The geometrical shape of mesenchymal stromal cells measured by quantitative shape descriptors is determined by the stiffness of the biomaterial and by cyclic tensile forces

Controlling mesenchymal stromal cell (MSC) shape is a novel method for investigating and directing MSC behaviour in vitro. it was hypothesized that specifigc MSC shapes can be generated by using stiffnessâ defined biomaterial surfaces and by applying cyclic tensile forces. Biomaterials used were thin and thick silicone sheets, fibronectin coating, and compacted collagen type I sheets. The MSC morphology was quantified by shape descriptors describing dimensions and membrane protrusions. Nanoscale stiffness was measured by atomic force microscopy and the expression of smooth muscle cell (SMC) marker genes (ACTA2, TAGLN, CNN1) by quantitative reverseâ transcription polymerase chain reaction. Cyclic stretch was applied with 2.5% or 5% amplitudes. Attachment to biomaterials with a higher stiffness yielded more elongated MSCs with fewer membrane protrusions compared with biomaterials with a lower stiffness. For cyclic stretch, compacted collagen sheets were selected, which were associated with the most elongated MSC shape across all investigated biomaterials. As expected, cyclic stretch elongated MSCs during stretch. One hour after cessation of stretch, however, MSC shape was rounder again, suggesting loss of stretchâ induced shape. Different shape descriptor values obtained by different stretch regimes correlated significantly with the expression levels of SMC marker genes. Values of approximately 0.4 for roundness and 3.4 for aspect ratio were critical for the highest expression levels of ACTA2 and CNN1. Thus, specific shape descriptor values, which can be generated using biomaterialâ associated stiffness and tensile forces, can serve as a template for the induction of specific gene expression levels in MSC. Copyright © 2017 John Wiley & Sons, Ltd.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141253/1/term2263.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141253/2/term2263_am.pd

Stress-vs-time signals allow the prediction of structurally catastrophic events during fracturing of immature cartilage and predetermine the biomechanical, biochemical, and structural impairment

Objective Trauma-associated cartilage fractures occur in children and adolescents with clinically significant incidence. Several studies investigated biomechanical injury by compressive forces but the injury-related stress has not been investigated extensively. In this study, we hypothesized that the biomechanical stress occurring during compressive injury predetermines the biomechanical, biochemical, and structural consequences. We specifically investigated whether the stress-vs-time signal correlated with the injurious damage and may allow prediction of cartilage matrix fracturing. Methods Superficial and deeper zones disks (SZDs, DZDs; immature bovine cartilage) were biomechanically characterized, injured (50% compression, 100%/s strain-rate), and re-characterized. Correlations of the quantified functional, biochemical and histological damage with biomechanical parameters were zonally investigated. Results Injured SZDs exhibited decreased dynamic stiffness (by 93.04 ± 1.72%), unresolvable equilibrium moduli, structural damage (2.0 ± 0.5 on a 5-point-damage-scale), and 1.78-fold increased sGAG loss. DZDs remained intact. Measured stress-vs-time-curves during injury displayed 4 distinct shapes, which correlated with histological damage (p < 0.001), loss of dynamic stiffness and sGAG (p < 0.05). Damage prediction in a blinded experiment using stress-vs-time grades was 100%-correct and sensitive to differentiate single/complex matrix disruptions. Correlations of the dissipated energy and maximum stress rise with the extent of biomechanical and biochemical damage reached significance when SZDs and DZDs were analyzed as zonal composites but not separately. Conclusions The biomechanical stress that occurs during compressive injury predetermines the biomechanical, biochemical, and structural consequences and, thus, the structural and functional damage during cartilage fracturing. A novel biomechanical method based on the interpretation of compressive yielding allows the accurate prediction of the extent of structural damage.National Institutes of Health (U.S.) (Grant R01-AR45779)Deutsche Forschungsgemeinschaft (Grant RO2511/1-1)Deutsche Forschungsgemeinschaft (Grant RO2511/2-1)Germany. Federal Ministry of Education and Research (Grant 01KQ0902B TP2

Zielgerichtete siRNA-Trägersysteme für die Behandlung der akuten myeloischen Leukämie (AML)

Active targeting is an important prerequisite for selective administration of therapeutic agents into tumour cells. In acute myeloid leukaemia (AML) a targeted drug delivery via antibody fragments directed against CD33 can be reached. CD33 is an internalizing receptor expressed on the surface of myeloid blasts. Several variants of recombinant anti-CD33 single chain Fv (scFv) fragments differing in the order of their variable domains (VHVL or VLVH) were expressed. Immunoliposomes (ILs) were generated by conventional coupling to malPEG2000-functionalized liposomes and by postinsertion of scFv-coupled micelles into preformed PEGylated liposomes, respectively. The exclusively high binding activity of anti-CD33 ILs with scFv molecules in the VHVL configuration is remarkable and provides evidence of a possible influence of domain order on binding activity. ILs containing 0.3 mol% of scFv-coupled lipid showed the highest binding and uptake activity on target cells. Binding activity remained constant for several days and required only a few scFv molecules per liposome. SiRNA was encapsulated passively, either as naked siRNA, or complexed with polyethylenimine (PEI) or protamine with loading efficiencies between 24 and 100 %. ILs had a diameter of approximately 130 to 140 nm after encapsulation of siRNA. In addition, immunopolyplexes (IPPs) were generated by coupling of scFv CD33 VHVL to the synthetic polymer PEI, forming complexes with siRNA. Size of IPPs was very heterogenous and ranged from 180 to 450 nm, with a slightly negative surface charge. Target cell specific binding and uptake of both carrier systems could be shown by flow cytometry and fluorescence microscopy studies. Transfection of the carrier systems and silencing of the target leukaemic fusion gene AML1/MTG8 by encapsulation of a specific siRNA showed a selective, quantitative silencing of AML1/MTG8 on protein level. Knockdown of AML1/MTG8 expression led to a decreased cell clonogenicity, demonstrated in colony formation assays. However, observed target gene knockdown effects were not very pronounced and, depending on the concentration of the carrier system, knockdown could also be insufficient or unspecific caused by a narrow window between inefficient and unspecific or even cytotoxic concentrations. Furthermore, a stimulating effect of ILs on the clonogenicity of the target cells was observed. In the present study, basic procedures for the targeted delivery of siRNA via ILs and IPPs were established, which should be improved by further approaches. As a first step towards therapeutic applications, humanization of the murine scFv VHVL molecule via CDR-grafting was performed, resulting in a humanized scFv with identical binding properties and improved stability.Aktives Targeting ist eine wichtige Voraussetzung für die selektive Verabreichung therapeutischer Agenzien in Tumorzellen. In der akuten myeloischen Leukämie (AML) kann eine zielgerichtete Wirkstoffverabreichung über Antikörperfragmente, die gegen CD33 gerichtet sind, erreicht werden. CD33 ist ein internalisierender Rezeptor, der auf der Zelloberfläche myeloischer Blasten exprimiert wird. Mehrere verschiedene Varianten rekombinanter anti-CD33 single chain Fv (scFv) Fragmente, die sich in der Anordnung ihrer variablen Domänen unterscheiden (VHVL bzw. VLVH), wurden produziert. Immunliposomen (IL) wurden mit Hilfe der konventionellen Kopplung an malPEG2000-funktionalisierte Liposomen oder durch die Postinsertion von scFv-gekoppelten Micellen in vorgeformte PEGylierte Liposomen hergestellt. Bemerkenswert ist die ausschließliche, starke Bindungsaktivität der anti-CD33 IL, die mit scFv Molekülen in der VHVL Konfiguration gekoppelt wurden. Dies weist auf einen möglichen Einfluss der Anordnung der Domänen auf die Bindungsaktivität der IL hin. IL mit 0,3 mol% scFv-gekoppelten Lipids zeigten die höchste Bindungsaktivität und Aufnahme in Zielzellen. Die Bindungsaktivität blieb über mehrere Tage stabil und erforderte nur wenige scFv Moleküle pro Liposom. Liposomen wurden passiv mit siRNA beladen, entweder in freier Form oder als Komplex mit Polyethylenimin (PEI) oder Protamin. Die Beladungseffizienz variierte zwischen 24 und 100 %. Die IL wiesen eine Größe von 130 bis 140 nm nach siRNA Beladung auf. Zusätzlich zu IL wurden Immunopolyplexe (IPP), durch Kopplung von scFv CD33 VHVL an das synthetische Polymer PEI und siRNA-Komplexierung, hergestellt. Die Größen der IPP waren sehr heterogen und reichten von 180 bis 450 nm bei leicht negativer Oberflächenladung. Zielzellspezifische Bindung und Aufnahme konnte für beide Trägersysteme in der Durchflusszytometrie und Fluoreszenzmikroskopie gezeigt werden. Transfektion der Trägersysteme und Silencing des leukämischen Fusionsgens AML1/MTG8 durch die Verpackung spezifischer siRNA zeigten ein selektives, quantitatives Silencing von AML1/MTG8 auf Proteinebene. AML1/MTG8 Knockdown führte zu einem verminderten Proliferationspotential, das im Colony Formation Assay gezeigt werden konnte. Die beobachteten Knockdown-Effekte waren jedoch nicht sehr ausgeprägt und konnten, je nach Konzentration des Trägersystems, unzureichend oder unspezifisch sein, verursacht durch ein schmales Fenster zwischen Insuffizienz und Unspezifität oder gar Toxizität. Ein stimulierender Effekt der IL auf die Proliferation der Zielzellen wurde gezeigt. In dieser Arbeit wurden die Grundlagen für eine zielgerichtete Verabreichung von siRNA über IL und IPP geschaffen, die in weiteren Ansätzen verbessert werden sollten. Ein erster Schritt in Richtung therapeutischer Anwendung, wurde durch die Humanisierung des murinen scFv VHVL Moleküls mittels CDR-grafting gemacht, was zu einem humansierten scFv mit identischen Bindungseigenschaften und verbesserter Stabilität führte

Surgeons’ participation in the development of collaboration and management competencies in undergraduate medical education

The teaching of professional roles in medical education is an interdisciplinary concern. However, surgeons require specific standards of professionalism for certain context-based situations. In addition to communication, studies require collaboration, leadership, error-/conflict-management, patient-safety and decision-making as essential competencies for surgeons. Standards for corresponding competencies are defined in special chapters of the German National Competency-based Learning Objectives for Undergraduate Medical Education (NKLM; chapter 8, 10). The current study asks whether these chapters are adequately taught in surgical curricula. Eight German faculties contributed to analysing mapping data considering surgical courses of undergraduate programs. All faculties used the MERlin mapping platform and agreed on procedures for data collection and processing. Sub-competency and objective coverage, as well as the achievement of the competency level were mapped. Overall counts of explicit citations were used for analysis. Collaboration within the medical team is a strongly represented topic. In contrast, interprofessional cooperation, particularly in healthcare sector issues is less represented. Patient safety and dealing with errors and complications is most emphasized for the Manager/Leader, while time management, career planning and leadership are not addressed. Overall, the involvement of surgery in teaching the competencies of the Collaborator and Manager/Leader is currently low. However, there are indications of a curricular development towards explicit teaching of these roles in surgery. Moreover, implicitly taught roles are numerous, which indicates a beginning awareness of professional roles