Are we preparing for collaboration, advocacy and leadership? Targeted multi-site analysis of collaborative intrinsic roles implementation in medical undergraduate curricula.

BACKGROUND: The Collaborator, Health Advocate and Leader/Manager roles are highly relevant for safe patient management and optimization of healthcare system in rehabilitation and prevention. They are defined in competency-based frameworks and incorporate competencies empowering physicians to master typical daily tasks in interdisciplinary, interprofessional and institutional collaboration. However, appropriate implementation of roles remains difficult in undergraduate medical education (UME) and needs to be closely monitored. The aim of this cross-institutional mapping study was to examine for the roles of Collaborator, Health Advocate and Leader/Manager: (1) To what extent do German UME programs explicitly meet the given standards after 5 years of study? (2) Which information may be obtained from multi-site mapping data for evidence-based reflection on curricula and framework? METHODS: In a joint project of eight German UME programs, 80 to 100% of courses were mapped from teachers' perspective against given national standards: (sub-)competency coverage, competency level attainment and assessment. All faculties used a common tool and consented procedures for data collection and processing. The roles' representation was characterized by the curricular weighting of each role content expressed by the percentage of courses referring to it (citations). Data were visualized in a benchmarking approach related to a general mean of the intrinsic roles as reference line. RESULTS: (Sub-)competencies of the Health Advocate are consistently well-integrated in curricula with a wide range of generally high curricular weightings. The Collaborator reveals average curricular representation, but also signs of ongoing curricular development in relevant parts and clear weaknesses regarding assessment and achieved outcomes. The Leader/Manager displays consistently lowest curricular weightings with several substantial deficiencies in curricular representation, constructive alignment and/or outcome level. Our data allow identifying challenges to be considered by local curriculum developers or framework reviewers (e.g. non-achievement of competency levels, potential underrepresentation, lacking constructive alignment). CONCLUSION: Our non-normative, process-related benchmarking approach provides a differentiated crosscut snapshot to compare programs in the field of others, thus revealing shortcomings in role implementation, especially for Leader/Manager and Collaborator. The synopsis of multi-site data may serve as an external reference for program self-assessment and goal-oriented curriculum development. It may also provide practical data for framework review.

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.

Onset of preclinical osteoarthritis: the angular spatial organization permits early diagnosis.

OBJECTIVE: Superficial articular chondrocytes display distinct spatial remodeling processes in response to the onset of distant osteoarthritis (OA). Such processes may be used to diagnose early events before manifest OA results in tissue destruction and clinical symptoms. Using a novel method of spatial quantification by calculating the angles between a chondrocyte and its surrounding neighbors, we compared maturational and degenerative changes of the cellular organizations in rat and human cartilage specimens. METHODS: The nuclei of superficial chondrocytes obtained from intact rat cartilage and from human knee cartilage, as well as from cartilage with focal and severe OA, were digitally recorded in top-down views. Their Cartesian coordinates were used to determine the nearest neighbor for each chondrocyte and the angle between these 2 cells and a reference. These angles, cellularity, nearest neighbor distances, and aggregation were analyzed as a function of location and OA severity. RESULTS: Neighboring rat chondrocytes exhibited intricate angular patterns with 4 dominant angles that were maintained during maturation and during the onset and progression of OA. Within intact cartilage, human chondrocytes demonstrated 1 dominant angle and, thus, a significantly different angular organization. With early OA onset, human chondrocytes that were located within intact cartilage displayed an increased occurrence of 4 angles; the resulting angular patterns were indistinguishable from those observed in rats. The angular remodeling was associated with location- and OA severity-dependent changes in cellularity and aggregation. CONCLUSION: This study is the first to identify the presence of angular characteristics of spatial chondrocyte organization and species-specific remodeling processes correlating with OA onset. The appearance of distinct angular and spatial patterns between neighboring chondrocytes can identify the onset of distant OA prior to microscopically visible tissue damage and possibly before clinical onset. With further development, this novel concept may become suitable for the diagnosis and followup of patients susceptible to OA.

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.

Targeting of epidermal growth factor receptor (EGFR)-expressing tumor cells with sterically stabilized affibody liposomes (SAL).

Affibody molecules are small and stable antigen-binding molecules derived from the B domain of protein A. We applied a bivalent, high-affinity epidermal growth factor receptor (EGFR)-specific affibody molecule for the generation of targeted PEGylated liposomes. These sterically stabilized affibody liposomes (SAL) were produced by chemical coupling of the cysteine-modified affibody molecule to maleimide-PEG(2000)-DSPE and subsequent insertion into PEGylated liposomes. These SAL showed strong and selective binding to EGFR-expressing tumor cell lines. Binding was dependent on the amount of inserted affibody molecule-lipid conjugates and could be blocked by soluble EGF. Approximately 30% of binding activity was still retained after 6 days of incubation in human plasma at 37 degrees C. Binding of SAL to cells led to efficient internalization of the liposomes. Using mitoxantrone-loaded liposomes, we observed for SAL, compared to untargeted liposomes, an enhanced cytotoxicity toward EGFR-expressing cells. In summary, we show that SAL can be easily prepared from affibody molecules and thus may be suitable for the development of carrier systems for targeted delivery of drugs.

Human osteoarthritic chondrons outnumber patient- and joint-matched chondrocytes in hydrogel culture-Future application in autologous cell-based OA cartilage repair?

Autologous chondrocyte implantation (ACI) is used in 34-60% for osteoarthritic (OA) cartilage defects, although ACI is neither recommended nor designed for OA. Envisioning a hydrogel-based ACI for OA that uses chondrons instead of classically used chondrocytes, we hypothesized that human OA chondrons may outperform OA chondrocytes. We compared patient- and joint surface-matched human OA chondrons with OA chondrocytes cultured for the first time in a hydrogel, using a self-assembling peptide system. We determined yield, viability, cell numbers, mRNA expression, GAPDH mRNA enzyme activity, Collagen II synthesis (CPII) and degradation (C2C), and sulfated glycosaminoglycan. Ex vivo, mRNA expression was comparable. Over time, significant differences in survival led to 3.4-fold higher OA chondron numbers in hydrogels after 2 weeks (p = .002). Significantly, more enzymatically active GAPDH protein indicated higher metabolic activity. The number of cultures that expressed mRNA for Collagen Types I and VI, COMP, aggrecan, VEGF, TGF-ß1, and FGF-2 (but not Collagen Types II and X) was different, resulting in a 3.5-fold higher number of expression-positive OA chondron cultures (p < .05). Measuring CPII and C2C per hydrogel, OA chondron hydrogels synthesized more than they degraded Collagen Type II, the opposite was true for OA chondrocytes. Per cell, OA chondrons but not OA chondrocytes displayed more synthesis than degradation. Thus, OA chondrons displayed superior biosynthesis and mRNA expression of tissue engineering and phenotype-relevant genes. Moreover, human OA chondrons displayed a significant survival advantage in hydrogel culture, whose presence, drastic extent, and timescale was novel and is clinically significant. Collectively, these data highlight the high potential of human OA chondrons for OA ACI, as they would outnumber and, thus, surpass OA chondrocytes.

Engineering the geometrical shape of mesenchymal stromal cells through defined cyclic stretch regimens.

Stem cells have been predicted to improve disease outcomes and patient lives. Steering stem cell fate - through controlling cell shape - may substantially accelerate progress towards this goal. As mesenchymal stromal cells (MSCs) are continuously exposed in vivo to a dynamically changing biomechanical environment, we hypothesized that exogenous forces can be applied for engineering a variety of significantly different MSC shapes. We applied specific cyclic stretch regimens to human MSCs and quantitatively measured the resulting cell shape, alignment, and expression of smooth muscle (SMC) differentiation markers, as those have been associated with elongated morphology. As proof of principle, a range of different shapes, alignments, and correlating SMC marker levels were generated by varying strain, length, and repetition of stretch. However, the major determinant of biomechanically engineering cellular shape was the repetition of a chosen stretch regimen, indicating that the engineered shape and associated differentiation were complex non-linear processes relying on sustained biomechanical stimulation. Thus, forces are key regulators of stem cell shape and the targeted engineering of specific MSC shapes through biomechanical forces represents a novel mechanobiology concept that could exploit naturally occurring in vivo forces for improving stem cell fate in clinical regenerative therapies.

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 stiffness-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 reverse-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 stretch-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 biomaterial-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.

Stretching human mesenchymal stromal cells on stiffness-customized collagen type I generates a smooth muscle marker profile without growth factor addition.

Using matrix elasticity and cyclic stretch have been investigated for inducing mesenchymal stromal cell (MSC) differentiation towards the smooth muscle cell (SMC) lineage but not in combination. We hypothesized that combining lineage-specific stiffness with cyclic stretch would result in a significantly increased expression of SMC markers, compared to non-stretched controls. First, we generated dense collagen type I sheets by mechanically compressing collagen hydrogels. Atomic force microscopy revealed a nanoscale stiffness range known to support myogenic differentiation. Further characterization revealed viscoelasticity and stable biomechanical properties under cyclic stretch with >99% viable adherent human MSC. MSCs on collagen sheets demonstrated a significantly increased mRNA but not protein expression of SMC markers, compared to on culture flasks. However, cyclic stretch of MSCs on collagen sheets significantly increased both mRNA and protein expression of α-smooth muscle actin, transgelin, and calponin versus plastic and non-stretched sheets. Thus, lineage-specific stiffness and cyclic stretch can be applied together for inducing MSC differentiation towards SMCs without the addition of recombinant growth factors or other soluble factors. This represents a novel stimulation method for modulating the phenotype of MSCs towards SMCs that could easily be incorporated into currently available methodologies to obtain a more targeted control of MSC phenotype.

Antibody targeting of nanoparticles to tumor-specific receptors: immunoliposomes.

Immunoliposomes generated by coupling of antibodies to the liposomal surface allow for an active tissue targeting, e.g., through binding to tumor cell-specific receptors. Instead of whole antibodies, single-chain Fv fragments (scFv), which represent the smallest part of an antibody containing the entire antigen-binding site, find increasing usage as targeting moiety. Here we provide protocols for the preparation of type II scFv immunoliposomes by the conventional coupling method as well as the post-insertion method. Furthermore protocols to analyze binding of these immunoliposomes to antigen-expressing cells as well as internalization through receptor-mediated endocytosis are included.

Targeted delivery of SiRNA to CD33-positive tumor cells with liposomal carrier systems.

SiRNA molecules represent promising therapeutic molecules, e.g. for cancer therapy. However, efficient delivery into tumor cells remains a major obstacle for treatment. Here, we describe a liposomal siRNA carrier system for targeted delivery of siRNA to CD33-positive acute myeloid leukemia cells. The siRNA is directed against the t(8;21) translocation resulting in the AML1/MTG8 fusion protein. The siRNA was encapsulated in free or polyethylene imine (PEI)-complexed form into PEGylated liposomes endowed subsequently with an anti-CD33 single-chain Fv fragment (scFv) for targeted delivery. The resulting siRNA-loaded immunoliposomes (IL) and immunolipoplexes (ILP) showed specific binding and internalization by CD33-expressing myeloid leukemia cell lines (SKNO-1, Kasumi-1). Targeted delivery of AML1/MTG8 siRNA, but not of mismatch control siRNA, reduced AML1/MTG8 mRNA and protein levels and decreased leukemic clonogenicity, a hallmark of leukemic self-renewal. Although this study revealed that further modifications are necessary to increase efficacy of siRNA delivery and silencing, we were able to establish a targeted liposomal siRNA delivery system combining recombinant antibody fragments for targeted delivery with tumor cell-specific siRNA molecules as therapeutic agents.