Advancing Precision Medicine in Myocarditis: Current Status and Future Perspectives in Endomyocardial Biopsy-Based Diagnostics and Therapeutic Approaches.

The diagnosis and specific and causal treatment of myocarditis and inflammatory cardiomyopathy remain a major clinical challenge. Despite the rapid development of new imaging techniques, endomyocardial biopsies remain the gold standard for accurate diagnosis of inflammatory myocardial disease. With the introduction and continued development of immunohistochemical inflammation diagnostics in combination with viral nucleic acid testing, myocarditis diagnostics have improved significantly since their introduction. Together with new technologies such as miRNA and gene expression profiling, quantification of specific immune cell markers, and determination of viral activity, diagnostic accuracy and patient prognosis will continue to improve in the future. In this review, we summarize the current knowledge on the pathogenesis and diagnosis of myocarditis and inflammatory cardiomyopathies and highlight future perspectives for more in-depth and specialized biopsy diagnostics and precision, personalized medicine approaches.

Asymmetric Condensation Characteristics during Dropwise Condensation in the Presence of Non-condensable Gas: A Lattice Boltzmann Study.

In this work, the condensation characteristics of droplets considering the non-condensable gas with different interaction effects are numerically studied utilizing a multicomponent multiphase thermal lattice Boltzmann (LB) model, with a special focus on the asymmetric nature induced by the interaction effect. The results demonstrate that for isolated-like growth with negligible interactions, the condensation characteristics, that is, the concentration profile, the temperature distribution, and the flow pattern, are typically symmetric in nature. For the growth regime in a pattern, the droplet has to compete with its neighbors for catching vapor, which leads to an overlapping concentration profile (namely the interaction effect). The distribution of the condensation flux on the droplet surface is consequently modified, which contributes to the asymmetric flow pattern and temperature profile. The condensation characteristics for droplet growth in a pattern present an asymmetric nature. Significantly, the asymmetric condensation flux resulting from the interaction effect can induce droplet motion. The results further demonstrate that the interaction strongly depends on the droplet's spatial and size distribution, including two crucial parameters, namely the inter-distance and relative size of droplets. The asymmetric condensation characteristics are consequently dependent on the difference in the interaction intensities on both sides of the droplet. Finally, we demonstrate numerically and theoretically that the evolution of the droplet radius versus time can be suitably described by a power law; the corresponding exponent is kept at a constant of 0.50 for isolated-like growth and is strongly sensitive to the interaction effect for the growth in a pattern.

Intramyocardial Inflammation after COVID-19 Vaccination: An Endomyocardial Biopsy-Proven Case Series.

Myocarditis in response to COVID-19 vaccination has been reported since early 2021. In particular, young male individuals have been identified to exhibit an increased risk of myocardial inflammation following the administration of mRNA-based vaccines. Even though the first epidemiological analyses and numerous case reports investigated potential relationships, endomyocardial biopsy (EMB)-proven cases are limited. Here, we present a comprehensive histopathological analysis of EMBs from 15 patients with reduced ejection fraction (LVEF = 30 (14-39)%) and the clinical suspicion of myocarditis following vaccination with Comirnaty® (Pfizer-BioNTech) (n = 11), Vaxzevria® (AstraZenica) (n = 2) and Janssen® (Johnson & Johnson) (n = 2). Immunohistochemical EMB analyses reveal myocardial inflammation in 14 of 15 patients, with the histopathological diagnosis of active myocarditis according the Dallas criteria (n = 2), severe giant cell myocarditis (n = 2) and inflammatory cardiomyopathy (n = 10). Importantly, infectious causes have been excluded in all patients. The SARS-CoV-2 spike protein has been detected sparsely on cardiomyocytes of nine patients, and differential analysis of inflammatory markers such as CD4+ and CD8+ T cells suggests that the inflammatory response triggered by the vaccine may be of autoimmunological origin. Although a definitive causal relationship between COVID-19 vaccination and the occurrence of myocardial inflammation cannot be demonstrated in this study, data suggest a temporal connection. The expression of SARS-CoV-2 spike protein within the heart and the dominance of CD4+ lymphocytic infiltrates indicate an autoimmunological response to the vaccination.

Transcriptional Active Parvovirus B19 Infection Predicts Adverse Long-Term Outcome in Patients with Non-Ischemic Cardiomyopathy.

Parvovirus B19 (B19V) is the predominant cardiotropic virus currently found in endomyocardial biopsies (EMBs). However, direct evidence showing a causal relationship between B19V and progression of inflammatory cardiomyopathy are still missing. The aim of this study was to analyze the impact of transcriptionally active cardiotropic B19V infection determined by viral RNA expression upon long-term outcomes in a large cohort of adult patients with non-ischemic cardiomyopathy in a retrospective analysis from a prospective observational cohort. In total, the analyzed study group comprised 871 consecutive B19V-positive patients (mean age 50.0 ± 15.0 years) with non-ischemic cardiomyopathy who underwent EMB. B19V-positivity was ascertained by routine diagnosis of viral genomes in EMBs. Molecular analysis of EMB revealed positive B19V transcriptional activity in n = 165 patients (18.9%). Primary endpoint was all-cause mortality in the overall cohort. The patients were followed up to 60 months. On the Cox regression analysis, B19V transcriptional activity was predictive of a worse prognosis compared to those without actively replicating B19V (p = 0.01). Moreover, multivariable analysis revealed transcriptional active B19V combined with inflammation [hazard ratio 4.013, 95% confidence interval 1.515-10.629 (p = 0.005)] as the strongest predictor of impaired survival even after adjustment for age and baseline LVEF (p = 0.005) and independently of viral load. The study demonstrates for the first time the pathogenic clinical importance of B19V with transcriptional activity in a large cohort of patients. Transcriptionally active B19V infection is an unfavourable prognostic trigger of adverse outcome. Our findings are of high clinical relevance, indicating that advanced diagnostic differentiation of B19V positive patients is of high prognostic importance.

Dropwise condensation: From fundamentals of wetting, nucleation, and droplet mobility to performance improvement by advanced functional surfaces.

As a ubiquitous vapor-liquid phase-change process, dropwise condensation has attracted tremendous research attention owing to its remarkable efficiency of energy transfer and transformative industrial potential. In recent years, advanced functional surfaces, profiting from great progress in modifying micro/nanoscale features and surface chemistry on surfaces, have led to exciting advances in both heat transfer enhancement and fundamental understanding of dropwise condensation. In this review, we discuss the development of some key components for achieving performance improvement of dropwise condensation, including surface wettability, nucleation, droplet mobility, and growth, and discuss how they can be elaborately controlled as desired using surface design. We also present an overview of dropwise condensation heat transfer enhancement on advanced functional surfaces along with the underlying mechanisms, such as jumping condensation on nanostructured superhydrophobic surfaces, and new condensation characteristics (e.g., Laplace pressure-driven droplet motion, hierarchical condensation, and sucking flow condensation) on hierarchically structured surfaces. Finally, the durability, cost, and scalability of specific functional surfaces are focused on for future industrial applications. The existing challenges, alternative strategies, as well as future perspectives, are essential in the fundamental and applied aspects for the practical implementation of dropwise condensation.

Scleral Inflammation around Collector Channels in Eyes with Primary Open-Angle Glaucoma.

Purpose: Investigating the existence of inflammation in the distal outflow system posterior of the Schlemm's canal in primary open-angle glaucoma (POAG).Methods: Scleral biopsies (n = 62) from POAG-patients were taken during deep sclerectomy and fixed either in formalin or RNAlater®. Histologic (hematoxylin & eosin) and immunohistological staining for CD 3 and CD 45RO were performed.Results: Cellular infiltration of immunocompetent cells (CD 3 and CD 45RO positive cells) exists around collector channels (CC). This inflammation is limited to the area around the CCs. Ninety-two percent of the biopsies are positive for inflammation. Untreated, dysgenetic glaucoma eyes and 8% of POAG eyes were negative for inflammation. Neither the use of benzalkonium chloride nor the number and type of preoperative antiglaucomatous medication correlated to the immunohistological result.Conclusion: In POAG a diverse cellular infiltration exists around the CCs in the vast majority of biopsies. This could have major diagnostic and therapeutic consequences for the treatment of POAG.Abbreviations: POAG: primary open-angle glaucoma; CC: collector channel; AH: aqueous humor; TM: trabecular meshwork; SC: Schlemm's canal; HE: hematoxylin & eosin; APC: antigen-presenting cell.

Evaluation of Myocardial Gene Expression Profiling for Superior Diagnosis of Idiopathic Giant-Cell Myocarditis and Clinical Feasibility in a Large Cohort of Patients with Acute Cardiac Decompensation.

AIMS: The diagnostic approach to idiopathic giant-cell myocarditis (IGCM) is based on identifying various patterns of inflammatory cell infiltration and multinucleated giant cells (GCs) in histologic sections taken from endomyocardial biopsies (EMBs). The sampling error for detecting focally located GCs by histopathology is high, however. The aim of this study was to demonstrate the feasibility of gene profiling as a new diagnostic method in clinical practice, namely in a large cohort of patients suffering from acute cardiac decompensation. Methods and Results: In this retrospective multicenter study, EMBs taken from n = 427 patients with clinically acute cardiac decompensation and suspected acute myocarditis were screened (mean age: 47.03 ± 15.69 years). In each patient, the EMBs were analyzed on the basis of histology, immunohistology, molecular virology, and gene-expression profiling. Out of the total of n = 427 patient samples examined, GCs could be detected in 26 cases (6.1%) by histology. An established myocardial gene profile consisting of 27 genes was revealed; this was narrowed down to a specified profile of five genes (CPT1, CCL20, CCR5, CCR6, TLR8) which serve to identify histologically proven IGCM with high specificity in 25 of the 26 patients (96.2%). Once this newly established profiling approach was applied to the remaining patient samples, an additional n = 31 patients (7.3%) could be identified as having IGCM without any histologic proof of myocardial GCs. In a subgroup analysis, patients diagnosed with IGCM using this gene profiling respond in a similar fashion to immunosuppressive therapy as patients diagnosed with IGCM by conventional histology alone. Conclusions: Myocardial gene-expression profiling is a promising new method in clinical practice, one which can predict IGCM even in the absence of any direct histologic proof of GCs in EMB sections. Gene profiling is of great clinical relevance in terms of a) overcoming the sampling error associated with purely histologic examinations and b) monitoring the effectiveness of therapy.

Detection of viral SARS-CoV-2 genomes and histopathological changes in endomyocardial biopsies.

AIMS: Since December 2019, the novel coronavirus SARS-CoV-2 has spread rapidly throughout China and keeps the world in suspense. Cardiovascular complications with myocarditis and embolism due to COVID-19 have been reported. SARS-CoV-2 genome detection in the heart muscle has not been demonstrated so far, and the underlying pathophysiological mechanisms remain to be investigated. METHODS AND RESULTS: Endomyocardial biopsies (EMBs) of 104 patients (mean age: 57.90 ± 16.37 years; left ventricular ejection fraction: 33.7 ± 14.6%, sex: n = 79 male/25 female) with suspected myocarditis or unexplained heart failure were analysed. EMB analysis included histology, immunohistochemistry, and detection of SARS-CoV-2 genomes by real-time reverse transcription polymerase chain reaction in the IKDT Berlin, Germany. Among 104 EMBs investigated, five were confirmed with SARS-CoV-2 infected by reverse real-time transcriptase polymerase chain reaction. We describe patients of different history of symptoms and time duration. Additionally, we investigated histopathological changes in myocardial tissue showing that the inflammatory process in EMBs seemed to permeate vascular wall leading to small arterial obliteration and damage. CONCLUSIONS: This is the first report that established the evidence of SARS-CoV-2 genomes detection in EMBs. In these patients, myocardial injury ischaemia may play a role, which could explain the ubiquitous troponin increases. EMB-based identification of the cause of myocardial injury may contribute to explain the different evolution of complicated SARS-CoV-2-infection and to design future specific and personalized treatment strategies.

Intramyocardial inflammation predicts adverse outcome in patients with cardiac AL amyloidosis.

AIMS: To evaluate the influence of endomyocardial biopsy (EMB)-proven intramyocardial inflammation on mortality in patients with cardiac transthyretin amyloid (ATTR) or amyloid light-chain (AL) amyloidosis. METHODS AND RESULTS: We included 54 consecutive patients (mean age 68.83 ± 9.59 years; 45 men) with EMB-proven cardiac amyloidosis. We followed up patients from first diagnostic biopsy to as long as 36 months (mean 11.5 ± 12 months) and compared their outcome with information on all-cause mortality with or without proof of inflammation on EMB. Intramyocardial inflammation was assessed by quantitative immunohistology. Patients suffering from amyloidosis revealed a significant poor prognosis with proof of intramyocardial inflammation in contrast to those without inflammation (log-rank P = 0.019). Re-grouping of patients indicated AL amyloidosis to have a significant impact on all-cause mortality (log-rank P = 0.012). The detailed subgroup analysis showed that patients suffering from AL amyloidosis with intramyocardial inflammation have a significantly worse prognosis compared with AL amyloidosis without inflammation and ATTR with or without inflammation, respectively (log-rank P = 0.014, contingency Fisher's exact test, P = 0.008). CONCLUSION: Our study reports for the first time a high incidence (48.1%) of intramyocardial inflammation in a series of patients with EMB-proven cardiac amyloidosis and could show that in patients with AL amyloidosis, intramyocardial inflammation correlated significantly with increased mortality. Our data have a direct clinical impact because one can hypothesize that additional immunomodulating/anti-inflammatory treatment regimens in patients with biopsy-proven inflammation of heart muscle tissue could be beneficial for patients suffering from cardiac AL amyloidosis.

Soft tissue profile changes after Functional Mandibular Advancer or Herbst appliance treatment in class II patients.

OBJECTIVE: The objective of the present study is to compare the effects on soft tissue profile in class II patients after treatment with either "Functional Mandibular Advancer" (FMA) or Herbst appliance. MATERIALS AND METHODS: The study included n = 42 patients treated with either FMA (n = 21) or Herbst appliance (n = 21) by the same experienced orthodontist. The treatment followed a single-step advancement protocol. Lateral cephalograms were analyzed through a set of customized measurements. The actual therapeutic effect was calculated using data from a growth survey. After testing for normal distribution and homogeneity of variance, data were analyzed by one-sample Student's t tests and independent Student's t tests. Statistical significance was set at p < 0.05. RESULTS: For both FFAs, significant upper lip retrusion, increase in lower lip's thickness, and length of the lower face occurred. Additionally, significant lower lip retrusion and straightening of the profile were found in FMA and Herbst appliance patients. All remaining variables revealed no significant differences. CONCLUSIONS: Treatment-related changes on the facial soft tissue profile could be regarded similar in class II patients treated with FMA or Herbst appliance. No treatment-related changes that were specific for FMA or Herbst appliance could be identified. Only moderate changes were noted comparing pre- and posttreatment soft tissue profiles. CLINICAL RELEVANCE: Despite proven differences in skeletal and dental treatment effects, the facial profile has not to be taken into consideration when choosing between FMA and Herbst appliance for class II treatment.

A retrospective cephalometric investigation of two fixed functional orthodontic appliances in class II treatment: Functional Mandibular Advancer vs. Herbst appliance.

OBJECTIVE: The objective of the study is to compare skeletal and dental changes in class II patients treated with fixed functional appliances (FFA) that pursue different biomechanical concepts: (1) FMA (Functional Mandibular Advancer) from first maxillary molar to first mandibular molar through inclined planes and (2) Herbst appliance from first maxillary molar to lower first bicuspid through a rod-and-tube mechanism. MATERIALS AND METHODS: Forty-two equally distributed patients were treated with FMA (21) and Herbst appliance (21), following a single-step advancement protocol. Lateral cephalograms were available before treatment and immediately after removal of the FFA. The lateral cephalograms were analyzed with customized linear measurements. The actual therapeutic effect was then calculated through comparison with data from a growth survey. Additionally, the ratio of skeletal and dental contributions to molar and overjet correction for both FFA was calculated. Data was analyzed by means of one-sample Student's t tests and independent Student's t tests. Statistical significance was set at p < 0.05. RESULTS: Although differences between FMA and Herbst appliance were found, intergroup comparisons showed no statistically significant differences. Almost all measurements resulted in comparable changes for both appliances. Statistically significant dental changes occurred with both appliances. Dentoalveolar contribution to the treatment effect was ≥70%, thus always resulting in ≤30% for skeletal alterations. CONCLUSION: FMA and Herbst appliance usage results in comparable skeletal and dental treatment effects despite different biomechanical approaches. CLINICAL RELEVANCE: Treatment leads to overjet and molar relationship correction that is mainly caused by significant dentoalveolar changes.

High Perforin-Positive Cardiac Cell Infiltration and Male Sex Predict Adverse Long-Term Mortality in Patients With Inflammatory Cardiomyopathy.

BACKGROUND: The authors analyzed the effects of perforin-dependent infiltration on long-term mortality in patients with inflammatory cardiomyopathy (CMi). We previously demonstrated that left ventricular function deteriorates and progresses to substantial cardiac dysfunction in patients with perforin-positive cardiac cell infiltration. METHODS AND RESULTS: Between 2003 and 2013, 2389 consecutive patients with clinically suspected CMi who underwent endomyocardial biopsies were enrolled. Endomyocardial biopsies were performed at first admission after exclusion of ischemic or valvular heart disease, and CMi was confirmed in 1717 patients. Follow-up was up to 10.1 years (median 0.47 years; interquartile range, 0.03-2.56 years) and information on vital status was obtained from official resident data files. Multivariable statistical analysis was conducted for all patients with CMi regarding significant predictors of all-cause mortality or need for heart transplantation. Multiple Cox regression analysis revealed perforin above the calculated cutoff point of 2.9 cells/mm² as a strong predictor of impaired survival with a hazard ratio of 1.881 (95% confidence interval, 1.177-3.008; P=0.008), independent of left ventricular function and other myocardial inflammation markers (CD3, macrophage-1 antigen, leukocyte function-associated antigen-1, human leukocyte antigen-1, and intercellular cell adhesion molecule-1). Unexpectedly, male sex emerged as another strong adverse predictor of survival in CMi (hazard ratio, 1.863; confidence interval, 1.096-3.168 [P=0.022]). Whereas left ventricular ejection fraction course is adversely affected by myocardial perforin, multivariate analysis indicates that left ventricular ejection fraction explains only part of the observed overall mortality. CONCLUSIONS: High perforin-positive cardiac cell infiltration and male sex are independent adverse predictors of long-term mortality in CMi. Furthermore, exact quantification of immunohistochemically detected infiltrates is necessary to assess the prognosis.

Immunosuppressive treatment in fulminant myocarditis and gene expression pattern associated with, but no histological confirmation of giant cell myocarditis.

A healthy woman with acute onset of pulmonary oedema and severely depressed left ventricular function underwent endomyocardial biopsy under the clinical suspicion of fulminant myocarditis. While awaiting the results of biopsy, the situation deteriorated to Interagency Registry for Mechanically Assisted Circulatory Support (INTERMACS) and extracorporeal membrane oxygenation was implanted. Finally, immunohistochemistry in biopsy specimen corresponded to fulminant lymphocytic myocarditis, although active myocarditis was excluded. Furthermore, gene expression profiling identified giant cell myocarditis although multinuclear cells were absent. This prompted the start of immunosuppression with cortisone and cyclosporine. The patient fully recovered.

Chromosomally integrated human herpesvirus 6 in heart failure: prevalence and treatment.

AIMS: Human herpesvirus 6 (HHV-6) A and B are two betaherpesviruses that are associated with many conditions including roseola, drug-induced hypersensitivity syndrome, limbic encephalitis, and myocarditis. HHV-6 is integrated in the germline [chromosomically integrated HHV-6 (ciHHV-6)] in ∼0.8% of the human population. To date, the prevalence, species distribution, and treatment responses of ciHHV-6 are unknown for cardiac patients. METHODS AND RESULTS: We determined the prevalence of HHV-6 and ciHHV-6 genotypes in 1656 endomyocardial biopsies of patients with persisting unexplained symptoms of heart failure. Infection of cardiac tissue was identified by nested PCR, electron microscopy, and immunohistochemistry. Virus load and mRNA levels were followed in ciHHV-6 patients treated with ganciclovir. HHV-6 was detected in 273 of 1656 cardiac tissues (16.5%; HHV-6B, 98.2%, HHV-6A, 1.8%) by PCR. Nineteen of the 1656 patients (1.1%) presented with persistently high HHV-6 copy numbers indicative of ciHHV-6. Sequencing confirmed ciHHV-6A in seven patients (36.8%) which was considerably higher than detected in non-ciHHV-6 patients. Inheritance was demonstrated in three selected families, confirming ciHHV-6 chromosomal integration by PCR and fluorescence in situ hybridization. HHV-6 reactivation and chromosomal integration were confirmed in peripheral blood mononuclear cells and heart tissue. Virus particles were identified in degenerating myocytes and interstitial cells. Antiviral treatment abolished viral mRNA and ameliorated cardiac symptoms. CONCLUSION: Virus replication in cardiac tissue of ciHHV-6 heart failure patients suggests that ciHHV-6 reactivation causes persistence of unexplained heart failure symptoms. We demonstrated that antiviral treatment, effective in decreasing viral transcripts and clinical complaints of cardiomyopathies, is a new therapeutic option for ciHHV-6-associated diseases.

Effects of fixed appliances in correcting Angle Class II on the depth of the posterior airway space: FMA vs. Herbst appliance--a retrospective cephalometric study.

OBJECTIVE: The objective of this retrospective study based on the metric evaluation of lateral cephalograms was to investigate the extent to which treatment with two different fixed appliances for the correction of Angle Class II influenced the morphology of the extrathoracic airway space (the posterior airway space, PAS). PATIENTS AND METHODS: A total of 43 patients with Angle Class II malocclusion were classified into two groups according to the appliance used for treatment: the functional mandibular advancer (FMA; n = 18) or the Herbst appliance (n = 25). Lateral cephalograms were taken of each patient at the start of functional jaw orthopedic treatment (time point T1) and at its completion (time point T2). Specific distances and angles were measured and analyzed in a cephalometric analysis. RESULTS: We observed major differences among the 43 patients in the depth of the posterior airway space during treatment with fixed appliances for Angle Class II correction. Regression analysis revealed that changes in sagittal and vertical positions had different effects on the depth of specific PAS sections: increases in anterior facial height are associated proportionately with increases in PAS width, particularly in the upper region. On the other hand, increases in posterior facial height and in the mandible's forward displacement correlated inversely to the decreases in depth, particularly in the central and lower PAS regions. The two treatment appliances (FMA, Herbst appliance) had the same effects on extrathoracic airway depth. CONCLUSIONS: Analyses of lateral cephalograms indicate that Angle Class II treatment with fixed appliances does not prevent sleep apnea in patients at risk. Nevertheless, this study does not permit absolutely reliable conclusions about the dimensions of the pharyngeal airway space. As the lateral cephalogram provides good images of structures in the midsagittal plane but is incapable of imaging the transverse dimension, there is an automatic lack of information concerning the precise width and volume of the extrathoracic airway space.

Effects of Class II treatment with a banded Herbst appliance on root lengths in the posterior dentition.

INTRODUCTION: The aim of this study was a retrospective analysis of posterior teeth in 20 adolescents and young adults with Angle Class II Division 1 malocclusion treated with a banded Herbst appliance to check for inhibited root development and apical root resorption. METHODS: Panoramic radiographs were taken of every patient at the beginning and after the completion of functional orthopedic treatment. The magnification of the area of the posterior teeth was determined individually for every radiograph. Then the vestibular lengths of the molars and premolars were assessed metrically. To assess root-length changes, the difference between the lengths of the teeth before and after treatment was calculated. RESULTS: After treatment with a banded Herbst appliance, tooth length generally increased in the area of the anchorage. But there was a tendency toward root-length decrease in teeth immediately adjacent to the Herbst fittings in the vestibular roots of both the maxillary first molars (distovestibular, -0.02 ± 2.31 mm; mesiovestibular, -0.06 ± 2.05 mm) and the mandibular first premolars (-0.46 ± 3.53 mm). CONCLUSIONS: The banded Herbst appliance might deliver unphysiologic forces to immediate anchor teeth, thereby exposing these to a higher risk of root resorption than in other teeth incorporated into the anchorage either directly via bands or indirectly via occlusal or approximal contacts. Looking at uncompromised root morphology in the area of the anchorage, we believe that early treatment with fixed functional appliances can be predicted to yield better outcomes than late treatment because of the higher biologic tolerance expressed by teeth with an apical latency.

From 2D slices to 3D volumes: image based reconstruction and morphological characterization of hippocampal cells on charged and uncharged surfaces using FIB/SEM serial sectioning.

3D imaging at a subcellular resolution is a powerful tool in the life sciences to investigate cells and their interactions with native tissues or artificial objects. While a tomographic experimental setup achieving a sufficient structural resolution can be established with either X-rays or electrons, the use of electrons is usually limited to very thin samples in transmission electron microscopy due to the poor penetration depths of electrons. The combination of a serial sectioning approach and scanning electron microscopy in state of the art dual beam experimental setups therefore offers a means to image highly resolved spatial details using a focused ion beam for slicing and an electron beam for imaging. The advantage of this technique over X-ray µCT or X-ray microscopy attributes to the fact that absorption is not a limiting factor in imaging and therefore even strong absorbing structures can be spatially reconstructed with a much higher possible resolution. This approach was used in this study to elucidate the effect of an electric potential on the morphology of cells from a hippocampal cell line (HT22) deposited on gold microelectrodes. While cells cultivated on two different controls (gold and polymer substrates) did show the expected stretched morphology, cells on both the anode and the cathode differed significantly. Cells deposited on the anode part of the electrode exhibited the most extreme deviation, being almost spherical and showed signs of chromatin condensation possibly indicating cell death. Furthermore, EDX was used as supplemental methodology for combined chemical and structural analyses.

Going beyond histology. Synchrotron micro-computed tomography as a methodology for biological tissue characterization: from tissue morphology to individual cells.

Current light microscopic methods such as serial sectioning, confocal microscopy or multiphoton microscopy are severely limited in their ability to analyse rather opaque biological structures in three dimensions, while electron optical methods offer either a good three-dimensional topographic visualization (scanning electron microscopy) or high-resolution imaging of very thin samples (transmission electron microscopy). However, sample preparation commonly results in a significant alteration and the destruction of the three-dimensional integrity of the specimen. Depending on the selected photon energy, the interaction between X-rays and biological matter provides semi-transparency of the specimen, allowing penetration of even large specimens. Based on the projection-slice theorem, angular projections can be used for tomographic imaging. This method is well developed in medical and materials science for structure sizes down to several micrometres and is considered as being non-destructive. Achieving a spatial and structural resolution that is sufficient for the imaging of cells inside biological tissues is difficult due to several experimental conditions. A major problem that cannot be resolved with conventional X-ray sources are the low differences in density and absorption contrast of cells and the surrounding tissue. Therefore, X-ray monochromatization coupled with a sufficiently high photon flux and coherent beam properties are key requirements and currently only possible with synchrotron-produced X-rays. In this study, we report on the three-dimensional morphological characterization of articular cartilage using synchrotron-generated X-rays demonstrating the spatial distribution of single cells inside the tissue and their quantification, while comparing our findings to conventional histological techniques.

Cyclooxygenases (COX-1 and COX-2) for tissue engineering of articular cartilage--from a developmental model to first results of tissue and scaffold expression.

Tissue engineering of articular cartilage remains an ongoing challenge. Since tissue regeneration recapitulates ontogenetic processes the growth plate can be regarded as an innovative model to target suitable signalling molecules and growth factors for the tissue engineering of cartilage. In the present study we analysed the expression of cyclooxygenases (COX) in a short-term chondrocyte culture in gelatin-based scaffolds and in articular cartilage of rats and compared it with that in the growth plate. Our results demonstrate the strong cellular expression of COX-1 but only a focal weak expression of COX-2 in the seeded scaffolds. Articular cartilage of rats expresses homogeneously COX-1 and COX-2 with the exception of the apical cell layer. Our findings indicate a functional role of COX in the metabolism of articular chondrocytes. The expression of COX in articular cartilage and in the seeded scaffolds opens interesting perspectives to improve the proliferation and differentiation of chondrocytes in scaffold materials by addition of specific receptor ligands of the COX system.