In vitro evaluation of demineralized freeze-dried bone allograft in combination with enamel matrix derivative

BACKGROUND Preclinical and clinical studies suggest that a combination of enamel matrix derivative (EMD) with demineralized freeze-dried bone allograft (DFDBA) may improve periodontal wound healing and regeneration. To date, no single study has characterized the effects of this combination on in vitro cell behavior. The aim of this study is to test the ability of EMD to adsorb to the surface of DFDBA particles and determine the effect of EMD coating on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells. METHODS DFDBA particles were precoated with EMD or human blood and analyzed for protein adsorption patterns via scanning electron microscopy. Cell attachment and proliferation were quantified using a commercial assay. Cell differentiation was analyzed using real-time polymerase chain reaction for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen 1α1, and mineralization was assessed using alizarinred staining. RESULTS Analysis of cell attachment revealed no significant differences among control, blood-coated, and EMD-coated DFDBA particles. EMD significantly increased cell proliferation at 3 and 5 days after seeding for both osteoblasts and PDL cells compared to control and blood-coated samples. Moreover, there were significantly higher messenger ribonucleic acid levels of osteogenic differentiation markers, including collagen 1α1, alkaline phosphatase, and osteocalcin, in osteoblasts and PDL cells cultured on EMD-coated DFDBA particles at 3, 7, and 14 days. CONCLUSION The results suggest that the addition of EMD to DFDBA particles may influence periodontal regeneration by stimulating PDL cell and osteoblast proliferation and differentiation.

Immunotoxic effects of environmental toxicants in fish - how to assess them?

Numerous environmental chemicals, both long-known toxicants such as persistent organic pollutants as well as emerging contaminants such as pharmaceuticals, are known to modulate immune parameters of wildlife species, what can have adverse consequences for the fitness of individuals including their capability to resist pathogen infections. Despite frequent field observations of impaired immunocompetence and increased disease incidence in contaminant-exposed wildlife populations, the potential relevance of immunotoxic effects for the ecological impact of chemicals is rarely considered in ecotoxicological risk assessment. A limiting factor in the assessment of immunotoxic effects might be the complexity of the immune system what makes it difficult (1) to select appropriate exposure and effect parameters out of the many immune parameters which could be measured, and (2) to evaluate the significance of the selected parameters for the overall fitness and immunocompetence of the organism. Here, we present - on the example of teleost fishes - a brief discussion of how to assess chemical impact on the immune system using parameters at different levels of complexity and integration: immune mediators, humoral immune effectors, cellular immune defenses, macroscopical and microscopical responses of lymphoid tissues and organs, and host resistance to pathogens. Importantly, adverse effects of chemicals on immunocompetence may be detectable only after immune system activation, e.g., after pathogen challenge, but not in the resting immune system of non-infected fish. Current limitations to further development and implementation of immunotoxicity assays and parameters in ecotoxicological risk assessment are not primarily due to technological constraints, but are related from insufficient knowledge of (1) possible modes of action in the immune system, (2) the importance of intra- and inter-species immune system variability for the response against chemical stressors, and (3) deficits in conceptual and mechanistic assessment of combination effects of chemicals and pathogens.

Influence of enamel matrix derivative on cells at different maturation stages of differentiation

Enamel matrix derivative (EMD), a porcine extract harvested from developing porcine teeth, has been shown to promote formation of new cementum, periodontal ligament and alveolar bone. Despite its widespread use, an incredibly large variability among in vitro studies has been observed. The aim of the present study was to determine the influence of EMD on cells at different maturation stages of osteoblast differentiation by testing 6 cell types to determine if cell phenotype plays a role in cell behaviour following treatment with EMD. Six cell types including MC3T3-E1 pre-osteoblasts, rat calvarial osteoblasts, human periodontal ligament (PDL) cells, ROS cells, MG63 cells and human alveolar osteoblasts were cultured in the presence or absence of EMD and proliferation rates were quantified by an MTS assay. Gene expression of collagen1(COL1), alkaline phosphate(ALP) and osteocalcin(OC) were investigated by real-time PCR. While EMD significantly increased cell proliferation of all cell types, its effect on osteoblast differentiation was more variable. EMD significantly up-regulated gene expression of COL1, ALP and OC in cells early in their differentiation process when compared to osteoblasts at later stages of maturation. Furthermore, the effect of cell passaging of primary human PDL cells (passage 2 to 15) was tested in response to treatment with EMD. EMD significantly increased cell proliferation and differentiation of cells at passages 2-5 however had completely lost their ability to respond to EMD by passages 10+. The results from the present study suggest that cell stimulation with EMD has a more pronounced effect on cells earlier in their differentiation process and may partially explain why treatment with EMD primarily favors regeneration of periodontal defects (where the periodontal ligament contains a higher number of undifferentiated progenitor cells) over regeneration of pure alveolar bone defects containing no periodontal ligament and a more limited number of osteoprogenitor cells.

Sphingosine 1-phosphate (S1P) induces COX-2 expression and PGE2 formation via S1P receptor 2 in renal mesangial cells

Understanding the mechanisms of sphingosine 1-phosphate (S1P)-induced cyclooxygenase (COX)-2 expression and prostaglandin E2 (PGE2) formation in renal mesangial cells may provide potential therapeutic targets to treat inflammatory glomerular diseases. Thus, we evaluated the S1P-dependent signaling mechanisms which are responsible for enhanced COX-2 expression and PGE2 formation in rat mesangial cells under basal conditions. Furthermore, we investigated whether these mechanisms are operative in the presence of angiotensin II (Ang II) and of the pro-inflammatory cytokine interleukin-1β (IL-1β). Treatment of rat and human mesangial cells with S1P led to concentration-dependent enhanced expression of COX-2. Pharmacological and molecular biology approaches revealed that the S1P-dependent increase of COX-2 mRNA and protein expression was mediated via activation of S1P receptor 2 (S1P2). Further, inhibition of Gi and p42/p44 MAPK signaling, both downstream of S1P2, abolished the S1P-induced COX-2 expression. In addition, S1P/S1P2-dependent upregulation of COX-2 led to significantly elevated PGE2 levels, which were further potentiated in the presence of Ang II and IL-1β. A functional consequence downstream of S1P/S1P2 signaling is mesangial cell migration that is stimulated by S1P. Interestingly, inhibition of COX-2 by celecoxib and SC-236 completely abolished the migratory response. Overall, our results demonstrate that extracellular S1P induces COX-2 expression via activation of S1P2 and subsequent Gi and p42/p44 MAPK-dependent signaling in renal mesangial cells leading to enhanced PGE2 formation and cell migration that essentially requires COX-2. Thus, targeting S1P/S1P2 signaling pathways might be a novel strategy to treat renal inflammatory diseases.

Right ventricle best predicts the race performance in amateur ironman athletes

PURPOSE The ironman (IM) triathlon is a popular ultraendurance competition, consisting of 3.8 km of swimming, 180.2 km of cycling, and 42.2 km of running. The aim of this study was to investigate the predictors of IM race time, comparing echocardiographic findings, anthropometric measures, and training characteristics. METHODS Amateur IM athletes (ATHL) participating in the Zurich IM race in 2010 were included. Participants were examined the day before the race by a comprehensive echocardiographic examination. Moreover, anthropometric measurements were obtained the same day. During the 3 months before the race, each IM-ATHL maintained a detailed training diary. Recorded data were related to total IM race time. RESULTS Thirty-eight IM finishers (mean ± SD age = 38 ± 9 yr, 32 men [84%]) were evaluated. Total race time was 684 ± 89 min (mean ± SD). For right ventricular fractional area change (45% ± 7%, Spearman ρ = -0.33, P = 0.05), a weak correlation with race time was observed. Race performance exhibited stronger associations with percent body fat (15.2 ± 5.6%, ρ = 0.56, P = 0.001), speed in running training (11.7 ± 1.2 km · h(-1), ρ = -0.52, P = 0.002), and left ventricular myocardial mass index (98 ± 24 g · m(-2), ρ = -0.42, P = 0.009). The strongest association was found between race time and right ventricular end-diastolic area (22 ± 4 cm2, ρ = -0.64, P < 0.0001). In multivariate analysis, right ventricular end-diastolic area (β = -16.7, 95% confidence interval = -27.3 to -6.1, P = 0.003) and percent body fat (β = 6.8, 95% confidence interval = 1.1-12.6, P = 0.02) were independently predictive of IM race time. CONCLUSIONS In amateur IM-ATHL, RV end-diastolic area and percent body fat were independently related to race performance. RV end-diastolic area was the strongest predictor of race time. The role of the RV in endurance exercise may thus be more important than previously thought and needs to be further studied.

Trigger activity more than three years after left atrial linear ablation without pulmonary vein isolation in patients with atrial fibrillation.

OBJECTIVES The aim of this study was to analyze trigger activity in the long-term follow-up after left atrial (LA) linear ablation. BACKGROUND Interventional strategies for curative treatment of atrial fibrillation (AF) are targeted at the triggers and/or the maintaining substrate. After substrate modification using nonisolating linear lesions, the activity of triggers is unknown. METHODS With the LA linear lesion concept, 129 patients were treated using intraoperative ablation with minimal invasive surgical techniques. Contiguous radiofrequency energy-induced lesion lines involving the mitral annulus and the orifices of the pulmonary veins without isolation were placed under direct vision. RESULTS After a mean follow-up of 3.6 +/- 0.4 years, atrial ectopy, atrial runs, and reoccurrence of AF episodes were analyzed by digital 7-day electrocardiograms in 30 patients. Atrial ectopy was present in all patients. Atrial runs were present in 25 of 30 patients (83%), with a median number of 9 runs per patient/week (range 1 to 321) and a median duration of 1.2 s/run (range 0.7 to 25), without a significant difference in atrial ectopy and atrial runs between patients with former paroxysmal (n = 17) or persistent AF (n = 13). Overall, 87% of all patients were completely free from AF without antiarrhythmic drugs. CONCLUSIONS A detailed rhythm analysis late after specific LA linear lesion ablation shows that trigger activity remains relatively frequent but short and does not induce AF episodes in most patients. The long-term success rate of this concept is high in patients with paroxysmal or persistent AF.

Differentiating atrioventricular nodal reentrant tachycardia from tachycardia via concealed accessory pathway.

Studies analyzing the diagnostic value of 12-lead electrocardiographic criteria differentiating slow-fast atrioventricular nodal reentrant tachycardia (AVNRT) from atrioventricular reentrant tachycardia (AVRT) due to concealed accessory pathway have shown inconsistent results. In 97 patients (50 with AVNRT, 47 with AVRT) 12-lead electrocardiograms (ECGs) were recorded during sinus rhythm and tachycardia (QRS <120 ms). The ECGs were blinded for diagnosis and patient and analyzed independently by 2 electrophysiologists. The studied criteria differentiating AVNRT from AVRT included pseudo-r'/S, the presence of a retrograde P wave, RP interval, ST-segment depression >/=2 mm with the number and location of the affected leads, QRS amplitude, and cycle length alternans.

Outflow tract tachycardia with R/S transition in lead V3: six different anatomic approaches for successful ablation.

OBJECTIVES The aim of this study was to analyze different anatomic mapping approaches for successful ablation of outflow tract tachycardia with R/S transition in lead V(3). BACKGROUND Idiopathic ventricular tachycardia can originate from different areas in the outflow tract, including the right and left ventricular endocardium, the epicardium, the pulmonary artery, and the aortic sinus of Valsalva. Although electrocardiographic criteria may be helpful in predicting the area of origin, sometimes the focus is complex to determine, especially when QRS transition in precordial leads is in V(3). METHODS We analyzed surface electrocardiograms of 33 successfully ablated patients with outflow tract tachycardia: 20 from the right ventricular outflow tract (RVOT) and 13 from different sites. The R/S transition was determined, and the different anatomic approaches needed for successful catheter ablation were studied. RESULTS Overall, R/S transition in lead V(3) was present in 19 (58%) of all patients. In these patients, mapping was started and successfully completed in the RVOT in 11 of 19 (58%) patients. The remaining eight patients with R/S transition in lead V(3) needed five additional anatomic accesses for successful ablation: from the left ventricular outflow tract (n = 3), aortic sinus of Valsalva (n = 2), coronary sinus (n = 1), the epicardium via pericardial puncture (n = 1), and the trunk of the pulmonary artery (n = 1), respectively. CONCLUSIONS A R/S transition in lead V(3) is common. In patients with outflow tract tachycardia with R/S transition in lead V(3), a stepwise endocardial and epicardial mapping through up to six anatomic approaches can lead to successful radiofrequency catheter ablation.

Topographic variability of the esophageal left atrial relation influencing ablation lines in patients with atrial fibrillation.

UNLABELLED Topography of the esophagus in atrial fibrillation ablation. INTRODUCTION The close anatomic relationship of the posterior wall of the left atrium (LA) and the thermosensitive esophagus creates a potential hazard in catheter ablation procedures. METHODS AND RESULTS In 30 patients (pts) with atrial fibrillation (AF) undergoing catheter ablation, we prospectively studied the course and contact of the esophagus in relation to LA and the topographic proximity to ablation lines encircling the right-sided and left-sided pulmonary veins (PV) as well as to the posterior line connecting the encircling lines using the electromagnetic mapping system for reconstruction of LA and for tagging of the esophagus. This new technique of anatomic tagging of the esophagus was validated against the CT scan as a standard imaging procedure. The esophageal course was highly variable, extending from courses in direct vicinity to the left- or right-sided PV as well as in the midportion of the posterior LA. In order to avoid energy application in direct proximity to the esophagus, adjustments of the left and right PV encircling lines were necessary in 14/30 pts (47%) and 3/30 (10%). In 30 pts (100%), the mid- to inferior areas of the posterior LA revealed contact with the esophagus. Therefore, posterior and inferior linear ablation lines were abandoned and shifted to superior in 29 pts (97%). CONCLUSIONS Anatomic tagging of esophagus revealed a highly variable proximity to different areas of the posterior LA suggesting individual adjustment of encircling and linear ablation lines in AF ablation procedures to avoid the life threatening complication of esophagus perforation.

Paraseptal accessory pathway in Wolff-Parkinson- White-Syndrom: ablation from the right, from the left or within the coronary sinus/middle cardiac vein?

AIMS In 1999 the consensus statement "living anatomy of the atrioventricular junctions" was published. With that new nomenclature the former posteroseptal accessory pathway (APs) are termed paraseptal APs. The aim of this study was to identify ECG features of manifest APs located in this complex paraseptal space. METHODS AND RESULTS ECG characteristics of all patients who underwent radiofrequency ablation of an AP during a 3 year period were analyzed. Of the 239 patients with one or more APs, 30 patients had a paraseptal AP with preexcitation. Compared to APs within the coronary sinus (CS) or the middle cardiac vein (MCV) the right sided paraseptal APs significantly more often showed an isoelectric delta wave in lead II and/or a negative delta wave in aVR. The left sided paraseptal APs presented a negative delta wave in II significantly more often compared to the right sided APs. CONCLUSIONS According to the site of radiofrequency ablation, paraseptal APs are classified into 4 subgroups: paraseptal right, paraseptal left, inside the CS or inside the MCV. Subtle differences in preexcitation patterns of the delta wave as well as of the QRS complex exist. However, the definitive localization of APs remains reserved to the periinterventional intracardiac electrogram analysis.

Late recurrent arrhythmias after ablation of atrial fibrillation: incidence, mechanisms, and treatment.

OBJECTIVES The aim of the study was to determine the incidence of atrial flutter and other arrhythmia recurrences (other than atrial fibrillation [AF]) during long-term follow-up after left atrial substrate modification by percutaneous radiofrequency (RF) ablation of AF. BACKGROUND RF ablation is an effective treatment for patients with AF. However, late recurrent arrhythmias may complicate the patient's course. METHODS One hundred fifty consecutive patients with paroxysmal or persistent AF were included in this prospective study. The incidence of arrhythmia recurrences after AF ablation was analyzed during long-term follow-up using repetitive 7-day ECG recording. RESULTS In 28 of 150 patients (18.7%), stable regular arrhythmias other than AF were detected during follow-up. Left atrial flutter observed in 10 patients (6.7%) was treated by recompletion of the ablation lines in all 10 patients. Left atrial flutter was associated with recurrence of AF in all 10 patients. Nine of 10 patients (90%) were free from atrial flutter and 6 of 10 patients were free from AF after the second intervention. Typical right atrial flutter occurred in 10 patients (6.7%) and was treated successfully by percutaneous RF ablation without recurrence in all patients. Additionally, atrial flutter was documented during follow-up in 7 patients (4.7%); however, invasive electrophysiologic evaluation was not performed due to various reasons. CONCLUSIONS Left atrial flutter is a relevant complication after RF catheter ablation of AF and was always associated with AF recurrence in our study population. Prevention of left atrial flutter can be achieved by induction of ablation lines as continuous and transmural as possible. However, left atrial flutter that does occur late after ablation is amenable to interventional treatment with good prospects of success.

Stable secondary arrhythmias late after intraoperative radiofrequency ablation of atrial fibrillation: incidence, mechanism, and treatment.

INTRODUCTION Intraoperative radiofrequency (RF) ablation is an effective treatment of atrial fibrillation (AF). However, secondary arrhythmias late after ablation may complicate the patient's course. We report on the incidence, mechanisms, and treatment of gap-related atrial flutter and other secondary arrhythmias during long-term follow-up. METHODS AND RESULTS In 129 patients who underwent intraoperative RF ablation with placement of left atrial linear lesions using minimally invasive surgical techniques, secondary arrhythmias were analyzed during long-term follow-up (20 +/- 6 months). Transient atrial arrhythmias during the first 3 postoperative months were excluded. In 8 (6.2%) of 129 patients, sustained stable secondary arrhythmias were documented. Left atrial, gap-related atrial flutter was observed in 4 patients (3.1%). The flutter was treated by percutaneous RF ablation in 3 patients (2.3%) and with drugs in 1 patient (0.8%). In 2 patients (1.6%), right atrial isthmus-dependent atrial flutter occurred and was treated successfully by percutaneous RF ablation. In 2 patients (1.6%), ectopic right atrial tachycardias occurred and were treated with percutaneous RF ablation. CONCLUSION Late after intraoperative RF ablation of atrial fibrillation, three types of stable secondary arrhythmias were observed in 6% of patients: left atrial gap-related atrial flutter, right atrial isthmus-dependent atrial flutter, and ectopic atrial tachycardia. Gaps after intraoperative RF ablation due to noncontinuous or nontransmural linear lesions may lead to stable left atrial macroreentrant tachycardias, requiring new interventional therapy.