Probing the glioma micro-environment: analysis using biopsy in combination with ultra-fast cyclic immunolabeling.

The interaction between gliomas and the immune system is poorly understood and thus hindering development of effective immunotherapies for glioma patients. The immune response is highly variable during tumor development, and affected by therapies such as surgery, radiation, and chemotherapy. Currently, analysis of these local changes is difficult due to poor accessibility of the tumor and high-morbidity of sampling. In this study, we developed a model for repeat-biopsy in mice to study these local immunological changes over time. Using fine needle biopsy we were able to safely and repeatedly collect cells from intracranial tumors in mice. Ultra-fast cycling technology (FAST) was used for multi-cycle immunofluorescence of retrieved cells, and provided insights in the changing immune response over time. The combination of these techniques can be utilized to study changes in the immune response in glioma or other intracranial diseases over time, and in response to treatment within the same animal. Teaser: Fine-needle biopsy and ultra-fast cycling technology techniques were developed to allow for repeat sampling and analysis of glial tumors in mice.

Subjective Health Status, Health-Related Behavior, and Health Literacy of Health Professional Students: Results from a Cross-Sectional Study.

The importance of health promotion for health professional students is increasingly crucial, as the rising requirements for those students can have a negative impact on their health. Despite this awareness, there is still limited knowledge in Germany about the specific health needs of this group of students. This study's aim was, therefore, to assess the subjective health of first-year students and to identify health needs. Thus, this study is based on an online survey using standardized measurement instruments. First-year students from three degree programs were included. The data were analyzed descriptively. A total of n = 98 (72.6%) participated in the survey. The results showed that a major proportion of participants (80.3%) rated their health positively, but a significant proportion reported weight disorders (24%) and pre-existing health conditions (62.1%) at baseline. Interestingly, a high proportion (59%) reported a high level of mental well-being at the time of the survey. However, worrying findings regarding lifestyle behaviors, including physical inactivity (40.6%), smoking (20%), risky alcohol use (24%), and unhealthy eating habits (37%) were determined. In terms of health literacy, around 45% of students rated their health competencies as problematic. Furthermore, it was found that students with low health literacy had a significantly higher prevalence of low mental well-being (53.3% vs. 30.8%, p = 0.036) and unhealthy eating habits (48.8% vs. 26.5%, p = 0.027) compared to students with sufficient health literacy. Health professional students should be considered a relevant target group for health and health competence promotion from the beginning of their studies. The identified fields of action should be addressed in the context of health promotion. This is of particular importance as they are not only aimed at improving the students' well-being but also will later work directly with patients and, therefore, have a direct influence on the health of others.

Health of (dual) health professional students in German-speaking countries: a scoping review.

University education marks a new stage in life, which is associated with unknown demands and challenges and can have a negative impact on students' health. Therefore, health promotion in the university setting is becoming increasingly important. In this context, scientific data on the health situation play a crucial role in improving students' health. Thus, the aim of the scoping review was to highlight the current scope of research on the health of health professional students. It also explored problems and outlined key future challenges and solutions. The review was conducted using the Joanna Briggs Institute (JBI) methodology for a scoping review. A total of nine databases (PubMed, CINAHL, CareLit, LIVIVO, Scopus, Psyndex, PEDro, OTseeker, Google Scholar) were systematically searched. The following search criteria were defined: health professional students, health, Germany, German-speaking countries, all types of sources from 2012 to present are selected. The research studies were mapped in a table and health evidence of included studies was summarized narratively. The initial search resulted in 23,938 records. Seven records met the inclusion criteria and were included in the review. Six cross-sectional studies were conducted in Germany, and one cross-sectional study was conducted in Switzerland. In fact, one study included a representative population. Qualitative studies were not found. The most studies investigated health status, health behavior, and personal resources. Most of the studies examined female nursing students. The included studies indicated that the young students reported physical or mental health conditions. In addition, the studies also identified health resources of the students that need to be improved. In summary, there is currently limited health evidence on this group of students in German-speaking countries. Therefore, further research is needed to generate knowledge and comprehensively describe the health situation.

Monitoring a cohort of trainees: changes over time and associations between health literacy, health behaviour and health.

BACKGROUND: For many entrants, the initial stages of professional training are a challenge. Demands at work can lead to new physical and psychological stress, as well as new social requirements. These new requirements can influence the health behaviour and the state of health of young employees. In recent years, there have been many studies on health literacy (HL). HL represents resources and potentials that allow individuals to achieve improved control of their health and of factors that influence health. Thus, HL can influence both well-being and health. In the present study, the health of trainees in different branches (health and welfare services, office, sales, technology) is monitored over time ending in the period of the COVID-19 pandemic. Furthermore, the association between health literacy and health or health behaviour has been examined. METHODS: In 2017/18, a baseline survey (T0) was performed on trainees in various sectors (office, sales, teaching, nursing and social welfare, engineering, hairdressers), who had been contacted through vocational colleges in four federal states in north Germany. The trainees were surveyed again in the in the first year after training in 2021 (T3). Demographic data were collected, as well as information on health literacy (HLS-EU-16), health behaviour (physical exercise, nutrition, smoking and alcohol) and state of health (BMI, psychological well-being and subjective state of health). Recognition, satisfaction at work and thoughts of leaving the profession were surveyed with the Copenhagen Psychosocial Questionnaire (COPSOQ). Statistical analysis was performed with tests for paired samples and multivariate regression analysis in SPSS 26. RESULTS: 129 data sets were evaluated, with a follow-up rate of 10.2%. 85% of the trainees were female. The mean age at follow-up was 25.6 years. 56% were employed in the health service or social welfare. 35% worked in the office, sales or engineering. At T3, 47% of the employees exhibited limited health literacy, 67% low levels of exercise and 30% risky alcohol use. 42% exhibited overweight and 42% poor psychological well-being. An association between health literacy (HL) and psychological well-being was only observed in cross-section (HL inadequate vs. HL adequate OR: 3.2 95% CI: 1.07-9.49, p = 0.037). The odds ratio relative to subjective state of health was also increased, although the association was not statistically significant (HL inadequate vs. HL adequate OR: 2.7 95% CI: 0.72-9.78, p = 0.143). In the sector for health service and social welfare, there was statistically significant deterioration over time in all COPSOQ variables (recognition, satisfaction at work, thoughts of leaving the profession). CONCLUSIONS: For a group of trainees in the first year of work during the covid-19 epidemic, the present findings show that there is a need to prevent risky health behaviour, overweight and poor psychological well-being. Health literacy was shown to be a modifiable parameter, that apparently is associated in cross - but not in longitudinal section with the health of young employees. It would appear to be reasonable to modify developing health literacy in the setting of work and school.

What's the Harm in Cardiopulmonary Resuscitation?

In clinical ethics, there remains a great deal of uncertainty regarding the appropriateness of attempting cardiopulmonary resuscitation (CPR) for certain patients. Although the issue continues to receive ample attention and various frameworks have been proposed for navigating such cases, most discussions draw heavily on the notion of harm as a central consideration. In the following, I use emerging philosophical literature on the notion of harm to argue that the ambiguities and disagreement about harm create important and oft-overlooked challenges for the ethics of CPR. I begin by elucidating the standard account of harm, called the Counterfactual Comparative Account (CCA). I then show that three challenges to the CCA-preemptive harms, the harm of death, and non-experiential harms-are particularly salient when assessing potential harms for candidates of CPR and likely impact-related decision-making and communication. I extend this argument to explore how the ambiguities of harm might extend to other realms of clinical decision-making, such as the use and limitations of life-sustaining treatments. To address these challenges, I propose two strategies for identifying and minimizing the impact of such uncertainty: first, clinicians and ethicists ought to promote pluralistic conversations that account for different understandings of harm; second, they ought to invoke harm-independent considerations when discussing the ethics of CPR in order to reflect the nuances of such conversations. These strategies, coupled with a richer philosophical understanding of harm, promise to help clinicians and ethicists navigate the prevalent and difficult cases involving patient resuscitation and many other harm-based decisions in the clinical setting.

Effects of TP63 mutations on keratinocyte adhesion and migration.

The goal of this study was to investigate the molecular mechanisms responsible for the formation of skin erosions in patients affected by Ankyloblepharon-ectodermal defects-cleft lip/palate syndrome (AEC). This ectodermal dysplasia is caused by mutations in the TP63 gene, which encodes several transcription factors that control epidermal development and homeostasis. We generated induced pluripotent stem cells (iPSC) from AEC patients and corrected the TP63 mutations using genome editing tools. Three pairs of the resulting conisogenic iPSC lines were differentiated into keratinocytes (iPSC-K). We identified a significant downregulation of key components of hemidesmosomes and focal adhesions in AEC iPSC-K compared to their gene-corrected counterparts. Further, we demonstrated reduced AEC iPSC-K migration, suggesting the possibility that a process critical for cutaneous wound healing might be impaired in AEC patients. Next, we generated chimeric mice expressing a TP63-AEC transgene and confirmed a downregulation of these genes in transgene-expressing cells in vivo. Finally, we also observed these abnormalities in AEC patient skin. Our findings suggest that integrin defects in AEC patients might weaken the adhesion of keratinocytes to the basement membrane. We propose that reduced expression of extracellular matrix adhesion receptors, potentially in conjunction with previously identified desmosomal protein defects, contribute to skin erosions in AEC.

Effects of TP63 Mutations on Keratinocyte Adhesion and Migration.

The goal of this study was to investigate the molecular mechanisms responsible for the formation of skin erosions in patients affected by Ankyloblepharon-ectodermal defects-cleft lip/palate syndrome (AEC). This ectodermal dysplasia is caused by mutations in the TP63 gene, which encodes several transcription factors that control epidermal development and homeostasis. We generated induced pluripotent stem cells (iPSC) from AEC patients and corrected the TP63 mutations using genome editing tools. Three pairs of the resulting conisogenic iPSC lines were differentiated into keratinocytes (iPSC-K). We identified a significant downregulation of key components of hemidesmosomes and focal adhesions in AEC iPSC-K compared to their gene-corrected counterparts. Further, we demonstrated reduced iPSC-K migration, suggesting the possibility that a process critical for cutaneous wound healing might be impaired in AEC patients. Next, we generated chimeric mice expressing a TP63-AEC transgene and confirmed a downregulation of these genes in transgene-expressing cells in vivo. Finally, we also observed these abnormalities in AEC patient skin. Our findings suggest that integrin defects in AEC patients might weaken the adhesion of keratinocytes to the basement membrane. We propose that reduced expression of extracellular matrix adhesion receptors, potentially in conjunction with previously identified desmosomal protein defects, contribute to skin erosions in AEC.

A neutrophil response linked to tumor control in immunotherapy.

Neutrophils accumulate in solid tumors, and their abundance correlates with poor prognosis. Neutrophils are not homogeneous, however, and could play different roles in cancer therapy. Here, we investigate the role of neutrophils in immunotherapy, leading to tumor control. We show that successful therapies acutely expanded tumor neutrophil numbers. This expansion could be attributed to a Sellhi state rather than to other neutrophils that accelerate tumor progression. Therapy-elicited neutrophils acquired an interferon gene signature, also seen in human patients, and appeared essential for successful therapy, as loss of the interferon-responsive transcription factor IRF1 in neutrophils led to failure of immunotherapy. The neutrophil response depended on key components of anti-tumor immunity, including BATF3-dependent DCs, IL-12, and IFNγ. In addition, we found that a therapy-elicited systemic neutrophil response positively correlated with disease outcome in lung cancer patients. Thus, we establish a crucial role of a neutrophil state in mediating effective cancer therapy.

Biomechanical Comparison of Anterior Cruciate Ligament Reconstruction Using a Single-Bundle Round or Ribbon-like Hamstring Tendon Graft.

BACKGROUND: Persistent instability of the knee is reported in up to 30% of patients after anterior cruciate ligament (ACL) reconstruction. Based on anatomic findings showing that ACL is a flat ribbon-like structure that twists during knee flexion, a new surgical ACL reconstruction technique using a ribbon-like graft has been developed. However the effect of this surgical technique on knee kinematics has not yet been evaluated. PURPOSE: To compare the anteroposterior and rotational stability of the knee after ACL reconstruction using single-bundle (SB) round and ribbon-like grafts in anterolateral-intact/deficient knees. STUDY DESIGN: Controlled laboratory study. METHODS: Twelve human fresh-frozen cadaveric knees were tested with a 6 degrees of freedom robotic system. Internal rotation and anterior translation of the knee were recorded from 0° to 90° of flexion. A full kinematic assessment was performed in each of the following conditions: (1) intact knee, (2) after sectioning of the ACL, (3) after ACL reconstruction using a SB hamstring tendon graft in a round configuration and a ribbon-like configuration, and (4) after sectioning of the anterolateral structures. One-way analysis of variance and post hoc Tukey tests were used for statistical analyses. RESULTS: When compared with the intact knee, the ACL-deficient knee demonstrated a mean ± SD increase in anterior translation and internal rotation of 6.3 ± 2.5 mm (P < .01) and 5.8°± 2.3° (P < .01), respectively. After ACL reconstruction using a SB ribbon-like graft, the mean difference in anterior translation and internal rotation as compared with the intact knee was -0.1 ± 1.5 mm (P = .842) and 0.0°± 1.1° (P = .999). These differences from the intact knee were also not significant after ACL reconstruction using a round graft (-0.1 ± 1.3 mm, P = .999; -0.5°± 1.5°, P = .401). In the ACL-reconstructed knee using either a ribbon-like or round graft, sectioning of the anterolateral structures did not induce a significant increase of anterior translation and internal rotation of the knee. CONCLUSION: ACL reconstruction using a SB ribbon-like or round graft restored the kinematics of the intact knee at time zero. Secondary sectioning of the anterolateral structures in the ACL-reconstructed knee using both types of graft did not significantly affect the anterior translation and internal rotation of the knee. CLINICAL RELEVANCE: This is the first biomechanical study on the new ACL reconstruction technique using a ribbon-like graft.

Structure, evolution and expression of zebrafish cartilage oligomeric matrix protein (COMP, TSP5). CRISPR-Cas mutants show a dominant phenotype in myosepta.

COMP (Cartilage Oligomeric Matrix Protein), also named thrombospondin-5, is a member of the thrombospondin family of extracellular matrix proteins. It is of clinical relevance, as in humans mutations in COMP lead to chondrodysplasias. The gene encoding zebrafish Comp is located on chromosome 11 in synteny with its mammalian orthologs. Zebrafish Comp has a domain structure identical to that of tetrapod COMP and shares 74% sequence similarity with murine COMP. Zebrafish comp is expressed from 5 hours post fertilization (hpf) on, while the protein is first detectable in somites of 11 hpf embryos. During development and in adults comp is strongly expressed in myosepta, craniofacial tendon and ligaments, around ribs and vertebra, but not in its name-giving tissue cartilage. As in mammals, zebrafish Comp forms pentamers. It is easily extracted from 5 days post fertilization (dpf) whole zebrafish. The lack of Comp expression in zebrafish cartilage implies that its cartilage function evolved recently in tetrapods. The expression in tendon and myosepta may indicate a more fundamental function, as in evolutionary distant Drosophila muscle-specific adhesion to tendon cells requires thrombospondin. A sequence encoding a calcium binding motif within the first TSP type-3 repeat of zebrafish Comp was targeted by CRISPR-Cas. The heterozygous and homozygous mutant Comp zebrafish displayed a patchy irregular Comp staining in 3 dpf myosepta, indicating a dominant phenotype. Electron microscopy revealed that the endoplasmic reticulum of myosepta fibroblasts is not affected in homozygous fish. The disorganized extracellular matrix may indicate that this mutation rather interferes with extracellular matrix assembly, similar to what is seen in a subgroup of chondrodysplasia patients. The early expression and easy detection of mutant Comp in zebrafish points to the potential of using the zebrafish model for large scale screening of small molecules that can improve secretion or function of disease-associated COMP mutants.

Exploring the subtle factors that control the structural preferences in Cu7Te4.

In the quest for materials suited as components in future technologies, the copper-rich regions of the binary Cu-Te system have been of great interest. In this context, several explorative efforts were also focused on Cu7Te4which was reported to crystallize with different types of structure. To explore the structural preferences for two Cu7Te4structure models, both experimental as well as quantum-chemical means were employed. The crystal structures of both Cu7Te4types are composed of hexagonal closest packed layers of tellurium atoms, and differ in the respective distributions of the copper atoms between these layers. The analysis of the electronic structures was accomplished based on the densities-of-states, Mulliken charges, projected crystal orbital Hamilton populations, and electron localization functions of both structure models, and its outcome indicates that the factors that control the formation of a respective type of structure are rather subtle.

Dissecting the treatment-naive ecosystem of human melanoma brain metastasis.

Melanoma brain metastasis (MBM) frequently occurs in patients with advanced melanoma; yet, our understanding of the underlying salient biology is rudimentary. Here, we performed single-cell/nucleus RNA-seq in 22 treatment-naive MBMs and 10 extracranial melanoma metastases (ECMs) and matched spatial single-cell transcriptomics and T cell receptor (TCR)-seq. Cancer cells from MBM were more chromosomally unstable, adopted a neuronal-like cell state, and enriched for spatially variably expressed metabolic pathways. Key observations were validated in independent patient cohorts, patient-derived MBM/ECM xenograft models, RNA/ATAC-seq, proteomics, and multiplexed imaging. Integrated spatial analyses revealed distinct geography of putative cancer immune evasion and evidence for more abundant intra-tumoral B to plasma cell differentiation in lymphoid aggregates in MBM. MBM harbored larger fractions of monocyte-derived macrophages and dysfunctional TOX+CD8+ T cells with distinct expression of immune checkpoints. This work provides comprehensive insights into MBM biology and serves as a foundational resource for further discovery and therapeutic exploration.

Self-Examination Low-Cost Full-Field Optical Coherence Tomography (SELFF-OCT) for neovascular age-related macular degeneration: a cross-sectional diagnostic accuracy study.

OBJECTIVES: Self-Examination Low-Cost Full-Field Optical Coherence Tomography (SELFF-OCT) is a novel OCT technology that was specifically designed for home monitoring of neovascular age-related macular degeneration (AMD). First clinical findings have been reported before. This trial investigates an improved prototype for patients with AMD and focusses on device operability and diagnostic accuracy compared with established spectral-domain OCT (SD-OCT). DESIGN: Prospective single-arm diagnostic accuracy study. SETTING: Tertiary care centre (University Eye Clinic). PARTICIPANTS: 46 patients with age-related macular degeneration. INTERVENTIONS: Patients received short training in device handling and then performed multiple self-scans with the SELFF-OCT according to a predefined protocol. Additionally, all eyes were examined with standard SD-OCT, performed by medical personnel. All images were graded by at least 2 masked investigators in a reading centre. PRIMARY OUTCOME MEASURE: Rate of successful self-measurements. SECONDARY OUTCOME MEASURES: Sensitivity and specificity of SELFF-OCT versus SD-OCT for different biomarkers and necessity for antivascular endothelial growth factor (anti-VEGF) treatment. RESULTS: In 86% of all examined eyes, OCT self-acquisition resulted in interpretable retinal OCT volume scans. In these patients, the sensitivity for detection of anti-VEGF treatment necessity was 0.94 (95% CI 0.79 to 0.99) and specificity 0.95 (95% CI 0.82 to 0.99). CONCLUSIONS: SELFF-OCT was used successfully for retinal self-examination in most patients, and it could become a valuable tool for retinal home monitoring in the future. Improvements are in progress to reduce device size and to improve handling, image quality and success rates. TRIAL REGISTRATION NUMBER: DRKS00013755, CIV-17-12-022384.

ISB clinical biomechanics award winner 2021: Tibio-femoral kinematics of natural versus replaced knees - A comparison using dynamic videofluoroscopy.

BACKGROUND: A comparison of natural versus replaced tibio-femoral kinematics in vivo during challenging activities of daily living can help provide a detailed understanding of the mechanisms leading to unsatisfactory results and lay the foundations for personalised implant selection and surgical implantation, but also enhance further development of implant designs towards restoring physiological knee function. The aim of this study was to directly compare in vivo tibio-femoral kinematics in natural versus replaced knees throughout complete cycles of different gait activities using dynamic videofluoroscopy. METHODS: Twenty-seven healthy and 30 total knee replacement subjects (GMK Sphere, GMK PS, GMK UC) were assessed during multiple complete gait cycles of level walking, downhill walking, and stair descent using dynamic videofluoroscopy. Following 2D/3D registration, tibio-femoral rotations, condylar antero-posterior translations, and the location of the centre of rotation were compared. FINDINGS: The total knee replacement groups predominantly experienced reduced tibial internal/external rotation and altered medial and lateral condylar antero-posterior translations compared to natural knees. An average medial centre of rotation was found for the natural and GMK sphere groups in all three activities, whereas the GMK PS and UC groups experienced a more central to lateral centre of rotation. INTERPRETATION: Each total knee replacement design exhibited characteristic motion patterns, with the GMK Sphere most closely replicating the medial centre of rotation found for natural knees. Despite substantial similarities between the subject groups, none of the implant geometries was able to replicate all aspects of natural tibio-femoral kinematics, indicating that different implant geometries might best address individual functional needs.

Differentiation of Human Induced Pluripotent Stem Cells into Keratinocytes.

Investigating basic biological mechanisms underlying human diseases relies on the availability of sufficient quantities of patient cells. As most primary somatic cells have a limited lifespan, obtaining sufficient material for biological studies has been a challenge. The development of induced pluripotent stem cell (iPSC) technology has been a game changer, especially in the field of rare genetic disorders. iPSC are essentially immortal, can be stored indefinitely, and can thus be used to generate defined somatic cells in unlimited quantities. Further, the availability of genome editing technologies, such as CRISPR/CAS, has provided us with the opportunity to create "designer" iPSC lines with defined genetic characteristics. A major advancement in biological research stems from the development of methods to direct iPSC differentiation into defined cell types. In this article, we provide the basic protocol for the generation of human iPSC-derived keratinocytes (iPSC-K). These cells have the characteristics of basal epidermal keratinocytes and represent a tool for the investigation of normal epidermal biology, as well as genetic and acquired skin disorders. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Directed differentiation of human iPSC into keratinocytes Support Protocol 1: Coating cell culture dishes or plates with Vitronectin XF™ Support Protocol 2: Freezing iPSC Support Protocol 3: Preparing AggreWell™ 400 6-well plates for EB formation Support Protocol 4: Coating cell culture dishes or plates with Collagen IV Support Protocol 5: Immunofluorescence staining of cells.

High-Content Screening and Computational Prediction Reveal Viral Genes That Suppress the Innate Immune Response.

Suppression of the host innate immune response is a critical aspect of viral replication. Upon infection, viruses may introduce one or more proteins that inhibit key immune pathways, such as the type I interferon pathway. However, the ability to predict and evaluate viral protein bioactivity on targeted pathways remains challenging and is typically done on a single-virus or -gene basis. Here, we present a medium-throughput high-content cell-based assay to reveal the immunosuppressive effects of viral proteins. To test the predictive power of our approach, we developed a library of 800 genes encoding known, predicted, and uncharacterized human virus genes. We found that previously known immune suppressors from numerous viral families such as Picornaviridae and Flaviviridae recorded positive responses. These include a number of viral proteases for which we further confirmed that innate immune suppression depends on protease activity. A class of predicted inhibitors encoded by Rhabdoviridae viruses was demonstrated to block nuclear transport, and several previously uncharacterized proteins from uncultivated viruses were shown to inhibit nuclear transport of the transcription factors NF-κB and interferon regulatory factor 3 (IRF3). We propose that this pathway-based assay, together with early sequencing, gene synthesis, and viral infection studies, could partly serve as the basis for rapid in vitro characterization of novel viral proteins. IMPORTANCE Infectious diseases caused by viral pathogens exacerbate health care and economic burdens. Numerous viral biomolecules suppress the human innate immune system, enabling viruses to evade an immune response from the host. Despite our current understanding of viral replications and immune evasion, new viral proteins, including those encoded by uncultivated viruses or emerging viruses, are being unearthed at a rapid pace from large-scale sequencing and surveillance projects. The use of medium- and high-throughput functional assays to characterize immunosuppressive functions of viral proteins can advance our understanding of viral replication and possibly treatment of infections. In this study, we assembled a large viral-gene library from diverse viral families and developed a high-content assay to test for inhibition of innate immunity pathways. Our work expands the tools that can rapidly link sequence and protein function, representing a practical step toward early-stage evaluation of emerging and understudied viruses.

Comparative Cost Analysis of Four Different Computer-Assisted Technologies to Implant a Total Knee Arthroplasty over Conventional Instrumentation.

Several computer-assisted technologies, such as navigation and robotics, have been introduced to Total Knee Arthroplasty (TKA) in order to increase surgical precision and reduce complications. However, these technologies are often criticized due to the increased costs and effort associated with them; however, comparative data are missing. The aim of the present study was to evaluate differences in intraoperative workflows and the related perioperative cost-profiles of four current computer-assisted technologies, used to implant a TKA, in order to gain a comparison to conventional instrumentation. For the cost analysis, additional preoperative imaging and instruments, increased operating room (OR) and planning-time, and expenditures for technical support of the equipment and disposals were calculated, in comparison to conventional TKA, for (1) standard computer-navigation, (2) patient specific instruments (PSI), (3) image-based robotic assistance, and (4) imageless robotic assistance. Workflows at four expert centers which use these technologies were reviewed by an independent observer. The total cost calculation was based on a 125 TKA per year unit in Switzerland. Computer-navigation resulted in 14 min (+23%) increased surgery time and, overall, USD 650 in additional costs. PSI technology saved 5 min (8%) OR time but it created USD 1520 in expenditures for imaging and disposals. The image-based robotic system was the most expensive technology; it created overall additional costs of USD 2600, which predominately resulted from technical support, disposals, the CT-Scan, and 14 min of increased OR time. The imageless robotic assistance resulted in the largest increase in OR-time, as it resulted in an additional 25 min (+42%) on average. Overall, additional costs of USD 1530 were calculated. Every one of the assistive technologies in this study increased the total cost of TKA when compared to a conventional technique, and the most important variables, related to cost, were technical support and additional disposables. The longer surgical times and additional surgical trays required for the techniques had a marginal effect on overall costs. This comparative cost analysis gives valuable information for future efforts to calculate the real costs of these technologies and the subsequent return on investment of each technique.