Three-Dimensional Bioprinting of Ovine Aortic Valve Endothelial and Interstitial Cells for the Development of Multicellular Tissue Engineered Tissue Constructs.

To investigate the pathogenic mechanisms of calcified aortic valve disease (CAVD), it is necessary to develop a new three-dimensional model that contains valvular interstitial cells (VIC) and valvular endothelial cells (VEC). For this purpose, ovine aortic valves were processed to isolate VIC and VEC that were dissolved in an alginate/gelatin hydrogel. A 3D-bioprinter (3D-Bioplotter® Developer Series, EnvisionTec, Gladbeck, Germany) was used to print cell-laden tissue constructs containing VIC and VEC which were cultured for up to 21 days. The 3D-architecture, the composition of the culture medium, and the hydrogels were modified, and cell viability was assessed. The composition of the culture medium directly affected the cell viability of the multicellular tissue constructs. Co-culture of VIC and VEC with a mixture of 70% valvular interstitial cell and 30% valvular endothelial cell medium components reached the cell viability best tested with about 60% more living cells compared to pure valvular interstitial cell medium (p = 0.02). The tissue constructs retained comparable cell viability after 21 days (p = 0.90) with different 3D-architectures, including a "sandwich" and a "tube" design. Good long-term cell viability was confirmed even for thick multilayer multicellular tissue constructs. The 3D-bioprinting of multicellular tissue constructs with VEC and VIC is a successful new technique to design tissue constructs that mimic the structure of the native aortic valve for research applications of aortic valve pathologies.

3D-bioprinting of aortic valve interstitial cells: impact of hydrogel and printing parameters on cell viability.

Calcific aortic valve disease (CAVD) is a frequent cardiac pathology in the aging society. Although valvular interstitial cells (VICs) seem to play a crucial role, mechanisms of CAVD are not fully understood. Development of tissue-engineered cellular models by 3D-bioprinting may help to further investigate underlying mechanisms of CAVD. VIC were isolated from ovine aortic valves and cultured in Dulbecco's modified Eagle's Medium (DMEM). VIC of passages six to ten were dissolved in a hydrogel consisting of 2% alginate and 8% gelatin with a concentration of 2 × 106VIC ml-1. Cell-free and VIC-laden hydrogels were printed with an extrusion-based 3D-bioprinter (3D-Bioplotter®Developer Series, EnvisionTec, Gladbeck, Germany), cross-linked and incubated for up to 28 d. Accuracy and durability of scaffolds was examined by microscopy and cell viability was tested by cell counting kit-8 assay and live/dead staining. 3D-bioprinting of scaffolds was most accurate with a printing pressure ofP< 400 hPa, nozzle speed ofv< 20 mm s-1, hydrogel temperature ofTH= 37 °C and platform temperature ofTP= 5 °C in a 90° parallel line as well as in a honeycomb pattern. Dissolving the hydrogel components in DMEM increased VIC viability on day 21 by 2.5-fold compared to regular 0.5% saline-based hydrogels (p< 0.01). Examination at day 7 revealed dividing and proliferating cells. After 21 d the entire printed scaffolds were filled with proliferating cells. Live/dead cell viability/cytotoxicity staining confirmed beneficial effects of DMEM-based cell-laden VIC hydrogel scaffolds even 28 d after printing. By using low pressure printing methods, we were able to successfully culture cell-laden 3D-bioprinted VIC scaffolds for up to 28 d. Using DMEM-based hydrogels can significantly improve the long-term cell viability and overcome printing-related cell damage. Therefore, future applications 3D-bioprinting of VIC might enable the development of novel tissue engineered cellular 3D-models to examine mechanisms involved in initiation and progression of CAVD.

Degeneration of biological heart valve grafts in a rat model of superoxide dismutase-3 deficiency.

While oxidative stress is known as key element in the pathogenesis of atherosclerosis and calcific aortic valve disease, its role in the degeneration of biological cardiovascular grafts has not been clarified yet. Therefore, the present study aimed to examine the impact of oxidative stress on the degeneration of biological cardiovascular allografts in a standardized chronic implantation model realized in rats exhibiting superoxide dismutase 3 deficiency (SOD3(-) ). Rats with SOD3 loss-of-function mutation (n = 24) underwent infrarenal implantation of cryopreserved valved aortic conduits, while SOD3-competent recipients served as controls (n = 28). After a follow-up period of 4 or 12 weeks, comparative analyses addressed degenerative processes, hemodynamics, and evaluation of the oxidative stress model. SOD3(-) rats presented decreased circulating SOD activity (p = .0079). After 12 weeks, 58% of the implant valves in SOD3(-) rats showed regurgitation (vs. 31% in controls, p = .2377). Intima hyperplasia and chondro-osteogenic transformation contributed to progressive graft calcification (p = .0024). At 12 weeks, hydroxyapatite deposition (p = .0198) and the gene expression of runt-related transcription factor-2 (Runx2) (p = .0093) were significantly enhanced in group SOD3(-) . This study provides the first in vivo evidence that impaired systemic antioxidant activity contributes to biological cardiovascular graft degeneration.

Ventricular stabilization with a customized decellularized cardiac ECM-based scaffold after myocardial infarction alters gene expression in a rodent LAD-ligation model.

Objectives: Decellularized extracellular matrix (dECM) is increasingly used in a wide range of regenerative medicine applications and may also offer the potential to support injured myocardium. Here, we evaluated the myocardial gene expression pattern after myocardial infarction (MI) in a standardized rodent LAD-ligation model with and without ventricular stabilization with a customized, cardiac dECM-based scaffold (cdECM). Methods: MI was induced in male Wistar rats by standard LAD-ligation and confirmed 14 days post-intervention by echocardiographic parameters (FAS<40%). Cardiac ECM from donor rats was used to generate individual cdECM-scaffolds (tissue engineered myocardial sleeve, TEMS), which were epicardially implanted after confirmed MI for ventricular stabilization. After 4 and 8 weeks heart function was assessed by echocardiography, rats were sacrificed and explanted hearts were analyzed. In addition to histological analysis, standardized anterior left ventricular wall myocardial tissue samples were assessed by quantitative real-time PCR evaluating the specific gene expression pattern for immunomodulatory (IL-10, TGFBR2, TNFα), pro-angiogenic (VEGFA, FGF2, PGF, PDGFB), pro-survival (HGF, SDF1, IGF1, AKT1), remodeling-associated (TIMP1, MMP2, MMP9) and infarction-specific (NPPA, NPPB) markers. Results: Ventricular stabilization led to integration of the TEMS-scaffold into the myocardial scar with varying degrees of cellular infiltration, as well as significantly improved echocardiographic parameters demonstrating attenuation of maladaptive cardiac remodeling. Further, TEMS implantation after MI altered the myocardial gene expression pattern. Differences in gene expression were most striking after 4 weeks with significantly reduced expression of NPPA (0.36 ± 0.26 vs 0.75 ± 0.40; p < 0.05), NPPB (0.47 ± 0.25 vs 0.91 ± 0.429; p < 0.01), TGFBR2 (0.68 ± 0.16 vs 0.90 ± 0.14; p < 0.01) and PDGFB (0.81 ± 0.13 vs 1.06 ± 0.14; p < 0.01) as well as increased expression of IL-10 (5.93 ± 5.67 vs 1.38 ± 0.60; p < 0.05), PGF (1.48 ± 0.38 vs 1.09 ± 0.25; p < 0.05) and IGF1 (1.67 ± 0.70 vs 1.03 ± 0.42; p < 0.05). However, after 8 weeks differences in the gene expression patterns of remodeling-associated, and pro-angiogenic markers could still be observed between groups. Conclusion: Ventricular stabilization via TEMS implantation after MI did not only led to biological integration of the cdECM-scaffolds into the host tissue and improved functional cardiac parameters, but also altered 4 and 8 week gene expression of infarcted myocardium, possibly contributing to reducing chronic deteriorating effects while increasing the potential for myocardial regeneration.

Reproducible In Vitro Tissue Culture Model to Study Basic Mechanisms of Calcific Aortic Valve Disease: Comparative Analysis to Valvular Interstitials Cells.

The hallmarks of calcific aortic valve disease (CAVD), an active and regulated process involving the creation of calcium nodules, lipoprotein accumulation, and chronic inflammation, are the significant changes that occur in the composition, organization, and mechanical properties of the extracellular matrix (ECM) of the aortic valve (AV). Most research regarding CAVD is based on experiments using two-dimensional (2D) cell culture or artificially created three-dimensional (3D) environments of valvular interstitial cells (VICs). Because the valvular ECM has a powerful influence in regulating pathological events, we developed an in vitro AV tissue culture model, which is more closely able to mimic natural conditions to study cellular responses underlying CAVD. AV leaflets, isolated from the hearts of 6-8-month-old sheep, were fixed with needles on silicon rubber rings to achieve passive tension and treated in vitro under pro-degenerative and pro-calcifying conditions. The degeneration of AV leaflets progressed over time, commencing with the first visible calcified domains after 14 d and winding up with the distinct formation of calcium nodules, heightened stiffness, and clear disruption of the ECM after 56 d. Both the expression of pro-degenerative genes and the myofibroblastic differentiation of VICs were altered in AV leaflets compared to that in VIC cultures. In this study, we have established an easily applicable, reproducible, and cost-effective in vitro AV tissue culture model to study pathological mechanisms underlying CAVD. The valvular ECM and realistic VIC-VEC interactions mimic natural conditions more closely than VIC cultures or 3D environments. The application of various culture conditions enables the examination of different pathological mechanisms underlying CAVD and could lead to a better understanding of the molecular mechanisms that lead to VIC degeneration and AS. Our model provides a valuable tool to study the complex pathobiology of CAVD and can be used to identify potential therapeutic targets for slowing disease progression.

Coca-Cola®-Lyse eines Dünndarmileus durch intestinalen Phytobezoar.

Phytobezoars are rare causes for intestinal obstruction. Therapy options include surgical removal, enzyme treatment, endoscopic removal and lyis with Coca-Cola®. While there is ample literature about successful Coca-Cola®-lysis, little is to be found concerning intestinal beozars. In our case, neither surgical nor endoscopic treatment of the intestinal obstruction was possible, which is why we opted for a Coca-Cola®-lysis.

Influence of Laminin Coating on the Autologous In Vivo Recellularization of Decellularized Vascular Protheses.

Decellularization of non-autologous biological implants reduces the immune response against foreign tissue. Striving for in vivo repopulation of aortic prostheses with autologous cells, thereby improving the graft biocompatibility, we examined surface coating with laminin in a standardized rat implantation model. Detergent-decellularized aortic grafts from donor rats (n = 37) were coated with laminin and systemically implanted into Wistar rats. Uncoated implants served as controls. Implant re-colonization and remodeling were examined by scanning electron microscopy (n = 10), histology and immunohistology (n = 18). Laminin coating persisted over eight weeks. Two weeks after implantation, no relevant neoendothelium formation was observed, whereas it was covering the whole grafts after eight weeks, with a significant acceleration in the laminin group (p = 0.0048). Remarkably, the intima-to-media ratio, indicating adverse hyperplasia, was significantly diminished in the laminin group (p = 0.0149). No intergroup difference was detected in terms of medial recellularization (p = 0.2577). Alpha-smooth muscle actin-positive cells originating from the adventitial surface invaded the media in both groups to a similar extent. The amount of calcifying hydroxyapatite deposition in the intima and the media did not differ between the groups. Inflammatory cell markers (CD3 and CD68) proved negative in coated as well as uncoated decellularized implants. The coating of decellularized aortic implants with bioactive laminin caused an acceleration of the autologous recellularization and a reduction of the intima hyperplasia. Thereby, laminin coating seems to be a promising strategy to enhance the biocompatibility of tissue-engineered vascular implants.

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis.

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released from many cell types and play a pivotal role in regulating cell-cell communication and a diverse range of biological processes. Many different methods for the characterization of EVs have been described. However, most of these methods have the disadvantage that the preparation and characterization of the samples are very time-consuming, or it is extremely difficult to analyze specific markers of interest due to their small size and due to the lack of discrete populations. While methods for analysis of EVs have been considerably improved over the last decade, there is still no standardized method for characterization of single EVs. Here, we demonstrate a semi-automated method for characterization of single EVs by fluorescence-based nanoparticle-tracking analysis. The protocol that is presented addresses the common problem of many researchers in this field and provides the complete workflow for rapid isolation of EVs and characterization with PKH67, a general cell membrane linker, as well as with specific surface markers such as CD63, CD9, vimentin, and lysosomal-associated membrane protein 1 (LAMP-1). The presented results show a high level of reproducibility, as confirmed by other methods, such as Western blotting. In the conducted experiments, we exclusively used EVs isolated from human serum samples, but this method is also suitable for plasma or other body fluids and can be adjusted for characterization of EVs from cell culture supernatants. Irrespective of the future progress of research on EV biology, the protocol that is presented here provides a rapid and reliable method for rapid characterization of single EVs with specific markers.

Síndrome da Morte Súbita Infantil em Pelotas de 2006 a 2013: uma análise descritiva / Sudden Infant Death Syndrome in Pelotas from 2006 to 2013: a descriptive analysis

Introdução e objetivo: A Síndrome da Morte Súbita Infantil (SMSI) ocupa a oitava posição entre as causas de anos potenciais de vida perdidos e as primeiras posições como causa de mortalidade infantil pós-neonatal em países desenvolvidos. O presente estudo objetiva conhecer as características socioepidemiológicas das crianças que foram a óbito por SMSI no município de Pelotas. Métodos: Estudo observacional, retrospectivo, descritivo baseado nos dados da Secretaria Municipal de Saúde, coletados através da aplicação de Fichas de Investigação de óbitos padronizadas pelo Ministério da Saúde de todos os casos de SMSI que ocorreram do ano de 2006 a 2013 em Pelotas/RS. Resultados: Houve 37 óbitos registrados no período, o que representa um coefi ciente de mortalidade por SMSI de 1,5 por mil. A média de idade materna foi de 23,5 anos (dp=5,2), 29 (78%) eram fumantes e 23 (62%) concederam aleitamento materno exclusivo até a data do óbito, 28 (76%) tiverem seus bebês nascidos a termo. Dentre os 37 casos, 16 (43%) vieram a falecer com menos de 1 mês de vida, 26 (70%) dormiam junto aos pais e 23 (61%) em decúbito lateral, enquanto que apenas 2 (5%) em decúbito ventral e 16 (43%) dos casos de SMSI ocorreram durante o inverno. Conclusão: O presente estudo é o único que abrange tamanha amostra (37 casos) de SMSI na cidade de Pelotas, a qual apresenta um coefi ciente de mortalidade por essa patologia semelhante aos mais altos encontrados na literatura. Portanto, políticas públicas que visem à prevenção de SMSI em Pelotas são necessárias.

Introduction and aim: Sudden Infant Death Syndrome (SIDS) ranks eighth among the causes of potential years of life lost and is among the leading causes of post-neonatal infant mortality in developed countries. This study aimed to evaluate the social and epidemiological characteristics of children who died of SIDS in the city of Pelotas. Methods: An observational, retrospective, and descriptive study based on Municipal Health Department data collected by applying Research Sheets standardized by the Ministry of Health to all cases of SIDS occurring from 2006 to 2013 in Pelotas, South Brazil. Results: A total of 37 deaths were recorded in the studied period, placing the SIDS mortality rate at 1.5 per thousand. The mean maternal age was 23.5 years (SD = 5.2), 29 (78%) were smokers, 23 (62%) granted exclusive breastfeeding until the date of death, 28 (76%) had term infants. From the 37 cases, 16 (43%) died under 1 month of age, 26 (70%) were sleeping with their parents, and 23 (61%) in the lateral position, while only 2 (5%) in the prone position, and 16 cases (43%) of SIDS occurred during the winter. Conclusion: This study is the only one that covers such sample (37 cases) of SIDS in the city of Pelotas, whose mortality rate from this disorder is close to the highest in the literature. Therefore, public policies for the prevention of SIDS in Pelotas are required.

[Caffeine consumption during pregnancy and attention deficit hyperactivity disorder (ADHD): a systematic literature review]. / Consumo de cafeína durante a gestação e transtorno de déficit de atenção e hiperatividade (TDAH): uma revisão sistemática da literatura.

This aim of this study was to conduct a systematic literature review on the association between maternal caffeine intake during pregnancy and attention deficit hyperactivity disorder (ADHD) in childhood. The systematic multiple-stage literature search in PubMed, LILACS, BIREME, and PsycINFO was limited to research in human subjects and published in Portuguese, English, and Spanish. A total of 373 references were retrieved. Of these, only five met the study's objectives and were kept in the review. Most of the studies employed a longitudinal design, were conducted in developed countries, and were published in the last five years. Only one study found a positive association. Studies on caffeine consumption during pregnancy and ADHD are scarce, with conflicting results and several methodological difficulties such as lack of standardized outcome measures.

Consumo de cafeína durante a gestação e transtorno de déficit de atenção e hiperatividade (TDAH): uma revisão sistemática da literatura / Caffeine consumption during pregnancy and attention deficit hyperactivity disorder (ADHD): a systematic literature review / Consumo de cafeína durante el embarazo y trastorno por déficit de atención e hiperactividad (TDAH): una revisión sistemática de la literatura

O objetivo do presente estudo foi avaliar, mediante revisão sistemática da literatura, as evidências acerca da associação entre consumo materno de cafeína durante a gestação e transtorno de déficit de atenção e hiperatividade (TDAH) na infância. A busca na literatura ocorreu de forma sistemática, em múltiplas etapas, nas bases PubMed, LILACS, BIREME e PsycINFO, com limites para artigos publicados em português, inglês e espanhol, realizados em humanos. Foram encontradas 373 referências. Dessas, somente cinco foram mantidas, por atenderem ao objetivo deste estudo. Os cinco trabalhos foram realizados em países desenvolvidos; a maioria utilizou delineamento longitudinal e foi publicada nos últimos cinco anos. Apenas um estudo encontrou associação positiva. Estudos sobre o consumo de cafeína na gestação e TDAH são escassos, com resultados controversos e se deparam com várias dificuldades metodológicas, como falta de padronização na definição do desfecho.

This aim of this study was to conduct a systematic literature review on the association between maternal caffeine intake during pregnancy and attention deficit hyperactivity disorder (ADHD) in childhood. The systematic multiple-stage literature search in PubMed, LILACS, BIREME, and PsycINFO was limited to research in human subjects and published in Portuguese, English, and Spanish. A total of 373 references were retrieved. Of these, only five met the study's objectives and were kept in the review. Most of the studies employed a longitudinal design, were conducted in developed countries, and were published in the last five years. Only one study found a positive association. Studies on caffeine consumption during pregnancy and ADHD are scarce, with conflicting results and several methodological difficulties such as lack of standardized outcome measures.

El objetivo de este estudio fue evaluar, a través de una revisión sistemática de la literatura, evidencias sobre la asociación entre el consumo de cafeína durante el embarazo y el trastorno por déficit de atención e hiperactividad (TDAH) en la infancia. Se realizó una búsqueda sistemática en la literatura, por etapas múltiples, en PubMed, LILACS BIREME y PsycINFO, limitándose a artículos publicados en portugués, inglés y español, realizados en estudios sobre humanos. Fueron localizadas 373 referencias. De ellas, apenas se mantuvieron cinco, por cumplir el objetivo de este estudio. Los estudios se realizaron en países desarrollados; el diseño longitudinal fue el más utilizado y se trata de publicaciones de los últimos cinco años. Sólo un estudio encontró asociación positiva. Los estudios sobre el consumo de cafeína durante el embarazo y el TDAH son escasos, con resultados controvertidos, y enfrentan varias dificultades metodológicas, como la no estandarización de la evaluación del resultado.