Extracellular vesicles selective capture by peptide-functionalized hollow fiber membranes.

Recently, membrane devices and processes have been applied for the separation and concentration of subcellular components such as extracellular vesicles (EVs), which play a diagnostic and therapeutic role in many pathological conditions. However, the separation and isolation of specific EV populations from other components found in biological fluids is still challenging. Here, we developed a peptide-functionalized hollow fiber (HF) membrane module to achieve the separation and enrichment of highly pure EVs derived from the culture media of human cardiac progenitor cells. The strategy is based on the functionalization of PSf HF membrane module with BPt, a peptide sequence able to bind nanovesicles characterized by highly curved membranes. HF membranes were modified by a nanometric coating with a copoly azide polymer to limit non-specific interactions and to enable the conjugation with peptide ligand by click chemistry reaction. The BPt-functionalized module was integrated into a TFF process to facilitate the design, rationalization, and optimization of EV isolation. This integration combined size-based transport of species with specific membrane sensing ligands. The TFF integrated BPt-functionalized membrane module demonstrated the ability to selectively capture EVs with diameter < 200 nm into the lumen of fibers while effectively removing contaminants such as albumin. The captured and released EVs contain the common markers including CD63, CD81, CD9 and syntenin-1. Moreover, they maintained a round shape morphology and structural integrity highlighting that this approach enables EVs concentration and purification with low shear stress. Additionally, it achieved the removal of contaminants such as albumin with high reliability and reproducibility, reaching a removal of 93%.

N-glycosylation is crucial for trafficking and stability of SLC3A2 (CD98).

The type II glycoprotein CD98 (SLC3A2) is a membrane protein with pleiotropic roles in cells, ranging from modulation of inflammatory processes, host-pathogen interactions to association with membrane transporters of the SLC7 family. The recent resolution of CD98 structure in complex with LAT1 showed that four Asn residues, N365, N381, N424, N506, harbour N-glycosylation moieties. Then, the role of N-glycosylation on CD98 trafficking and stability was investigated by combining bioinformatics, site-directed mutagenesis and cell biology approach. Single, double, triple and quadruple mutants of the four Asn exhibited altered electrophoretic mobility, with apparent molecular masses from 95 to 70 kDa. The quadruple mutant displayed a single band of 70 kDa corresponding to the unglycosylated protein. The presence in the membrane and the trafficking of CD98 were evaluated by a biotinylation assay and a brefeldin assay, respectively. Taken together, the results highlighted that the quadruple mutation severely impaired both the stability and the trafficking of CD98 to the plasma membrane. The decreased presence of CD98 at the plasma membrane, correlated with a lower presence of LAT1 (SLC7A5) and its transport activity. This finding opens new perspectives for human therapy. Indeed, the inhibition of CD98 trafficking would act synergistically with LAT1 inhibitors that are under clinical trial for anticancer therapy.

Membrane and Membrane Bioreactors Applied to Health and Life Sciences.

The interest in membranes and membrane bioreactors for health and life sciences is rapidly growing thanks to their wide applications in advanced therapies and biotechnologies [...].

5-fluorouracil treatment of patient-derived scaffolds from colorectal cancer reveal clinically critical information.

BACKGROUND: Colorectal cancer is a commonly diagnosed cancer worldwide. Unfortunately, many patients do not respond to standard chemotherapy treatments and develop disease relapse and metastases. Besides cancer cell specific genetic changes, heterogeneity in the tumor microenvironment contribute to the clinical presentation of the disease and can potentially also influence drug resistance. By using a recently developed patient-derived scaffold method monitoring how a standardized reporter cancer cell line adapts to various microenvironments treated with chemotherapy, we wanted to clarify how individual patient specific microenvironments influence the chemotherapy response in colorectal cancer. METHODS: Surgically resected colorectal cancer specimens from 89 patients were decellularized to produce patient-derived scaffold, which were seeded with HT29 cells, cultured for 3 weeks, and treated with 5-fluorouracil. Gene expression changes of adapted and treated HT29 cells were monitored by qPCR and compared with clinical parameters including disease-free survival. RESULTS: The effects of 5-fluorouracil treatment varied between different patient-derived scaffold, but generally induced a reduced expression of proliferation genes and increased expression of pluripotency and epithelial-to-mesenchymal transition genes. Interestingly, patient-derived scaffold cultures obtained from patients with disease recurrences showed a significantly less pronounced anti-proliferative effect of 5-fluorouracil and more pronounced increase of pluripotency, with MKI67 and POU5F1 being among the most significant genes linked to disease relapse in colorectal cancer. CONCLUSIONS: Colorectal patient-derived scaffold can decode clinically relevant tumor microenvironmental influence of 5-fluorouracil treatment effects opening up for optimized precision medicine in colorectal cancer treatment.

Hollow Fiber and Nanofiber Membranes in Bioartificial Liver and Neuronal Tissue Engineering.

To date, the creation of biomimetic devices for the regeneration and repair of injured or diseased tissues and organs remains a crucial challenge in tissue engineering. Membrane technology offers advanced approaches to realize multifunctional tools with permissive environments well-controlled at molecular level for the development of functional tissues and organs. Membranes in fiber configuration with precisely controlled, tunable topography, and physical, biochemical, and mechanical cues, can direct and control the function of different kinds of cells toward the recovery from disorders and injuries. At the same time, fiber tools also provide the potential to model diseases in vitro for investigating specific biological phenomena as well as for drug testing. The purpose of this review is to present an overview of the literature concerning the development of hollow fibers and electrospun fiber membranes used in bioartificial organs, tissue engineered constructs, and in vitro bioreactors. With the aim to highlight the main biomedical applications of fiber-based systems, the first part reviews the fibers for bioartificial liver and liver tissue engineering with special attention to their multifunctional role in the long-term maintenance of specific liver functions and in driving hepatocyte differentiation. The second part reports the fiber-based systems used for neuronal tissue applications including advanced approaches for the creation of novel nerve conduits and in vitro models of brain tissue. Besides presenting recent advances and achievements, this work also delineates existing limitations and highlights emerging possibilities and future prospects in this field.

The Effect of Hypoxic and Normoxic Culturing Conditions in Different Breast Cancer 3D Model Systems.

The field of 3D cell cultures is currently emerging, and material development is essential in striving toward mimicking the microenvironment of a native tissue. By using the response of reporter cells to a 3D environment, a comparison between materials can be assessed, allowing optimization of material composition and microenvironment. Of particular interest, the response can be different in a normoxic and hypoxic culturing conditions, which in turn may alter the conclusion regarding a successful recreation of the microenvironment. This study aimed at determining the role of such environments to the conclusion of a better resembling cell culture model to native tissue. Here, the breast cancer cell line MCF7 was cultured in normoxic and hypoxic conditions on patient-derived scaffolds and compared at mRNA and protein levels to cells cultured on 3D printed scaffolds, Matrigel, and conventional 2D plastics. Specifically, a wide range of mRNA targets (40), identified as being regulated upon hypoxia and traditional markers for cell traits (cancer stem cells, epithelial-mesenchymal transition, pluripotency, proliferation, and differentiation), were used together with a selection of corresponding protein targets. 3D cultured cells were vastly different to 2D cultured cells in gene expression and protein levels on the majority of the selected targets in both normoxic and hypoxic culturing conditions. By comparing Matrigel and 3DPS-cultured cells to cells cultured on patient-derived scffolds, differences were also noted along all categories of mRNA targets while specifically for the GLUT3 protein. Overall, cells cultured on patient-derived scaffolds closely resembled cells cultured on 3D printed scaffolds, contrasting 2D and Matrigel-cultured cells, regardless of a normoxic or hypoxic culturing condition. Thus, these data support the use of either a normoxic or hypoxic culturing condition in assays using native tissues as a blueprint to optimize material composition.

Patient-derived scaffolds as a drug-testing platform for endocrine therapies in breast cancer.

Three-dimensional cell culture platforms based on decellularised patient-based microenvironments provide in vivo-like growth conditions allowing cancer cells to interact with intact structures and components of the surrounding tissue. A patient-derived scaffold (PDS) model was therefore evaluated as a testing platform for the endocrine therapies (Z)-4-Hydroxytamoxifen (4OHT) and fulvestrant as well as the CDK4/6-inhibitor palbociclib, monitoring the treatment responses in breast cancer cell lines MCF7 and T47D adapted to the patient-based microenvironments. MCF7 cells growing in PDSs showed increased resistance to 4OHT and fulvestrant treatment (100- and 20-fold) compared to 2D cultures. Quantitative PCR analyses of endocrine treated cancer cells in PDSs revealed upregulation of pluripotency markers further supported by increased self-renewal capacity in sphere formation assays. When comparing different 3D growth platforms including PDS, matrigel, gelatin sponges and 3D-printed hydrogels, 3D based cultures showed slightly varying responses to fulvestrant and palbociclib whereas PDS and matrigel cultures showed more similar gene expression profiles for 4OHT treatment compared to the other platforms. The results support that the PDS technique maximized to provide a multitude of smaller functional PDS replicates from each primary breast cancer, is an up-scalable patient-derived drug-testing platform available for gene expression profiling and downstream functional assays.

Nano- and Micro-Porous Chitosan Membranes for Human Epidermal Stratification and Differentiation.

The creation of partial or complete human epidermis represents a critical aspect and the major challenge of skin tissue engineering. This work was aimed at investigating the effect of nano- and micro-structured CHT membranes on human keratinocyte stratification and differentiation. To this end, nanoporous and microporous membranes of chitosan (CHT) were prepared by phase inversion technique tailoring the operational parameters in order to obtain nano- and micro-structured flat membranes with specific surface properties. Microporous structures with different mean pore diameters were created by adding and dissolving, in the polymeric solution, polyethylene glycol (PEG Mw 10,000 Da) as porogen, with a different CHT/PEG ratio. The developed membranes were characterized and assessed for epidermal construction by culturing human keratinocytes on them for up to 21 days. The overall results demonstrated that the membrane surface properties strongly affect the stratification and terminal differentiation of human keratinocytes. In particular, human keratinocytes adhered on nanoporous CHT membranes, developing the structure of the corneum epidermal top layer, characterized by low thickness and low cell proliferation. On the microporous CHT membrane, keratinocytes formed an epidermal basal lamina, with high proliferating cells that stratified and differentiated over time, migrating along the z axis and forming a multilayered epidermis. This strategy represents an attractive tissue engineering approach for the creation of specific human epidermal strata for testing the effects and toxicity of drugs, cosmetics and pollutants.

Optimized alginate-based 3D printed scaffolds as a model of patient derived breast cancer microenvironments in drug discovery.

The cancer microenvironment influences tumor progression and metastasis and is pivotal to consider when designingin vivo-like cancer models. Current preclinical testing platforms for cancer drug development are mainly limited to 2D cell culture systems that poorly mimic physiological environments and traditional, low throughput animal models. The aim of this work was to produce a tunable testing platform based on 3D printed scaffolds (3DPS) with a simple geometry that, by extracellular components and response of breast cancer reporter cells, mimics patient-derived scaffolds (PDS) of breast cancer. Here, the biocompatible polysaccharide alginate was used as base material to generate scaffolds consisting of a 3D grid containing periostin and hydroxyapatite. Breast cancer cell lines (MCF7 and MDA-MB-231) produced similar phenotypes and gene expression levels of cancer stem cell, epithelial-mesenchymal transition, differentiation and proliferation markers when cultured on 3DPS and PDS, contrasting conventional 2D cultures. Importantly, cells cultured on 3DPS and PDS showed scaffold-specific responses to cytotoxic drugs (doxorubicin and 5-fluorouracil) that were different from 2D cultured cells. In conclusion, the data presented support the use of a tunable alginate-based 3DPS as a tumor model in breast cancer drug discovery.

Patient-derived scaffolds as a model of colorectal cancer.

BACKGROUND: Colorectal cancer is the second most common cause of cancer-related death worldwide and standardized therapies often fail to treat the more aggressive and progressive types of colorectal cancer. Tumor cell heterogeneity and influence from the surrounding tumor microenvironment (TME) contribute to the complexity of the disease and large variability in clinical outcomes. METHODS: To model the heterogeneous nature of colorectal cancer, we used patient-derived scaffolds (PDS), which were obtained via decellularization of surgically resected tumor material, as a growth substrate for standardized cell lines. RESULTS: After confirmation of native cell absence and validation of the structural and compositional integrity of the matrix, 89 colorectal PDS were repopulated with colon cancer cell line HT29. After 3 weeks of PDS culture, HT29 cells varied their gene and protein expression profiles considerably compared to 2D-grown HT29 cells. Markers associated with proliferation were consistently decreased, while markers associated with pluripotency were increased in PDS-grown cells compared to their 2D counterparts. When comparing the PDS-induced changes in HT29 cells with clinically relevant tumor information from individual patients, we observed significant associations between stemness/pluripotency markers and tumor location, and between epithelial-to-mesenchymal transition (EMT) markers and cancer mortality. Kaplan-Meier analysis revealed that low PDS-induced EMT correlated with worse cancer-specific survival. CONCLUSIONS: The colorectal PDS model can be used as a simplified personalized tool that can potentially reveal important diagnostic and pathophysiological information related to the TME.

Poly(ε-Caprolactone) Hollow Fiber Membranes for the Biofabrication of a Vascularized Human Liver Tissue.

The creation of a liver tissue that recapitulates the micro-architecture and functional complexity of a human organ is still one of the main challenges of liver tissue engineering. Here we report on the development of a 3D vascularized hepatic tissue based on biodegradable hollow fiber (HF) membranes of poly(ε-caprolactone) (PCL) that compartmentalize human hepatocytes on the external surface and between the fibers, and endothelial cells into the fiber lumen. To this purpose, PCL HF membranes were prepared by a dry-jet wet phase inversion spinning technique tailoring the operational parameters in order to obtain fibers with suitable properties. After characterization, the fibers were applied to generate a human vascularized hepatic unit by loading endothelial cells in their inner surface and hepatocytes on the external surface. The unit was connected to a perfusion system, and the morpho-functional behavior was evaluated. The results demonstrated the large integration of endothelial cells with the internal surface of individual PCL fibers forming vascular-like structures, and hepatocytes covered completely the external surface and the space between fibers. The perfused 3D hepatic unit retained its functional activity at high levels up to 18 days. This bottom-up tissue engineering approach represents a rational strategy to create relatively 3D vascularized tissues and organs.

Two-photon excitation of FluoVolt allows improved interrogation of transmural electrophysiological function in the intact mouse heart.

BACKGROUND & AIMS: Two-photon excitation of voltage sensitive dyes (VSDs) can measure rapidly changing electrophysiological signals deep within intact cardiac tissue with improved three-dimensional resolution along with reduced photobleaching and photo-toxicity compared to conventional confocal microscopy. Recently, a category of VSDs has emerged which records membrane potentials by photo-induced electron transfer. FluoVolt is a novel VSD in this category which promises fast response and a 25% fractional change in fluorescence per 100 mV, making it an attractive optical probe for action potential (AP) recordings within intact cardiac tissue. The purpose of this study was to characterize the fluorescent properties of FluoVolt as well as its utility for deep tissue imaging. METHODS: Discrete tissue layers throughout the left ventricular wall of isolated perfused murine hearts loaded with FluoVolt or di-4-ANEPPS were sequentially excited with two-photon microscopy. RESULTS: FluoVolt loaded hearts suffered significantly fewer episodes of atrio-ventricular block compared to di-4-ANEPPS loaded hearts, indicating comparatively low toxicity of FluoVolt in the intact heart. APs recorded with FluoVolt were characterized by a lower signal-to-noise ratio and a higher dynamic range compared to APs recorded with di-4-ANEPPS. Although both depolarization and repolarization parameters were similar in APs recorded with either dye, FluoVolt allowed deeper tissue excitation with improved three-dimensional resolution due to reduced out-of-focus fluorescence generation under two-photon excitation. CONCLUSION: Our results demonstrate several advantages of two-photon excitation of FluoVolt in functional studies in intact heart preparations, including reduced toxicity and improved fluorescent properties.

Double porous poly (Ɛ-caprolactone)/chitosan membrane scaffolds as niches for human mesenchymal stem cells.

In this paper, we developed membrane scaffolds to mimic the biochemical and biophysical properties of human mesenchymal stem cell (hMSC) niches to help direct self-renewal and proliferation providing to cells all necessary chemical, mechanical and topographical cues. The strategy was to create three-dimensional membrane scaffolds with double porosity, able to promote the mass transfer of nutrients and to entrap cells. We developed poly (Ɛ-caprolactone) (PCL)/chitosan (CHT) blend membranes consisting of double porous morphology: (i) surface macrovoids (big pores) which could be easily accessible for hMSCs invasion and proliferation; (ii) interconnected microporous network to transfer essential nutrients, oxygen, growth factors between the macrovoids and throughout the scaffolds. We varied the mean macrovoid size, effective surface area and surface morphology by varying the PCL/CHT blend composition (100/0, 90/10, 80/20, 70/30). Membranes exhibited macrovoids connected with each other through a microporous network; macrovoids size increased by increasing the CHT wt%. Cells adhered on the surfaces of PCL/CHT 100/0 and PCL/CHT 90/10 membranes, that are characterized by a high effective surface area and small macrovoids while PCL/CHT 80/20 and PCL/CHT 70/30 membranes with large macrovoids and low effective surface area entrapped cells inside macrovoids. The scaffolds were able to create a permissive environment for hMSC adhesion and invasion promoting viability and metabolism, which are important for the maintenance of cell integrity. We found a relationship between hMSCs proliferation and oxygen uptake rate with surface mean macrovoid size and effective surface area. The macrovoids enabled the cell invasion into the membrane and the microporosity ensured an adequate diffusive mass transfer of nutrients and metabolites, which are essential for the long-term maintenance of cell viability and functions.

Membrane bioreactor for investigation of neurodegeneration.

To gain a better understanding of neurodegeneration mechanisms and for preclinical evaluation of new therapeutics more accurate models of neuronal tissue are required. Our strategy was based on the implementation of advanced engineered system, like membrane bioreactor, in which neurons were cultured in the extracapillary space of poly(l-lactic acid) (PLLA) microtube array (MTA) membranes within a dynamic device designed to recapitulate specific microenvironment of living neuronal tissue. The high membrane permeability and the optimized fluid dynamic conditions created by PLLA-MTA membrane bioreactor provide a 3D low-shear stress environment fully controlled at molecular level with enhanced diffusion of nutrients and waste removal that successfully develops neuronal-like tissue. This neuronal membrane bioreactor was employed as in vitro model of ß-amyloid -induced toxicity associated to Alzheimer's disease, to test for the first time the potential neuroprotective effect of the isoflavone glycitein. Glycitein protected neurons from the events induced by ß-amyloid aggregation, such as the production of ROS, the activation of apoptotic markers and ensuring the viability and maintenance of cellular metabolic activity. PLLA-MTA membrane bioreactor has great potential as investigational tool in preclinical research, contributing to expand the available in vitro devices for drug screening.

Corrigendum: Characterization of Electrical Activity in Post-myocardial Infarction Scar Tissue in Rat Hearts Using Multiphoton Microscopy.

[This corrects the article DOI: 10.3389/fphys.2018.01454.].

High Intensity Interval Training Ameliorates Mitochondrial Dysfunction in the Left Ventricle of Mice with Type 2 Diabetes.

Both human and animal studies have shown mitochondrial and contractile dysfunction in hearts of type 2 diabetes mellitus (T2DM). Exercise training has shown positive effects on cardiac function, but its effect on the mitochondria have been insufficiently explored. The aim of this study was to assess the effect of exercise training on mitochondrial function in T2DM hearts. We divided T2DM mice (db/db) into a sedentary and an interval training group at 8 weeks of age and used heterozygote db/+ as controls. After 8 weeks of training, we evaluated mitochondrial structure and function, as well as the levels of mRNA and proteins involved in key metabolic processes from the left ventricle. db/db animals showed decreased oxidative phosphorylation capacity and fragmented mitochondria. Mitochondrial respiration showed a blunted response to Ca2+ along with reduced protein levels of the mitochondrial calcium uniporter. Exercise training ameliorated the reduced oxidative phosphorylation in complex (C) I + II, CII and CIV, but not CI or Ca2+ response. Mitochondrial fragmentation was partially restored. mRNA levels of isocitrate, succinate and oxoglutarate dehydrogenase were increased in db/db mice and normalized by exercise training. Exercise training induced an upregulation of two transcripts of peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC1α1 and PGC1α4) previously linked to endurance training adaptations and strength training adaptations, respectively. The T2DM heart showed mitochondrial dysfunction at multiple levels and exercise training ameliorated some, but not all mitochondrial dysfunctions.

Characterization of Electrical Activity in Post-myocardial Infarction Scar Tissue in Rat Hearts Using Multiphoton Microscopy.

Background: The origin of electrical behavior in post-myocardial infarction scar tissue is still under debate. This study aims to examine the extent and nature of the residual electrical activity within a stabilized ventricular infarct scar. Methods and Results: An apical infarct was induced in the left ventricle of Wistar rats by coronary artery occlusion. Five weeks post-procedure, hearts were Langendorff-perfused, and optically mapped using di-4-ANEPPS. Widefield imaging of optical action potentials (APs) on the left ventricular epicardial surface revealed uniform areas of electrical activity in both normal zone (NZ) and infarct border zone (BZ), but only limited areas of low-amplitude signals in the infarct zone (IZ). 2-photon (2P) excitation of di-4-ANEPPS and Fura-2/AM at discrete layers in the NZ revealed APs and Ca2+ transients (CaTs) to 500-600 µm below the epicardial surface. 2P imaging in the BZ revealed superficial connective tissue structures lacking APs or CaTs. At depths greater than approximately 300 µm, myocardial structures were evident that supported normal APs and CaTs. In the IZ, although 2P imaging did not reveal clear myocardial structures, low-amplitude AP signals were recorded at discrete layers. No discernible Ca2+ signals could be detected in the IZ. AP rise times in BZ were slower than NZ (3.50 ± 0.50 ms vs. 2.23 ± 0.28 ms) and further slowed in IZ (9.13 ± 0.56 ms). Widefield measurements of activation delay between NZ and BZ showed negligible difference (3.37 ± 1.55 ms), while delay values in IZ showed large variation (11.88 ± 9.43 ms). Conclusion: These AP measurements indicate that BZ consists of an electrically inert scar above relatively normal myocardium. Discrete areas/layers of IZ displayed entrained APs with altered electrophysiology, but the structure of this tissue remains to be elucidated.

Bioengineering Organs for Blood Detoxification.

For patients with severe kidney or liver failure the best solution is currently organ transplantation. However, not all patients are eligible for transplantation and due to limited organ availability, most patients are currently treated with therapies using artificial kidney and artificial liver devices. These therapies, despite their relative success in preserving the patients' life, have important limitations since they can only replace part of the natural kidney or liver functions. As blood detoxification (and other functions) in these highly perfused organs is achieved by specialized cells, it seems relevant to review the approaches leading to bioengineered organs fulfilling most of the native organ functions. There, the culture of cells of specific phenotypes on adapted scaffolds that can be perfused takes place. In this review paper, first the functions of kidney and liver organs are briefly described. Then artificial kidney/liver devices, bioartificial kidney devices, and bioartificial liver devices are focused on, as well as biohybrid constructs obtained by decellularization and recellularization of animal organs. For all organs, a thorough overview of the literature is given and the perspectives for their application in the clinic are discussed.

Polymeric electrospun scaffolds for bone morphogenetic protein 2 delivery in bone tissue engineering.

HYPOTHESIS: The development of novel scaffolds based on biocompatible polymers is of great interest in the field of bone repair for fabrication of biodegradable scaffolds that mimic the extracellular matrix and have osteoconductive and osteoinductive properties for enhanced bone regeneration. EXPERIMENTS: Polycaprolactone (PCL) and polycaprolactone/polyvinyl acetate (PCL/PVAc) core-shell fibers were synthesised and decorated with poly(lactic-co-glycolic acid) [PLGA] particles loaded with bone morphogenetic protein 2 (BMP2) by simultaneous electrospinning and electrospraying. Hydroxyapatite nanorods (HAn) were loaded into the core of fibers. The obtained scaffolds were characterised by scanning and transmission electron microscopy, Fourier-transform infrared spectroscopy, and thermogravimetric analysis. The in vitro potential of these materials for bone regeneration was assessed in biodegradation assays, osteoblast viability assays, and analyses of expression of specific bone markers, such as alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). FINDINGS: PLGA particles were homogeneously distributed in the entire fibre mat. The growth factor load was 1.2-1.7 µg/g of the scaffold whereas the HAn load was in the 8.8-12.6 wt% range. These scaffolds were able to support and enhance cell growth and proliferation facilitating the expression of osteogenic and osteoconductive markers (OCN and OPN). These observations underline the great importance of the presence of BMP2 in scaffolds for bone remodelling as well as the good potential of the newly developed scaffolds for clinical use in tissue engineering.

Normal interventricular differences in tissue architecture underlie right ventricular susceptibility to conduction abnormalities in a mouse model of Brugada syndrome.

Aims: Loss-of-function of the cardiac sodium channel NaV1.5 is a common feature of Brugada syndrome. Arrhythmias arise preferentially from the right ventricle (RV) despite equivalent NaV1.5 downregulation in the left ventricle (LV). The reasons for increased RV sensitivity to NaV1.5 loss-of-function mutations remain unclear. Because ventricular electrical activation occurs predominantly in the transmural axis, we compare RV and LV transmural electrophysiology to determine the underlying cause of the asymmetrical conduction abnormalities in Scn5a haploinsufficient mice (Scn5a+/-). Methods and results: Optical mapping and two-photon microscopy in isolated-perfused mouse hearts demonstrated equivalent depression of transmural conduction velocity (CV) in the LV and RV of Scn5a+/- vs. wild-type littermates. Only RV transmural conduction was further impaired when challenged with increased pacing frequencies. Epicardial dispersion of activation and beat-to-beat variation in activation time were increased only in the RV of Scn5a+/- hearts. Analysis of confocal and histological images revealed larger intramural clefts between cardiomyocyte layers in the RV vs. LV, independent of genotype. Acute sodium current inhibition in wild type hearts using tetrodotoxin reproduced beat-to-beat activation variability and frequency-dependent CV slowing in the RV only, with the LV unaffected. The influence of clefts on conduction was examined using a two-dimensional monodomain computational model. When peak sodium channel conductance was reduced to 50% of normal the presence of clefts between cardiomyocyte layers reproduced the activation variability and conduction phenotype observed experimentally. Conclusions: Normal structural heterogeneities present in the RV are responsible for increased vulnerability to conduction slowing in the presence of reduced sodium channel function. Heterogeneous conduction slowing seen in the RV will predispose to functional block and the initiation of re-entrant ventricular arrhythmias.