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

Pancreatic steatosis and iron overload increases cardiovascular risk in non-alcoholic fatty liver disease

ObjectiveTo assess the prevalence of pancreatic steatosis and iron overload in non-alcoholic fatty liver disease (NAFLD) and their correlation with liver histology severity and the risk of cardiometabolic diseases.MethodA prospective, multicenter study including NAFLD patients with biopsy and paired Magnetic Resonance Imaging (MRI) was performed. Liver biopsies were evaluated according to NASH Clinical Research Network, hepatic iron storages were scored, and digital pathology quantified the tissue proportionate areas of fat and iron. MRI-biomarkers of fat fraction (PDFF) and iron accumulation (R2*) were obtained from the liver and pancreas. Different metabolic traits were evaluated, cardiovascular disease (CVD) risk was estimated with the atherosclerotic CVD score, and the severity of iron metabolism alteration was determined by grading metabolic hiperferritinemia (MHF). Associations between CVD, histology and MRI were investigated.ResultsIn total, 324 patients were included. MRI-determined pancreatic iron overload and moderate-to severe steatosis were present in 45% and 25%, respectively. Liver and pancreatic MRI-biomarkers showed a weak correlation (r=0.32 for PDFF, r=0.17 for R2*). Pancreatic PDFF increased with hepatic histologic steatosis grades and NASH diagnosis (p<0.001). Prevalence of pancreatic steatosis and iron overload increased with the number of metabolic traits (p<0.001). Liver R2* significantly correlated with MHF (AUC=0.77 [0.72-0.82]). MRI-determined pancreatic steatosis (OR=3.15 [1.63-6.09]), and iron overload (OR=2.39 [1.32-4.37]) were independently associated with high-risk CVD. Histologic diagnosis of NASH and advanced fibrosis were also associated with high-risk CVD.ConclusionPancreatic steatosis and iron overload could be of utility in clinical decision-making and prognostication of NAFLD

Ahora / Ara

La cinquena edició del microrelatari per l’eradicació de la violència contra les dones de l’Institut Universitari d’Estudis Feministes i de Gènere «Purificación Escribano» de la Universitat Jaume I vol ser una declaració d’esperança. Aquest és el moment en el qual les dones (i els homes) hem de fer un pas endavant i eliminar la violència sistèmica contra les dones. Ara és el moment de denunciar el masclisme i els micromasclismes començant a construir una societat més igualitària. Cadascun dels relats del llibre és una denúncia i una declaració que ens encamina cap a un món millor

HoLaMa: A Klenow sub-fragment lacking the 3′-5′ exonuclease domain

The design, construction, overexpression, and purification of a Klenow sub-fragment lacking the 30–50 exonuclease domain is presented here. In particular, a synthetic gene coding for the residues 515–928 of Escherichia coli DNA polymerase I was constructed. To improve the solubility and stability of the corresponding protein, the synthetic gene was designed to contain 11 site-specific substitutions. The gene was inserted into the pBADHis expression vector, generating 2 identical Klenow sub-fragments, bearing or not a hexahistidine tag. Both these Klenow sub-fragments, denominated HoLaMa and HoLaMaHis, were purified, and their catalytic properties were compared to those of Klenow enzyme. When DNA polymerase activity was assayed under processive conditions, the Klenow enzyme performed much better than HoLaMa and HoLaMaHis. However, when DNA polymerase activity was assayed under distributive conditions, the initial velocity of the reaction catalyzed by HoLaMa was comparable to that observed in the presence of Klenow enzyme. In particular, under distributive conditions HoLaMa was found to strongly prefer dsDNAs bearing a short template overhang, to the length of which the Klenow enzyme was relatively insensitive. Overall, our observations indicate that the exonuclease domain of the Klenow enzyme, besides its proofreading activity, does significantly contribute to the catalytic efficiency of DNA elongation

Digital Pathology Enables Automated and Quantitative Assessment of Inflammatory Activity in Patients with Chronic Liver Disease

Traditional histological evaluation for grading liver disease severity is based on subjective and semi-quantitative scores. We examined the relationship between digital pathology analysis and corresponding scoring systems for the assessment of hepatic necroinflammatory activity. A prospective, multicenter study including 156 patients with chronic liver disease (74% nonalcoholic fatty liver disease-NAFLD, 26% chronic hepatitis-CH etiologies) was performed. Inflammation was graded according to the Nonalcoholic Steatohepatitis (NASH) Clinical Research Network system and METAVIR score. Whole-slide digital image analysis based on quantitative (I-score: inflammation ratio) and morphometric (C-score: proportionate area of staining intensities clusters) measurements were independently performed. Our data show that I-scores and C-scores increase with inflammation grades (p ρ = 0.85–0.88), but only moderate for NAFLD (ρ = 0.5–0.53). I-score (p = 0.008) and C-score (p = 0.002) were higher for CH than NAFLD. Our MATLAB algorithm performed better than QuPath software for the diagnosis of low-moderate inflammation (p p < 0.001). In conclusion, quantitative and morphometric metrics of inflammatory burden obtained by digital pathology correlate well with pathologists’ scores, showing a higher accuracy for the evaluation of CH than NAFLD