3D Spheroid Cultivation Alters the Extent and Progression of Osteogenic Differentiation of Mesenchymal Stem/Stromal Cells Compared to 2D Cultivation.

Mesenchymal stem/stromal cells (MSC) are capable of progenitor cell fraction renewal or tissue-specific differentiation. These properties are maintained during in vitro cultivation, making them an interesting model system for testing biological and pharmacological compounds. Cell cultivation in 2D is commonly used to study cellular responses, but the 2D environment does not reflect the structural situation of most cell types. Therefore, 3D culture systems have been developed to provide a more accurate physiological environment in terms of cell-cell interactions. Since knowledge about the effects of 3D culture on specific differentiation processes is limited, we studied the effects on osteogenic differentiation and the release of factors affecting bone metabolism for up to 35 days and compared them with the effects in 2D culture. We demonstrated that the selected 3D model allowed the rapid and reliable formation of spheroids that were stable over several weeks and both accelerated and enhanced osteogenic differentiation compared with the 2D culture. Thus, our experiments provide new insights into the effects of cell arrangement of MSC in 2D and 3D. However, due to the different culture dimensions, various detection methods had to be chosen, which in principle limits the explanatory power of the comparison between 2D and 3D cultures.

Volumetric mass density measurements of mesenchymal stem cells in suspension using a density meter.

To use regeneratively active cells for cell therapeutic applications, the cells must be isolated from their resident tissues. Different isolation procedures subject these cells to varying degrees of mechanical strain, which can affect the yield of cell number and viability. Knowledge of cell volumetric mass density is important for experimental and numerical optimization of these procedures. Although methods for measuring cell volumetric mass density already exist, they either consume much time and cell material or require a special setup. Therefore, we developed a user-friendly method that is based on the use of readily available instrumentation. The newly developed method is predicated on the linear relationship between the volumetric mass density of the cell suspension and the volumetric mass density, number, and diameter of the cells in the suspension. We used this method to determine the volumetric mass density of mesenchymal stem cells (MSCs) and compared it to results from the established density centrifugation.

Neotropical ostracode oxygen and carbon isotope signatures: implications for calcification conditions.

Calcitic valves of non-marine ostracodes are important geochemical archives. Investigations of the relationship between the ranges of oxygen and carbon isotope values of modern ostracode populations and their host water provide important information on local or regional conditions and influences. Here we present the first δ18Oostracode and δ13C of the freshwater ostracode species Cytheridella ilosvayi along with the isotopic composition of the waters in which the ostracodes calcified, δDwater, δ18Owater, δ13CDIC values-covering a large geographical range (Florida to Brazil). With this data we extended a newly developed approach based on the estimation of δ18O values of monthly equilibrium calcites as references for the interpretation of δ18Oostracode values. The expected apparent oxygen isotope fractionation between CaCO3 and H2O is correlated with temperature with smaller values occurring at higher temperatures as valid at isotope equilibrium (δ18Ocalcite_eq). Uncertainties about the expected equilibrium calcites derive from incomplete knowledge of high-frequency variations of the water bodies caused by interplay of mixing, evaporation, and temperature. Coincidence between δ18Oostracode and δ18Ocalcite_eq is restricted to few months indicating a seasonal calcification of Cytheridella. There is a characteristic pattern in its difference between mean δ18Oostracode and δ18Ocalcite_eq which implies that Cytheridella provides a synchronous life cycle in its geographical range with two calcification periods in spring (May, June) and autumn (October). This ubiquitous life cycle of Cytheridella in the entire study area is considered to be phylogenetically inherited. It might have originally been adapted to environmental conditions but has been conserved during the migration and radiation of the group over the Neotropical realm. Supplementary Information: The online version contains supplementary material available at 10.1007/s10533-022-00917-9.

[The Impact of Parenthood, Support, and Short-Term Contracts on Work-Family Conflict of Female and Male Residents' Career]. / Der Einfluss von Elternstatus, erlebter Unterstützung und Befristung auf das Belastungserleben und die Karriereentwicklung von Ärztinnen und Ärzten am Ende der Weiterbildung.

BACKGROUND: Work factors and work-family interference play an important role in physicians leaving clinical practice. OBJECTIVES: The purpose of this study was to examine residents' work-family conflict and family-work conflict in association with parental status, perceived support, and short-term contracts. MATERIAL AND METHODS: Data acquisition was carried out within the multi-centric and prospective "KarMed" study in Germany at the end of the postgraduate training in 2016 (N=433). The Work-Family Conflict and Family-Work Conflict scales were used. Further independent variables were gender, parental status, short-term contracts, and perceived support from partner. Results Female physicians with children interrupted postgraduate training five times more often then female physicians without children and 18 times more often than male physicians with children. Female as well as male physicians with children showed greater family-work conflicts, and female physicians without children scored higher on work-family conflict. Male physicians did not show significant results on work-family conflict. Neither short-term contracts nor perceived support from the partner had a significant influence on work-family or family work conflict. CONCLUSIONS: There is a need to reduce work-family conflicts and their associated factors in female resident physicians.

[Analysis of career and life satisfaction of women residents with children during their postgraduate training compared to male colleagues: A longitudinal analysis as part of the KarMed study]. / Berufsverlaufs- und Lebenszufriedenheit von Ärztinnen in fachärztlicher Weiterbildung mit Kindern im Vergleich zu ihren männlichen Kollegen ­ Eine longitudinale Untersuchung im Rahmen der KarMed-Studie.

OBJECTIVE: Former results of the KarMed study revealed a significant decrease in the career satisfaction of female physicians with children in the course of their postgraduate training compared to male physicians with children. Yet, female physicians with children showed the highest scores on satisfaction with life at the fourth year of postgraduate training. The present study evaluates whether the different courses of career satisfaction and life satisfaction of female physicians compared to male physicians are caused by parental status. METHODS: Data were collected in the course of the KarMed study (2008-2016). A cross-lagged panel design was used to analyze data from T2 to T5. RESULTS: The results indicate an inverse association of career satisfaction with life satisfaction. Women physicians with a low level of career satisfaction were more likely to have children than those with a higher career satisfaction. Furthermore, we found a causal effect of parental status on career satisfaction in male physicians. For female physicians, yet not for male physicians, life satisfaction predicts the parental status. CONCLUSIONS: The structural improvement in postgraduate training needs to be adjusted in terms of parental status. Prospective studies should take medical leave during pregnancy as well as parental leave of male medical residents into account.

[Reflections on the attractiveness of General Internal Medicine and its Sub-Specialties in German Postgraduate Medical Education]. / Eine Abstimmung mit den Füßen gegen die Spezialisierung in der Inneren Medizin? ­ Zur Attraktivität der internistischen Fachrichtungen im Verlauf der ärztlichen Weiterbildung.

OBJECTIVE: The aim of the present study was to examine the number of physicians who chose internal medicine for their postgraduate training, their development over the 6-year period of training and whether there were changes in their preferences regarding future work sector and working hours, with a focus on gender-specific differences. METHODOLOGY: Annual postal surveys were conducted of a cohort of undergraduate students (N=1.012) in their final year of study 2008/09, and during the six years of their postgraduate training. Descriptive statistics were used for analysis. RESULTS: The study showed that internal medicine was a sought-after medical discipline, which recruited up to 25% of medical graduates. However, over the course of the six years of postgraduate training, the attractiveness shifted from the specialized sub-disciplines of internal medicine to general internal medicine, especially among male physicians. General internal medicine was particularly attractive to female physicians who intended to work part-time after completion of their specialization. CONCLUSIONS: Internal medicine in Germany is still the most frequently chosen field of specialization. However, the proportion of physicians intending to work ina hospital after specialization is decreasing. This fact, together with the decreasing attractiveness of sub-disciplines in this field, must be taken into account when planning the staff in hospitals.

Treatment of Grade 3 and 4 Osteoarthritis with Intraoperatively Separated Adipose Tissue-Derived Stromal Vascular Fraction: A Comparative Case Series.

Osteoarthritis (OA) is the most common form of arthritis of the joints. The stromal vascular fraction (SVF) is a regenerative cell population that can be isolated from adipose tissue. It is the immunomodulatory properties of the stromal vascular fraction that make it a promising candidate for the regenerative treatment of OA. Patients with grade 3 and 4 osteoarthritis were treated with the stromal vascular fraction with and without platelet-rich plasma (PRP) and followed up on their Knee Injury and Osteoarthritis Outcome Score (KOOS) score for 12 months, with MRI and subjective evaluation of the procedure. Magnetic resonance imaging (MRI) revealed a widening of the joint space, a restructuring of the cartilage, and an alleviation of effusions in the treated joints. In three of the four treatment groups, a substantial improvement of the KOOS scores was documented at the 12-month follow-up time point. According to the subjective evaluation, 67% of the patients were satisfied or very satisfied with the procedure and would recommend it to others. No serious adverse events or unwanted side effects related to the SVF treatment were observed or reported. Prior to an invasive artificial joint replacement, the treatment of arthritic knee joints with the intraarticular injection of autologous adipose tissue-derived SVF should be considered a regenerative treatment option.

Burnout is associated with work-family conflict and gratification crisis among German resident physicians.

BACKGROUND: Studies investigating the longitudinal predictive value of burnout on both effort-reward imbalance (within the working place) and work-family conflict (between work and private life) in residents are lacking. Former cross-sectional studies showed an association of effort-reward imbalance and work family conflict with an elevated burnout risk in physicians. METHODS: Data acquisition was carried out within the multi-centric, longitudinal, and prospective "KarMed" study in Germany from 2009 until 2016. Yearly surveys including validated scales: the Maslach Burnout Inventory with its three subscales (emotional exhaustion, personal accomplishment, depersonalisation), the Work-Family Conflict Scale, and the Effort-Reward Imbalance Inventory. Further independent variables were gender and parental status.The analyses were based on general linear models and general linear mixed models with repeated measures designs. RESULTS: Significant time-fixed effects were found for all three subscales of the Maslach Burnout Inventory, with gender effects on the subscales emotional exhaustion and depersonalisation. The parental status had no significant effect on burnout. All estimated means for burnout during 6 years of post-graduate training were higher when work-family conflict and gratification crisis were taken into account. Personal accomplishment increased continuously over time as well showing neither gender differences nor influences by the parental status. CONCLUSIONS: Personal accomplishments might act as a buffer compensating to some extent for the physicians' stress experience. Given that burnout may be associated with poor patient care, there is a need to reduce burnout rates and their associated factors in resident physicians.

[Longitudinal evaluation of female and male medical residents' career satisfaction]. / Longitudinale Untersuchung der Berufsverlaufszufriedenheit von Ärztinnen und Ärzten in der Facharztweiterbildung.

OBJECTIVE: Evaluation of longitudinal data of German medical residents' career satisfaction and its dependency on perceived delays in obtaining the degree as a medical specialist, as well as postgraduate training quality, controlled for gender, parental status, and specialty choice. METHODS: Data was collected within the KarMed study. The first data collection (T0) was conducted in 2008/2009 at the end of the practical year. Hierarchical linear models and path analysis were used to analyse longitudinal associations after three and five years (T3 to T5). RESULTS: A positive residents' career satisfaction had a small causal effect on the training quality. A delay in obtaining the degree as a medical specialist had a small negative effect on residents' career satisfaction. A high residents' career satisfaction was negatively associated with a delay in training. Gender predicted the career satisfaction of physicians with children. The career satisfaction of female physicians with children decreased significantly in the course of their postgraduate training compared to male physicians with children. The speciality choice had no significant impact on residents' career satisfaction. CONCLUSION: Residents who were satisfied with their job subjectively rated the quality of postgraduate training conditions more positively. The anticipated delays in obtaining the degree as a medical specialist and the residents' career satisfaction were reciprocally related. The improvement of career satisfaction in female physicians with children needs special attention in the future.

Differences in the Emission of Volatile Organic Compounds (VOCs) between Non-Differentiating and Adipogenically Differentiating Mesenchymal Stromal/Stem Cells from Human Adipose Tissue.

Metabolic characterization of human adipose tissue-derived mesenchymal stromal/stem cells (ASCs) is of importance in stem cell research. The monitoring of the cell status often requires cell destruction. An analysis of volatile organic compounds (VOCs) in the headspace above cell cultures might be a noninvasive and nondestructive alternative to in vitro analysis. Furthermore, VOC analyses permit new insight into cellular metabolism due to their view on volatile compounds. Therefore, the aim of our study was to compare VOC profiles in the headspace above nondifferentiating and adipogenically differentiating ASCs. To this end, ASCs were cultivated under nondifferentiating and adipogenically differentiating conditions for up to 21 days. At different time points the headspace samples were preconcentrated by needle trap micro extraction and analyzed by gas chromatography/mass spectrometry. Adipogenic differentiation was assessed at equivalent time points. Altogether the emissions of 11 VOCs showed relevant changes and were analyzed in more detail. A few of these VOCs, among them acetaldehyde, were significantly different in the headspace of adipogenically differentiating ASCs and appeared to be linked to metabolic processes. Furthermore, our data indicate that VOC headspace analysis might be a suitable, noninvasive tool for the metabolic monitoring of (mesenchymal stem) cells in vitro.

Human adipocytes and CD34+ cells from the stromal vascular fraction of the same adipose tissue differ in their energy metabolic enzyme configuration.

Adipose tissue plays a role in energy storage and metabolic balance and is composed of different cell types. The metabolic activity of the tissue itself has been a matter of research for a long time, but comparative data about the energy metabolism of different cell types of human subcutaneous adipose tissue are sparse. Therefore, we compared the activity of major energy metabolic pathways of adipocytes and CD34+ cells from the stromal vascular fraction (SVF) separated from the same tissue. This CD34+ cell fraction is enriched with adipose tissue-derived mesenchymal progenitors, as they account for the largest proportion of CD34+ cells of the SVF. Adipocytes displayed significantly higher mitochondrial enzyme capacities compared to CD34+ SVF-cells, as shown by the higher activities of isocitrate dehydrogenase and ß-hydroxyacyl-CoA dehydrogenase. Inversely, the CD34+ SVF-cells showed higher capacities for cytosolic carbohydrate metabolism, represented by the activity of glycolysis and the pentose phosphate pathway. Thus, the CD34+ SVF-cells may ensure the provision of pentose phosphates and reduction equivalents for the replication of DNA during proliferation. The data indicate that these two cell fractions of the human adipose tissue vary in their metabolic configuration adapted to their physiological demands regarding proliferation and differentiation in vivo.

Energy metabolic capacities of human adipose-derived mesenchymal stromal cells in vitro and their adaptations in osteogenic and adipogenic differentiation.

Mesenchymal stromal/stem cells (MSC) are important in tissue homeostasis and regeneration due to their ability for self-renewal and multipotent differentiation. Differentiation, as well as proliferation, requires adaptations in the cell metabolism. However, only few data exist concerning the energy metabolism of non-differentiating and differentiating MSC. In this study we compared capacities of major energy metabolic pathways of MSC from human adipose tissue (adMSC) in vitro in the non-differentiated state with those of osteogenically or adipogenically differentiating adMSC. To this end we quantified the proliferation and differentiation status of adMSC and analyzed maximum enzyme capacities and several enzyme isoforms of major energy metabolic pathways regarding their activity and gene expression. We could show that non-differentiating and osteogenic cultivation conditions induced proliferation and showed increasing capacities of the glycolytic marker enzyme phosphofructokinase as well as the marker enzyme of the pentose phosphate pathway glucose-6-phosphate dehydrogenase. Adipogenic stimulation, which was accompanied by the absence of proliferation, reduced the glycolytic capacity (e.g. decreased glyceraldehyde 3-phosphate dehydrogenase capacity) and induced an increase in mitochondrial enzyme capacities. These changes in energy metabolism might represent an adaptation of adMSC to the high energy demand during proliferation and to the specific cellular functions during osteogenic or adipogenic differentiation respectively.

Shape Variation in Neotropical Cytheridella (Ostracoda) Using Semilandmarks-Based Geometric Morphometrics: A Methodological Approach and Possible Biogeographical Implications.

Geometric morphometrics offer effective methods to obtain information about shape and shape variability. In ostracodology, landmark-based methods are, however, not well established. To test the applicability of geometric morphometric analyses for ostracods, we investigated shape variation among recent and fossil populations of the genus Cytheridella using a combination of landmarks and semilandmarks. The study focuses on the species' intraspecific morphological variability on a supra-regional scale, comparing living populations from Florida, Yucatán, Colombia and Brazil. We performed Generalized least-squares Procrustes Analysis on 508 adult and juvenile specimens (valves) including stages A-1 to A-4. The analyses show that the primary pattern in shape variation is ontogenetic allometry, supporting a clear separation of adults and juveniles. Shape changes are relatively small during ontogeny from A-4 to A-1. Greatest modification of valve shape occurs during the last molt phase. Insufficient differentiation of sexes is caused by females with less developed brood pouches. Disentangling size- and non-size-dependent shape changes reveals regional differences between populations of the species C. ilosvayi and supports its taxonomic distinction from a fossil relative (C. danielopoli). The distribution of regional morphotypes of C. ilosvayi in Florida, Mexico, and Brazil are congruent with fly ways of water birds.

Isolation and differentiation potential of human mesenchymal stem cells from adipose tissue harvested by water jet-assisted liposuction.

BACKGROUND: In recent years the therapeutic application of extracted adipose tissue for autologous fat grafting and the application of adipose tissue-derived mesenchymal stem cells (adMSC) isolated thereof has progressed. Water-jet assisted liposuction (WAL) is 1 procedure for harvesting adipose tissue and provides a favorable aesthetic outcome combined with high tissue protection. Tissue aspirated by WAL has been successfully applied in grafting procedures. OBJECTIVES: The aims of this study were to confirm the tissue viability and to understand the abundance and mesenchymal differentiation capacity of stem cells within the tissue. METHODS: We analyzed tissue integrity of WAL tissue particles via fluorescence microscopy. The adMSC content was determined by isolating the cells from the tissue. The mesenchymal differentiation capacity was confirmed with cytochemical staining methods. RESULTS: The stromal vascular fraction of WAL tissue showed high viability and contained an average of 2.6 × 105 CD34-positive cells per milliliter of tissue. Thus WAL tissue contains a high number of stem cells. Furthermore adMSC isolated from WAL tissue showed typical mesenchymal differentiation potential. CONCLUSIONS: WAL of adipose tissue is well suited for autologous fat grafting because it retains tissue viability. Furthermore it is a valid source for the subsequent isolation of adMSC with multipotent differentiation potential. LEVEL OF EVIDENCE: 3 Therapeutic.

Information needs and visitors' experience of an Internet expert forum on infertility.

BACKGROUND: Patients increasingly use health portals and Web-based expert forums (ask-the-doctor services), but little is known about the specific needs of Internet users visiting such websites, the nature of their requests, or how satisfied they are with Internet health experts. OBJECTIVE: The aim of this study was to analyze the information requests of (mostly female) patients visiting an Internet expert forum on involuntary childlessness and their satisfaction with the experts' feedback. METHODS: We posted an electronic questionnaire on a website hosting an expert forum on involuntary childlessness. The questionnaire was "activated" whenever a visitor sent a question or request to the expert forum. The survey focused on the reasons for visiting the expert forum and whether the visitors were satisfied with the experts' answers to previously posted questions. The free-text questions of visitors who answered the survey were analyzed using Atlas-ti, a software program for qualitative data analysis. RESULTS: Over a period of 6 months, 513 out of 610 visitors (84%) answered the questionnaire. The majority of respondents (65.5%) expected general information about involuntary childlessness, conception, or an evaluation of drugs. Others were concerned about their actual treatment (40.6%) and therapeutic options (28.8%). Out of 225 respondents who had previously contacted the forum, 223 has received an answer, and 123 (55.2%) were satisfied with the experts' answers. About half (105/223) of those users who had previously received an answer from the expert forum stated that they had discussed it with their own doctor. More of these users were satisfied with their subsequent care in fertility clinics than users who did not talk to their doctor about their Internet activities (93.9% vs 76.1%; P = .015). According to the qualitative analysis, many requests (n = 194) were more or less trivial, especially those for information on basic aspects of reproduction. More than one-third of visitors (n = 199) sent detailed results of diagnostic tests and asked for a first or second opinion. Requests to the expert forum were also sent in order to obtain emotional support (17%) or to complain about a doctor (15%). CONCLUSIONS: Visitors who sent their laboratory findings to receive a thorough evaluation or a second opinion had a good command of the opportunities that an expert forum offers. One important expectation of the forum was emotional support, indicating psychological needs that were not met by medical providers. Future websites must find a compromise in order to protect experts from being overwhelmed by general, nonspecific requests while supporting patients with individualized answers.