombi (stage 3). Wistar animals also demonstrated small vessel wall damage, but were free of any of the later HA stages. Nearly half of all SHRSP additionally displayed vascular Methoxy-X04 positivity indicative of cortical CAA. Vascular β-amyloid deposits were found in small vessels characterized by thrombotic occlusions (stage 2 or 3). Post-mortem analysis of the rat brains confirmed the findings derived from intravital 2PM microscopy. Our data thus overall suggest that advanced HA may play a role in CAA development with the two small vessel disease entities might be related to the same pathological spectrum of the aging brain.
KW  - cerebral amyloid angiopathy
KW  - cerebral small vessel disease
KW  - hypertensive arteriopathy
KW  - intravital imaging
KW  - spontaneously hypertensive stroke-prone rat
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-323279
VL  - 28
ER  - 
TY  - JOUR
A1  - Kwok, Chee Keong
A1  - Ueda, Yuichiro
A1  - Kadari, Asifiqbal
A1  - Günther, Katharina
A1  - Ergün, Süleyman
A1  - Heron, Antoine
A1  - Schnitzler, Aletta C.
A1  - Rook, Martha
A1  - Edenhofer, Frank
T1  - Scalable stirred suspension culture for the generation of billions of human induced pluripotent stem cells using single-use bioreactors
JF  - Journal of Tissue Engineering and Regenerative Medicine
N2  - The production of human induced pluripotent stem cells (hiPSCs) in quantities that are relevant for cell-based therapies and cell-loaded implants through standard adherent culture is hardly achievable and lacks process scalability. A promising approach to overcoming these hurdles is the culture of hiPSCs in suspension. In this study, stirred suspension culture vessels were investigated for their suitability in the expansion of two hiPSC lines inoculated as a single cell suspension, with a free scalability between volumes of 50 and 2400 ml. The simple and robust two-step process reported here first generates hiPSC aggregates of 324 ± 71 μm diameter in 7 days in 125 ml spinner flasks (100 ml volume). These are subsequently dissociated into a single cell suspension for inoculation in 3000 ml bioreactors (1000 ml volume), finally yielding hiPSC aggregates of 198 ± 58 μm after 7 additional days. In both spinner flasks and bioreactors, hiPSCs can be cultured as aggregates for more than 40 days in suspension, maintain an undifferentiated state as confirmed by the expression of pluripotency markers TRA-1-60, TRA-1-81, SSEA-4, OCT4, and SOX2, can differentiate into cells of all three germ layers, and can be directed to differentiate into specific lineages such as cardiomyocytes. Up to a 16-fold increase in hiPSC quantity at the 100 ml volume was achieved, corresponding to a fold increase per day of 2.28; at the 1000 ml scale, an additional 10-fold increase was achieved. Taken together, 16 × 106 hiPSCs were expanded into 2 × 109 hiPSCs in 14 days for a fold increase per day of 8.93. This quantity of hiPSCs readily meets the requirements of cell-based therapies and brings their clinical potential closer to fruition.
KW  - bioprocessing
KW  - human pluripotent stem cells
KW  - process optimization
KW  - single-use bioreactors
KW  - stirred suspension culture
KW  - scalable culture system
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-234545
VL  - 12
ER  - 
TY  - JOUR
A1  - Hettich, Georg
A1  - Schierjott, Ronja A.
A1  - Ramm, Heiko
A1  - Graichen, Heiko
A1  - Jansson, Volkmar
A1  - Rudert, Maximilian
A1  - Traina, Francesco
A1  - Grupp, Thomas M.
T1  - Method for quantitative assessment of acetabular bone defects
JF  - Journal of Orthopaedic Research
N2  - The objective of the study was to suggest a novel quantitative assessment of acetabular bone defects based on a statistical shape model, validate the method, and present preliminary results. Two exemplary CT-data sets with acetabular bone defects were segmented to obtain a solid model of each defect pelvis. The pathological areas around the acetabulum were excluded and a statistical shape model was fitted to the remaining healthy bone structures. The excluded areas were extrapolated such that a solid model of the native pelvis per specimen resulted (i.e., each pelvis without defect). The validity of the reconstruction was tested by a leave-one-out study. Validation results showed median reconstruction errors of 3.0 mm for center of rotation, 1.7 mm for acetabulum diameter, 2.1° for inclination, 2.5° for anteversion, and 3.3 mm3 for bone volume around the acetabulum. By applying Boolean operations on the solid models of defect and native pelvis, bone loss and bone formation in four different sectors were assessed. For both analyzed specimens, bone loss and bone formation per sector were calculated and were consistent with the visual impression. In specimen_1 bone loss was predominant in the medial wall (10.8 ml; 79%), in specimen_2 in the posterior column (15.6 ml; 46%). This study showed the feasibility of a quantitative assessment of acetabular bone defects using a statistical shape model-based reconstruction method. Validation results showed acceptable reconstruction accuracy, also when less healthy bone remains. The method could potentially be used for implant development, pre-clinical testing, pre-operative planning, and intra-operative navigation. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 9999:1–9, 2018.
KW  - acetabular bone defects
KW  - quantification
KW  - statistical shape model
KW  - volume analysis
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-320226
VL  - 37
ER  - 
TY  - JOUR
A1  - Greber, Johannes
A1  - Polat, Bülent
A1  - Flentje, Michael
A1  - Bratengeier, Klaus
T1  - Properties of the anisotropy of dose contributions: A planning study on prostate cases
JF  - Medical Physics
N2  - Purpose
To characterize the static properties of the anisotropy of dose contributions for different treatment techniques on real patient data (prostate cases). From this, we aim to define a class of treatment techniques with invariant anisotropy distribution carrying information of target coverage and organ-at-risk (OAR) sparing. The anisotropy presumably is a helpful quantity for plan adaptation problems.

Methods
The anisotropy field is analyzed for different intensity modulated radiotherapy (IMRT) and volumetric modulated arc therapy (VMAT) techniques for a total of ten planning CTs of prostate cases. Primary irradiation directions ranged from 5 to 15. The uniqueness of anisotropy was explored: In particular, the anisotropy distribution inside the planning treatment volume (PTV) and in its vicinity was investigated. Furthermore, deviations of the anisotropy under beam rotations were explored by direct plan comparison as an indicating the susceptibility of each planned technique to changes in the geometric plan configuration. In addition, plan comparisons enabled the categorization of treatment techniques in terms of their anisotropy distribution.

Results
The anisotropy profile inside the PTV and in the transition between OAR and PTV is independent of the treatment technique as long as a sufficient number of beams contribute to the dose distribution. Techniques with multiple beams constitute a class of almost identical and technique-independent anisotropy distribution. For this class of techniques, substructures of the anisotropy are particularly pronounced in the PTV, thus offering good options for applying adaptation rules. Additionally, the techniques forming the mentioned class fortunately allow a better OAR sparing at constant PTV coverage. Besides the characterization of the distribution, a pairwise plan comparison reveals each technique's susceptibility to deviations which decreases for an increasing number of primary irradiation directions.

Conclusions
Techniques using many irradiation directions form a class of almost identical anisotropy distributions which are assumed to provide a basis for improved adaptation procedures. Encouragingly, these techniques deliver quite invariant anisotropy distributions with respect to rotations correlated with good plan qualities than techniques using few gantry angles. The following will be the next steps toward anisotropy-based adaptation: first, the quantification of anisotropy regarding organ deformations; and second, establishing the interrelation between the anisotropy and beam shaping.
KW  - adaptation
KW  - IMRT
KW  - radiotherapy
KW  - VMAT
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-228237
VL  - 46
ER  - 
TY  - JOUR
A1  - de Ruijter, Mylène
A1  - Hrynevich, Andrei
A1  - Haigh, Jodie N.
A1  - Hochleitner, Gernot
A1  - Castilho, Miguel
A1  - Groll, Jürgen
A1  - Malda, Jos
A1  - Dalton, Paul D.
T1  - Out-of-Plane 3D-Printed Microfibers Improve the Shear Properties of Hydrogel Composites
JF  - Small
N2  - One challenge in biofabrication is to fabricate a matrix that is soft enough to elicit optimal cell behavior while possessing the strength required to withstand the mechanical load that the matrix is subjected to once implanted in the body. Here, melt electrowriting (MEW) is used to direct-write poly(ε-caprolactone) fibers “out-of-plane” by design. These out-of-plane fibers are specifically intended to stabilize an existing structure and subsequently improve the shear modulus of hydrogel–fiber composites. The stabilizing fibers (diameter = 13.3 ± 0.3 µm) are sinusoidally direct-written over an existing MEW wall-like structure (330 µm height). The printed constructs are embedded in different hydrogels (5, 10, and 15 wt% polyacrylamide; 65% poly(2-hydroxyethyl methacrylate) (pHEMA)) and a frequency sweep test (0.05–500 rad s−1, 0.01% strain, n = 5) is performed to measure the complex shear modulus. For the rheological measurements, stabilizing fibers are deposited with a radial-architecture prior to embedding to correspond to the direction of the stabilizing fibers with the loading of the rheometer. Stabilizing fibers increase the complex shear modulus irrespective of the percentage of gel or crosslinking density. The capacity of MEW to produce well-defined out-of-plane fibers and the ability to increase the shear properties of fiber-reinforced hydrogel composites are highlighted.
KW  - biofabrication
KW  - fiber reinforcement
KW  - hydrogels
KW  - mechanical properties
KW  - melt electrowriting
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-223666
VL  - 14
ER  - 
TY  - JOUR
A1  - Hrynevich, Andrei
A1  - Elçi, Bilge Ş.
A1  - Haigh, Jodie N.
A1  - McMaster, Rebecca
A1  - Youssef, Almoatazbellah
A1  - Blum, Carina
A1  - Blunk, Torsten
A1  - Hochleitner, Gernot
A1  - Groll, Jürgen
A1  - Dalton, Paul D.
T1  - Dimension-Based Design of Melt Electrowritten Scaffolds
JF  - Small
N2  - 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.
KW  - biofabrication
KW  - electrohydrodynamic
KW  - melt electrospinning writing
KW  - scaffold design
KW  - tissue engineering
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-322677
VL  - 14
ER  - 
TY  - JOUR
A1  - Steigerwald, Frank
A1  - Timmermann, Lars
A1  - Kühn, Andrea
A1  - Schnitzler, Alfons
A1  - Reich, Martin M.
A1  - Kirsch, Anna Dalal
A1  - Barbe, Michael Thomas
A1  - Visser-Vandewalle, Veerle
A1  - Hübl, Julius
A1  - van Riesen, Christoph
A1  - Groiss, Stefan Jun
A1  - Moldovan, Alexia-Sabine
A1  - Lin, Sherry
A1  - Carcieri, Stephen
A1  - Manola, Ljubomir
A1  - Volkmann, Jens
T1  - Pulse duration settings in subthalamic stimulation for Parkinson's disease
JF  - Movement Disorders
N2  - Background
Stimulation parameters in deep brain stimulation (DBS) of the subthalamic nucleus for Parkinson's disease (PD) are rarely tested in double-blind conditions. Evidence-based recommendations on optimal stimulator settings are needed. Results from the CUSTOM-DBS study are reported, comparing 2 pulse durations.

Methods
A total of 15 patients were programmed using a pulse width of 30 µs (test) or 60 µs (control). Efficacy and side-effect thresholds and unified PD rating scale (UPDRS) III were measured in meds-off (primary outcome). The therapeutic window was the difference between patients’ efficacy and side effect thresholds.

Results
The therapeutic window was significantly larger at 30 µs than 60 µs (P = ·0009) and the efficacy (UPDRS III score) was noninferior (P = .00008).

Interpretation
Subthalamic neurostimulation at 30 µs versus 60 µs pulse width is equally effective on PD motor signs, is more energy efficient, and has less likelihood of stimulation-related side effects. © 2017 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.
KW  - deep brain stimulation
KW  - Parkinson's disease;
KW  - pulse width
KW  - stimulation parameters
KW  - subthalamic
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-239402
VL  - 33
ER  - 
TY  - JOUR
A1  - Arlotti, Mattia
A1  - Palmisano, Chiara
A1  - Minafra, Brigida
A1  - Todisco, Massimiliano
A1  - Pacchetti, Claudio
A1  - Canessa, Andrea
A1  - Pozzi, Nicoló G.
A1  - Cilia, Roberto
A1  - Prenassi, Marco
A1  - Marceglia, Sara
A1  - Priori, Alberto
A1  - Rampini, Paolo
A1  - Barbieri, Sergio
A1  - Servello, Domenico
A1  - Volkmann, Jens
A1  - Pezzoli, Gianni
A1  - Isaias, Ioannis U.
T1  - Monitoring subthalamic oscillations for 24 hours in a freely moving Parkinson's disease patient
JF  - Movement Disorders
N2  - No abstract available
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-221249
VL  - 34
ER  - 
TY  - JOUR
A1  - Zeiner, Pia S.
A1  - Preusse, Corinna
A1  - Golebiewska, Anna
A1  - Zinke, Jenny
A1  - Iriondo, Ane
A1  - Muller, Arnaud
A1  - Kaoma, Tony
A1  - Filipski, Katharina
A1  - Müller-Eschner, Monika
A1  - Bernatz, Simon
A1  - Blank, Anna-Eva
A1  - Baumgarten, Peter
A1  - Ilina, Elena
A1  - Grote, Anne
A1  - Hansmann, Martin L.
A1  - Verhoff, Marcel A.
A1  - Franz, Kea
A1  - Feuerhake, Friedrich
A1  - Steinbach, Joachim P.
A1  - Wischhusen, Jörg
A1  - Stenzel, Werner
A1  - Niclou, Simone P.
A1  - Harter, Patrick N.
A1  - Mittelbronn, Michel
T1  - Distribution and prognostic impact of microglia/macrophage subpopulations in gliomas
JF  - Brain Pathology
N2  - While the central nervous system is considered an immunoprivileged site and brain tumors display immunosuppressive features, both innate and adaptive immune responses affect glioblastoma (GBM) growth and treatment resistance. However, the impact of the major immune cell population in gliomas, represented by glioma-associated microglia/macrophages (GAMs), on patients’ clinical course is still unclear. Thus, we aimed at assessing the immunohistochemical expression of selected microglia and macrophage markers in 344 gliomas (including gliomas from WHO grade I–IV). Furthermore, we analyzed a cohort of 241 IDH1R132H-non-mutant GBM patients for association of GAM subtypes and patient overall survival. Phenotypical properties of GAMs, isolated from high-grade astrocytomas by CD11b-based magnetic cell sorting, were analyzed by immunocytochemistry, mRNA microarray, qRT-PCR and bioinformatic analyses. A higher amount of CD68-, CD163- and CD206-positive GAMs in the vital tumor core was associated with beneficial patient survival. The mRNA expression profile of GAMs displayed an upregulation of factors that are considered as pro-inflammatory M1 (eg, CCL2, CCL3L3, CCL4, PTGS2) and anti-inflammatory M2 polarization markers (eg, MRC1, LGMN, CD163, IL10, MSR1), the latter rather being associated with phagocytic functions in the GBM microenvironment. In summary, we present evidence that human GBMs contain mixed M1/M2-like polarized GAMs and that the levels of different GAM subpopulations in the tumor core are positively associated with overall survival of patients with IDH1R132H-non-mutant GBMs.
KW  - glioma
KW  - glioma-associated microglia and macrophages;
KW  - immune polarization
KW  - tumor microenvironment
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-233897
VL  - 29
ER  - 
TY  - JOUR
A1  - Ingendoh-Tsakmakidis, Alexandra
A1  - Mikolai, Carina
A1  - Winkel, Andreas
A1  - Szafrański, Szymon P.
A1  - Flak, Christine S.
A1  - Rossi, Angela
A1  - Walles, Heike
A1  - Stiesch, Meike
T1  - Commensal and pathogenic biofilms differently modulate peri-implant oral mucosa in an organotypic model
JF  - Cellular Microbiology
N2  - The impact of oral commensal and pathogenic bacteria on peri-implant mucosa is not well understood, despite the high prevalence of peri-implant infections. Hence, we investigated responses of the peri-implant mucosa to Streptococcus oralis or Aggregatibacter actinomycetemcomitans biofilms using a novel in vitro peri-implant mucosa-biofilm model. Our 3D model combined three components, organotypic oral mucosa, implant material, and oral biofilm, with structural assembly close to native situation. S. oralis induced a protective stress response in the peri-implant mucosa through upregulation of heat shock protein (HSP70) genes. Attenuated inflammatory response was indicated by reduced cytokine levels of interleukin-6 (IL-6), interleukin-8 (CXCL8), and monocyte chemoattractant protein-1 (CCL2). The inflammatory balance was preserved through increased levels of tumor necrosis factor-alpha (TNF-α). A. actinomycetemcomitans induced downregulation of genes important for cell survival and host inflammatory response. The reduced cytokine levels of chemokine ligand 1 (CXCL1), CXCL8, and CCL2 also indicated a diminished inflammatory response. The induced immune balance by S. oralis may support oral health, whereas the reduced inflammatory response to A. actinomycetemcomitans may provide colonisation advantage and facilitate later tissue invasion. The comprehensive characterisation of peri-implant mucosa-biofilm interactions using our 3D model can provide new knowledge to improve strategies for prevention and therapy of peri-implant disease.
KW  - Aggregatibacter actinomycetemcomitans
KW  - dental implants
KW  - host modulation
KW  - organotypic oral mucosa
KW  - Streptococcus oralis
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-323077
VL  - 21
ER  - 
TY  - JOUR
A1  - Mazon, Melody
A1  - Larouche, Valérie
A1  - St-Louis, Maryse
A1  - Schindler, Detlev
A1  - Carreau, Madeleine
T1  - Elevated blood levels of Dickkopf-1 are associated with acute infections
JF  - Immunity, Inflammation and Disease
N2  - Introduction
Dickkopf-1 (DKK1) is a soluble protein and antagonist of the Wnt/β-catenin signaling pathway. DKK1 is found elevated in serum from patients affected with various types of cancers and in some instances, it is considered a diagnostic and prognostic biomarker. Elevated serum levels of DKK1 have also been detected in animal models of chronic inflammatory diseases. Previous work from our laboratory has demonstrated upregulation of DKK1 in cells and mouse models of the bone marrow failure (BMF) and cancer-prone disease Fanconi anemia (FA). The present study aimed to investigate whether DKK1 blood levels in patients are associated with FA or inflammatory responses to acute infections.

Methods
Plasma samples were collected from 58 children admitted to the Centre Mère-Enfant Soleil du Centre Hospitalier de Québec-Université Laval with signs of acute infections. Blood plasma specimens were also collected from healthy blood donors at the Héma-Québec blood donor clinic. Plasmas from patients diagnosed with FA were also included in the study. DKK1 levels in blood plasmas were assessed by standard ELISA.

Results
Patients with acute infections showed dramatically high levels of DKK1 (6072 ± 518 pg/ml) in their blood compared to healthy blood donors (1726 ± 95 pg/ml). No correlations were found between DKK1 levels and C reactive protein (CRP) concentration, platelet numbers, or white blood cell counts. Patients with FA showed higher DKK1 plasma levels (3419 ± 147.5 pg/ml) than healthy blood donors (1726 ± 95 pg/ml) but significantly lower than patients with acute infections.

Conclusion
These findings suggest that blood DKK1 is elevated in response to infections and perhaps to inflammatory responses.
KW  - blood plasma
KW  - Dickkopf-1
KW  - ELISA
KW  - Fanconi anemia
KW  - infections
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-222171
VL  - 6
ER  - 
TY  - JOUR
A1  - Van Assche, Nele
A1  - Fickl, Stefan
A1  - Francisco, Helena
A1  - Gurzawska, Katarzyna
A1  - Milinkovic, Iva
A1  - Navarro, Jose M.
A1  - Torsello, Ferruccio
A1  - Thoma, Daniel S.
T1  - Guidelines for development of Implant Dentistry in the next 10 years regarding innovation, education, certification, and associations
JF  - Clinical Oral Implants Research
N2  - Background
During the third Summer Camp of European Association of Osseointegration (EAO), 40 junior representatives from various European societies and associations were brought together to discuss and explore the following topics in Implant Dentistry in the next 10 years: (I) certification, (II) societies and associations, (III) continuing education, and (IV) innovations.

Aims
The aims of all working groups were to identify and outline the present situation in the area of the selected topic and to propose improvements and innovations to be implemented in the following 10 years.

Materials and methods
Four different groups were assigned randomly to one of the four working units. The method to discuss the selected topics was World Cafè. The summaries of four topics were then given to all participants for peer review.

Results and conclusions
All four groups presented the conclusions and guidelines accordingly: (I) The recognition for Implant Dentistry and accreditation of training programs would lead to an improvement of the quality of care to the benefit of the patients; (II) Dental associations and societies have to continuously improve communication to meet needs of dental students, professionals, and patients (III) European Dental Board should be installed and become responsible for continue dental education; (IV) dental engineering, peri-implant diseases, and digital workflow in dentistry currently have limited tools that do not guarantee predictable results.
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-232150
VL  - 29
ER  -