Convergence of melt electrowriting and extrusion-based bioprinting for vascular patterning of a myocardial construct

To progress cardiac tissue engineering strategies closer to the clinic, thicker constructs are required to meet the functional need following a cardiac event. Consequently, pre-vascularization of these constructs needs to be investigated to ensure survival and optimal performance of implantable engineered heart tissue. The aim of this research is to investigate the potential of combining extrusion-based bioprinting (EBB) and melt electrowriting for the fabrication of a myocardial construct with a precisely patterned pre-vascular pathway. Gelatin methacryloyl (GelMA) was investigated as a base hydrogel for the respective myocardial and vascular bioinks with collagen, Matrigel and fibrinogen as interpenetrating polymers to support myocardial functionality. Subsequently, extrusion-based printability and viability were investigated to determine the optimal processing parameters for printing into melt electrowritten meshes. Finally, an anatomically inspired vascular pathway was implemented in a dual EBB set-up into melt electrowritten meshes, creating a patterned pre-vascularized myocardial construct. It was determined that a blend of 5% GelMA and 0.8 mg·ml -1collagen with a low crosslinked density was optimal for myocardial cellular arrangement and alignment within the constructs. For the vascular fraction, the optimized formulation consisted of 5% GelMA, 0.8 mg·ml -1collagen and 1 mg·ml -1fibrinogen with a higher crosslinked density, which led to enhanced vascular cell connectivity. Printability assessment confirmed that the optimized bioinks could effectively fill the microfiber mesh while supporting cell viability (∼70%). Finally, the two bioinks were applied using a dual EBB system for the fabrication of a pre-vascular pathway with the shape of a left anterior descending artery within a myocardial construct, whereby the distinct cell populations could be visualized in their respective patterns up to D14. This research investigated the first step towards developing a thick engineered cardiac tissue construct in which a pre-vascularization pathway is fabricated within a myocardial construct. </p

Controlled delivery of gold nanoparticle-coupled miRNA therapeutics via an injectable self-healing hydrogel

Differential expression of microRNAs (miRNAs) plays a role in many diseases, including cancer and cardiovascular diseases. Potentially, miRNAs could be targeted with miRNA-therapeutics. Sustained delivery of these therapeutics remains challenging. This study couples miR-mimics to PEG-peptide gold nanoparticles (AuNP) and loads these AuNP-miRNAs in an injectable, shear thinning, self-assembling polymer nanoparticle (PNP) hydrogel drug delivery platform to improve delivery. Spherical AuNPs coated with fluorescently labelled miR-214 are loaded into an HPMC-PEG-b-PLA PNP hydrogel. Release of AuNP/miRNAs is quantified, AuNP-miR-214 functionality is shown in vitro in HEK293 cells, and AuNP-miRNAs are tracked in a 3D bioprinted human model of calcific aortic valve disease (CAVD). Lastly, biodistribution of PNP-AuNP-miR-67 is assessed after subcutaneous injection in C57BL/6 mice. AuNP-miRNA release from the PNP hydrogel in vitro demonstrates a linear pattern over 5 days up to 20%. AuNP-miR-214 transfection in HEK293 results in 33% decrease of Luciferase reporter activity. In the CAVD model, AuNP-miR-214 are tracked into the cytoplasm of human aortic valve interstitial cells. Lastly, 11 days after subcutaneous injection, AuNP-miR-67 predominantly clears via the liver and kidneys, and fluorescence levels are again comparable to control animals. Thus, the PNP-AuNP-miRNA drug delivery platform provides linear release of functional miRNAs in vitro and has potential for in vivo applications.publishersversionpublishe

Convergence of melt electrowriting and extrusion-based bioprinting for vascular patterning of a myocardial construct

To progress cardiac tissue engineering strategies closer to the clinic, thicker constructs are required to meet the functional need following a cardiac event. Consequently, pre-vascularization of these constructs needs to be investigated to ensure survival and optimal performance of implantable engineered heart tissue. The aim of this research is to investigate the potential of combining extrusion-based bioprinting (EBB) and melt electrowriting for the fabrication of a myocardial construct with a precisely patterned pre-vascular pathway. Gelatin methacryloyl (GelMA) was investigated as a base hydrogel for the respective myocardial and vascular bioinks with collagen, Matrigel and fibrinogen as interpenetrating polymers to support myocardial functionality. Subsequently, extrusion-based printability and viability were investigated to determine the optimal processing parameters for printing into melt electrowritten meshes. Finally, an anatomically inspired vascular pathway was implemented in a dual EBB set-up into melt electrowritten meshes, creating a patterned pre-vascularized myocardial construct. It was determined that a blend of 5% GelMA and 0.8 mg·ml -1collagen with a low crosslinked density was optimal for myocardial cellular arrangement and alignment within the constructs. For the vascular fraction, the optimized formulation consisted of 5% GelMA, 0.8 mg·ml -1collagen and 1 mg·ml -1fibrinogen with a higher crosslinked density, which led to enhanced vascular cell connectivity. Printability assessment confirmed that the optimized bioinks could effectively fill the microfiber mesh while supporting cell viability (∼70%). Finally, the two bioinks were applied using a dual EBB system for the fabrication of a pre-vascular pathway with the shape of a left anterior descending artery within a myocardial construct, whereby the distinct cell populations could be visualized in their respective patterns up to D14. This research investigated the first step towards developing a thick engineered cardiac tissue construct in which a pre-vascularization pathway is fabricated within a myocardial construct

Ablation of liver Fxr results in an increased colonic mucus barrier in mice

Background &amp; Aims: The interorgan crosstalk between the liver and the intestine has been the focus of intense research. Key in this crosstalk are bile acids, which are secreted from the liver into the intestine, interact with the microbiome, and upon absorption reach back to the liver. The bile acid-activated farnesoid X receptor (Fxr) is involved in the gut-to-liver axis. However, liver-to-gut communication and the roles of bile acids and Fxr remain elusive. Herein, we aim to get a better understanding of Fxr-mediated liver-to-gut communication, particularly in colon functioning. Methods: Fxr floxed/floxed mice were crossed with cre-expressing mice to yield Fxr ablation in the intestine (Fxr-intKO), liver (Fxr-livKO), or total body (Fxr-totKO). The effects on colonic gene expression (RNA sequencing), the microbiome (16S sequencing), and mucus barrier function by ex vivo imaging were analysed. Results: Despite relatively small changes in biliary bile acid concentration and composition, more genes were differentially expressed in the colons of Fxr-livKO mice than in those of Fxr-intKO and Fxr-totKO mice (3272, 731, and 1824, respectively). The colons of Fxr-livKO showed increased expression of antimicrobial genes, Toll-like receptors, inflammasome-related genes and genes belonging to the ‘Mucin-type O-glycan biosynthesis’ pathway. Fxr-livKO mice have a microbiome profile favourable for the protective capacity of the mucus barrier. The thickness of the inner sterile mucus layer was increased and colitis symptoms reduced in Fxr-livKO mice. Conclusions: Targeting of FXR is at the forefront in the battle against metabolic diseases. We show that ablation of Fxr in the liver greatly impacts colonic gene expression and increased the colonic mucus barrier. Increasing the mucus barrier is of utmost importance to battle intestinal diseases such as inflammatory bowel disease, and we show that this might be done by antagonising FXR in the liver. Lay summary: This study shows that the communication of the liver to the intestine is crucial for intestinal health. Bile acids are key players in this liver-to-gut communication, and when Fxr, the master regulator of bile acid homoeostasis, is ablated in the liver, colonic gene expression is largely affected, and the protective capacity of the mucus barrier is increased

MicroRNA-132/212 family enhances arteriogenesis after hindlimb ischaemia through modulation of the Ras-MAPK pathway

Arteriogenesis is a complicated process induced by increased local shear-and radial wall-stress, leading to an increase in arterial diameter. This process is enhanced by growth factors secreted by both inflammatory and endothelial cells in response to physical stress. Although therapeutic promotion of arteriogenesis is of great interest for ischaemic diseases, little is known about the modulation of the signalling cascades via microRNAs. We observed that miR-132/212 expression was significantly upregulated after occlusion of the femoral artery. miR-132/212 knockout (KO) mice display a slower perfusion recovery after hind-limb ischaemia compared to wildtype (WT) mice. Immunohistochemical analysis demonstrates a clear trend towards smaller collateral arteries in KO mice. Although Ex vivo aortic ring assays score similar number of branches in miR-132/212 KO mice compared to WT, it can be stimulated with exogenous miR-132, a dominant member of the miR-132/212 family. Moreover, in in vitro pericyte-endothelial co-culture cell assays, overexpression of miR-132 and mir-212 in endothelial cells results in enhanced vascularization, as shown by an increase in tubular structures and junctions. Our results suggested that miR-132/212 may exert their effects by enhancing the Ras-Mitogen-activated protein kinases MAPK signalling pathway through direct inhibition of Rasa1, and Spred1. The miR-132/212 cluster promotes arteriogenesis by modulating Ras-MAPK signalling via direct targeting of its inhibitors Rasa1 and Spred1

Engineering a 3D-Bioprinted Model of Human Heart Valve Disease Using Nanoindentation-Based Biomechanics

In calcific aortic valve disease (CAVD), microcalcifications originating from nanoscale calcifying vesicles disrupt the aortic valve (AV) leaflets, which consist of three (biomechanically) distinct layers: the fibrosa, spongiosa, and ventricularis. CAVD has no pharmacotherapy and lacks in vitro models as a result of complex valvular biomechanical features surrounding resident mechanosensitive valvular interstitial cells (VICs). We measured layer-specific mechanical properties of the human AV and engineered a three-dimensional (3D)-bioprinted CAVD model that recapitulates leaflet layer biomechanics for the first time. Human AV leaflet layers were separated by microdissection, and nanoindentation determined layer-specific Young’s moduli. Methacrylated gelatin (GelMA)/methacrylated hyaluronic acid (HAMA) hydrogels were tuned to duplicate layer-specific mechanical characteristics, followed by 3D-printing with encapsulated human VICs. Hydrogels were exposed to osteogenic media (OM) to induce microcalcification, and VIC pathogenesis was assessed by near infrared or immunofluorescence microscopy. Median Young’s moduli of the AV layers were 37.1, 15.4, and 26.9 kPa (fibrosa/spongiosa/ventricularis, respectively). The fibrosa and spongiosa Young’s moduli matched the 3D 5% GelMa/1% HAMA UV-crosslinked hydrogels. OM stimulation of VIC-laden bioprinted hydrogels induced microcalcification without apoptosis. We report the first layer-specific measurements of human AV moduli and a novel 3D-bioprinted CAVD model that potentiates microcalcification by mimicking the native AV mechanical environment. This work sheds light on valvular mechanobiology and could facilitate high-throughput drug-screening in CAVD

Production of Single W Bosons at LEP

We report on the observation of single W boson production in a data sample collected by the L3 detector at LEP2. The signal consists of large missing energy final states with a single energetic lepton or two hadronic jets. The cross-section is measured to be $0.61^{+0.43}_{-0.33} \pm 0.05 \; \rm{pb}$ at the centre of mass energy \sqrt{s}=172 \GeV{}, consistent with the Standard Model expectation. From this measurement the following limits on the anomalous $\gamma$WW gauge couplings are derived at 95\% CL: $\rm -3.6 \Delta \kappa_\gamma 1.5$ and $\rm -3.6 \lambda_\gamma 3.6$

Measurement of the Average Lifetime of b-Hadrons in Z Decays

We present a measurement of the average b-hadron lifetime ${\rm \tau_b}$ at the $\mathrm{e^+e^-} \,$ collider LEP. Using hadronic Z decays collected in the period from 1991 to 1994, two independent analyses have been performed. In the first one, the b-decay position is reconstructed as a secondary vertex of hadronic b-decay particles. The second analysis is an updated measurement of ${\rm \tau_b}$ using the impact parameter of leptons with high momentum and high transverse momentum. The combined result is \begin{center} ${\rm \tau_b= [ 1549 \pm 9 \, (stat) \, \pm 15 \, (syst) ] \; fs \,}$ . \end{center} In addition, we measure the average charged b-decay multiplicity ${\rm \langle n_{\rm b}} \rangle$ and the normalized average b-energy ${\rm \langle x_E \rangle_{\rm b}}$ at LEP to be \begin{center} ${\rm \langle n_{\rm b} \rangle = 4.90 \pm 0.04 \ (stat) \pm 0.11 \, (syst)}$ , \end{center} \begin{center} ${\rm \langle x_E \rangle_{\rm b} = 0.709 \pm 0.004 \, (stat + syst).}$ \end{center

The 2010 ACR/EULAR criteria are not sufficiently accurate in the early identification of autoantibody-negative rheumatoid arthritis: Results from the Leiden-EAC and ESPOIR cohorts

Pathophysiology and treatment of rheumatic disease

Evaluating relationships between symptom duration and persistence of rheumatoid arthritis: does a window of opportunity exist? Results on the Leiden early arthritis clinic and ESPOIR cohorts.

International audienceA prolonged symptom or disease duration at treatment initiation is associated with unfavourable outcomes in rheumatoid arthritis (RA). It is unknown whether this relation is linear, referring to a common 'the-earlier-the-better principle', or whether a transient time frame in which the disease is more susceptible to treatment exists, referring to a 'window of opportunity'. To elucidate this, we evaluated the shape of the associations of symptom duration with persistence of RA.Patients with 1987 RA treated with disease modifying antirheumatic drugs (DMARDs) in the Leiden Early Arthritis Clinic (EAC, n=738) and Evaluation et Suivi de POlyarthrites Indifférenciées Récentes (ESPOIR) (n=533) were studied. Cox proportional hazards regression models using natural cubic splines were performed; the log-HR on DMARD-free sustained remission (the opposite of RA persistence) during 5-year follow-up was plotted against symptom duration. Discrimination was measured using time-dependent receiver operator characteristic curves. Subanalyses were performed stratified for the DMARDs used (methotrexate or other conventional DMARDs) and for anticitrullinated peptide antibody (ACPA).11.5% (85/738) and 5.4% (29/533) of EAC and ESPOIR RA patients achieved DMARD-free sustained remission. In both cohorts and all analyses, the curves depicting the log-HRs on remission in relation to symptom duration were not linear. The symptom duration with optimal discriminative ability was 14.9 weeks (95% CI 12.3 to 16.0; area under the curve (AUC) 0.61) in the EAC and 19.1 weeks (95% CI 12.3 to 28.0; AUC 0.59) in ESPOIR. For ACPA-positive RA, this was 11.4 weeks (95% CI 7.7 to 79.0; AUC 0.56) and for ACPA-negative RA 15.0 weeks (95% CI 9.7 to 48.7; AUC 0.56).The association between symptom duration and RA persistence is not linear, suggesting the presence of a confined period in which RA is more susceptible to treatment