Silk-reinforced collagen hydrogels with raised multiscale stiffness for mesenchymal cells 3D culture

Type I collagen hydrogels are of high interest in tissue engineering. With the evolution of 3D bioprinting technologies, a high number of collagen-based scaffolds have been reported for the development of 3D cell cultures. A recent proposal was to mix collagen with silk fibroin derived from Bombyx mori silkworm. Nevertheless, due to the difficulties in the preparation and the characteristics of the protein, several problems such as phase separation and collagen denaturation appear during the procedure. Therefore, the common solution is to diminish the concentration of collagen although in that way the most biologically relevant component is reduced. In this study, we present a new, simple, and effective method to develop a collagen-silk hybrid hydrogel with high collagen concentration and with increased stiffness approaching that of natural tissues, which could be of high interest for the development of cardiac patches for myocardial regeneration and for preconditioning of mesenchymal stem cells (MSCs) to improve their therapeutic potential. Sericin in the silk was preserved by using a physical solubilizing procedure that results in a preserved fibrous structure of type I collagen, as shown by ultrastructural imaging. The macro- and micromechanical properties of the hybrid hydrogels measured by tensile stretch and atomic force microscopy, respectively, showed a more than twofold stiffening than the collagen-only hydrogels. Rheological measurements showed improved printability properties for the developed biomaterial. The suitability of the hydrogels for 3D cell culture was assessed by 3D bioprinting bone marrow-derived MSCs cultured within the scaffolds. The result was a biomaterial with improved printability characteristics that better resembled the mechanical properties of natural soft tissues while preserving biocompatibility owing to the high concentration of collagen. Impact statement In this study, we report the development of silk microfiber-reinforced type I collagen hydrogels for 3D bioprinting and cell culture. In contrast with previously reported studies, a novel physical method allowed the preservation of the silk sericin protein. Hydrogels were stable, showed no phase separation between the biomaterials, and they presented improved printability. An increase between two- and threefold of the multiscale stiffness of the scaffolds was achieved with no need of using additional crosslinkers or complex methods, which could be of high relevance for cardiac patches development and for preconditioning mesenchymal stem cells (MSCs) for therapeutic applications. We demonstrate that bone marrow-derived MSCs can be effectively bioprinted and 3D cultured within the stiffened structures.This work was supported in part by the Spanish Ministry of Sciences, Innovation and Universities (DPI2017-83721-P and PGC2018-097323-A-I00) and by the Marie Sklodowska- Curie Action, Innovative Training Networks 2018, EU Grant Agreement no. 812772.Peer ReviewedPostprint (published version

Intracoronary Administration of Allogeneic Adipose Tissue-Derived Mesenchymal Stem Cells Improves Myocardial Perfusion But Not Left Ventricle Function, in a Translational Model of Acute Myocardial Infarction

Background-Autologous adipose tissue-derived mesenchymal stem cells (ATMSCs) therapy is a promising strategy to improve post-myocardial infarction outcomes. In a porcine model of acute myocardial infarction, we studied the long-term effects and the mechanisms involved in allogeneic ATMSCs administration on myocardial performance. Methods and Results-Thirty-eight pigs underwent 50 minutes of coronary occlusion; the study was completed in 33 pigs. After reperfusion, allogeneic ATMSCs or culture medium (vehicle) were intracoronarily administered. Follow-ups were performed at short (2 days after acute myocardial infarction vehicle-treated, n=10; ATMSCs-treated, n=9) or long term (60 days after acute myocardial infarction vehicle-treated, n=7; ATMSCs-treated, n=7). At short term, infarcted myocardium analysis showed reduced apoptosis in the ATMSCs-treated animals (48.6 +/- 6\% versus 55.9 +/- 5.7\% in vehicle; P=0.017); enhancement of the reparative process with up-regulated vascular endothelial growth factor, granulocyte macrophage colony-stimulating factor, and stromal-derived factor-1 alpha gene expression; and increased M2 macrophages (67.2 +/- 10\% versus 54.7 +/- 10.2\% in vehicle; P=0.016). In long-term groups, increase in myocardial perfusion at the anterior infarct border was observed both on day-7 and day-60 cardiac magnetic resonance studies in ATMSCs-treated animals, compared to vehicle (87.9 +/- 28.7 versus 57.4 +/- 17.7 mL/min per gram at 7 days; P=0.034 and 99 +/- 22.6 versus 43.3 +/- 14.7 22.6 mL/min per gram at 60 days; P=0.0001, respectively). At day 60, higher vascular density was detected at the border zone in the ATMSCs-treated animals (118 +/- 18 versus 92.4 +/- 24.3 vessels/mm(2) in vehicle; P=0.045). Cardiac magnetic resonance-measured left ventricular ejection fraction of left ventricular volumes was not different between groups at any time point. Conclusions-In this porcine acute myocardial infarction model, allogeneic ATMSCs-based therapy was associated with increased cardioprotective and reparative mechanisms and with better cardiac magnetic resonance-measured perfusion. No effect on left ventricular volumes or ejection fraction was observed.This work was supported by grants from Fundacion la Marato de TV3 (122230); Fondo de Investigacion Sanitaria Instituto de Salud Carlos III and Fondo Europeo de Desarrollo Regional (FIS PI14/01682), (RD12/0042/0006), (RD12/0042/0047), (RD12/0019/0029) (TerCel RD16/0011/0006), CIBER Cardiovascular (CB16/11/00403) projects and Ministerio de Educacion y Ciencia (SAF2011-30067-C02-01) (SAF2014-59892). Fernaandez-Jimenez was the recipient of nonoverlapping grants from the Ministerio de Economia, Industria, y Competitividad through the Instituto de Salud Carlos III (Rio Hortega fellowship); and the Fundacion Jesus Serra, the Fundacion Interhospitalaria de Investigacion Cardiovascular (FIC), and the CNIC (FICNIC fellowship). The use of QMass software was partly supported by a scientific collaboration between the CNIC and Medis Medical Imaging Systems BV. The CNIC is supported by the Ministerio de Economia, Industria, y Competitividad (MINECO) and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). This work was also funded by ``la Caixa Banking Foundation, and the Generalitat de Catalunya (SGR 2014, CERCA Programme). This work has been developed in the context of AdvanceCat with the support of ACCIO (Catalonia Trade \& Investment; Generalitat de Catalunya) under the Catalonian ERDF operational program (European Regional Development Fund) 2014-2020.S

A simple low-cost electrocardiogram synchronizer

Electrocardiogram (ECG) synchronization is useful to avoid the effects of cardiac motion in medical measurements, and is widely used in standard medical imaging. A number of medical equipment include embedded commercial synchronizers. However, the use of independent synchronization modules is sometimes needed when several non-integrated instruments are used, or in the development of new medical instruments and procedures. We present a simple low-cost ECG synchronizer module based on an Arduino controller board that converts the ECG signal into a transistor-transistor-logic (TTL) one, allowing real-time medical measurements triggered at specific phases of the cardiac cycle. The device and conversion algorithm developed is optimized in vitro using synthetic and human ECG signals, and tested in vivo on three swine specimens. Error rates during the in vivo testing stage remain below the 2% of the cycles in all animals and critical false positives are less than 1%, which is sufficient for most applications. Possible algorithm updates are discussed if its performance needs to be improved.This research was funded by grants from the Instituto de Salud Carlos III [PI18/00256], the Fundació La MARATÓ de TV3 (20153530/31), the Sociedad Española de Cardiología, and the Generalitat de Catalunya [2017-SGR-483]. This work was also funded by CaixaImpulse (CI20/00230), and the Red de Terapia Celular—TerCel [RD16/0011/0006] and CIBER Cardiovascular [CB16/11/00403] projects, as part of the Plan Nacional de I+D+I, and cofunded by ISCIII-Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER). F.M. acknowledges support from the Catalan Government through COFUND and the Unidad de Excelencia Maria de Maeztu MDM-2016-0600.Peer ReviewedPostprint (published version

Multimarker strategy for heart failure prognostication. Value of neurohormonal biomarkers: neprilysin vs NT-proBNP.

INTRODUCTION AND OBJECTIVES: Neprilysin breaks down numerous vasoactive peptides. The soluble form of neprilysin, which was recently identified in heart failure, is associated with cardiovascular outcomes. Within a multibiomarker strategy, we directly compared soluble neprilysin and N-terminal pro-B-type natriuretic peptide as risk stratifiers in a real-life cohort of heart failure patients. METHODS: Soluble neprilysin, N-terminal pro-B-type natriuretic peptide, ST2, and high-sensitivity troponin T levels were measured in 797 consecutive ambulatory heart failure patients followed up for 4.7 years. Comprehensive multivariable analyses and soluble neprilysin vs N-terminal pro-B-type natriuretic peptide head-to-head assessments of performance were performed. A primary composite endpoint included cardiovascular death or heart failure hospitalization. A secondary endpoint explored cardiovascular death alone. RESULTS: Median soluble neprilysin and N-terminal pro-B-type natriuretic peptide concentrations were 0.64ng/mL and 1187 ng/L, respectively. Both biomarkers significantly correlated with age (P<.001) and ST2 (P<.001), but only N-terminal pro-B-type natriuretic peptide significantly correlated with estimated glomerular filtration rate (P<.001), body mass index (P<.001), left ventricular ejection fraction (P=.02) and high-sensitivity troponin T (P<.001). In multivariable Cox regression analyses, soluble neprilysin remained independently associated with the composite endpoint (hazard ratio=1.14; 95% confidence interval, 1.02-1.27; P=.03) and cardiovascular death (hazard ratio=1.15; 95% confidence interval, 1.01-1.31; P=.04), but N-terminal pro-B-type natriuretic peptide did not. The head-to-head soluble neprilysin vs N-terminal pro-B-type natriuretic peptide comparison showed good calibration and similar discrimination and reclassification for both neurohormonal biomarkers, but only soluble neprilysin improved overall goodness-of-fit. CONCLUSIONS: When added to a multimarker strategy, soluble neprilysin remained an independent prognosticator, while N-terminal pro-B-type natriuretic peptide lost significance as a risk stratifier in ambulatory patients with heart failure. Both biomarkers performed similarly in head-to-head analyses.We acknowledge Redes Temáticas de Investigación Cooperativa en Salud (RETICS), Red Cardiovascular (RD12/0042/0047; Postdoctoral RIC fellowship), Red de Terapia Celular–TerCel [RD12/0019/0029], and Ministerio de Economía y Competitividad (Juan de la Cierva, JCI-2012-14025