New scaffolds encapsulating TGF-β3/BMP-7 combinations driving strong chondrogenic differentiation

The regeneration of articular cartilage remains an unresolved question despite the current access to a variety of tissue scaffolds activated with growth factors relevant to this application. Further advances might result from combining more than one of these factors; here, we propose a scaffold composition optimized for the dual delivery of BMP-7 and TGF-β3, two proteins with described chondrogenic activity. First, we tested in a mesenchymal stem cell micromass culture with TGF-β3 whether the exposure to microspheres loaded with BMP-7 would improve cartilage formation. Histology and qRT-PCR data confirmed that the sustained release of BMP-7 cooperates with TGF-β3 towards the generation of chondrogenic differentiation. Then, we optimized a scaffold prototype for tissue culture and dual encapsulation of BMP-7 and TGF-β3. The scaffolds were prepared from poly(lactic-co-glycolic acid), and BMP-7/TGF-β3 were loaded as nanocomplexes with heparin and Tetronic 1107. The scaffolds showed the sustained release of both proteins over four weeks, with minimal burst effect. We finally cultured human mesenchymal stems cells on these scaffolds, in the absence of exogenous chondrogenic factor supplementation. The cells cultured on the scaffolds loaded with BMP-7 and TGF-β3 showed clear signs of cartilage formation macroscopically and histologically. RT-PCR studies confirmed a clear upregulation of cartilage markers SOX9 and Aggrecan. In summary, scaffolds encapsulating BMP-7 and TGF-β3 can efficiently deliver a cooperative growth factor combination that drives efficient cartilage formation in human mesenchymal stem cell cultures. These results open attractive perspectives towards in vivo translation of this technology in cartilage regeneration experimentsFundación Ramón Areces (CIVP16A1832), Xunta de Galicia (Proxectos de Investigación Desenvolvidos por Investigadores Emerxentes, EM2013/042), Fundación BBVA, Proyectos de Investigación en Biomedicina (2014- PO0110) y Ministerio de Economía y Competitividad (SAF2014-58189-R)S

Interaction of silver atomic quantum clusters with living organisms: bactericidal effect of Ag3 clusters mediated by disruption of topoisomerase–DNA complexes

Essential processes for living cells such as transcription and replication depend on the formation of specific protein–DNA recognition complexes. Proper formation of such complexes requires suitable fitting between the protein surface and the DNA surface. By adopting doxorubicin (DOX) as a model probe, we report here that Ag3 atomic quantum clusters (Ag-AQCs) inhibit the intercalation of DOX into DNA and have considerable influence on the interaction of DNA-binding proteins such as topoisomerase IV, Escherichia coli DNA gyrase and the restriction enzyme HindIII. Ag-AQCs at nanomolar concentrations inhibit enzyme activity. The inhibitory effect of Ag-AQCs is dose-dependent and occurs by intercalation into DNA. All these effects, not observed in the presence of Ag+ ions, can explain the powerful bactericidal activity of Ag-AQCs, extending the knowledge of silver bactericidal properties. Lastly, we highlight the interest of the interaction of Ag clusters with living organisms, an area that should be further explored due to the potential consequences that it might have, both beneficial and harmful.This work was supported by Obra Social“la Caixa” (OSLC-2012-007), European Commission through FEDER program (0681 InveNNta 1 E); Ministerio de Ciencia e Innovación, Spain (MAT2010-20442, MAT2011-28673-C02-01); MINECO, Spain (MAT2012-36754-C02-01 and CTQ2014-58812-C2-2-R), Xunta de Galicia, Spain (GRC2013-044, FEDER Funds). C. P.-A. is grateful for the FPU grant from Ministry of Education, Culture and Sports, Madrid, Spain (FPU13/00180)S

Silver clusters of five atoms as highly selective antitumoral agents through irreversible oxidation of thiols

Low atomicity clusters present properties dependent on the size, due to the quantum confinement, with well-defined electronic structures and high stability. Here it is shown that Ag5 clusters catalyze the complete oxidation of sulfur to S+6. Ag5 catalytic activity increases with different oxidant species in the order O2 ≪ H2O2 < OH•. Selective oxidation of thiols on the cysteine residues of glutathione and thioredoxin is the primary mechanism human cells have to maintain redox homeostasis. Contingent upon oxidant concentration, Ag5 catalyzes the irreversible oxidation of glutathione and thioredoxin, triggering apoptosis. Modification of the intracellular environment to a more oxidized state to mimic conditions within cancer cells through the expression of an activated oncogene (HRASG12V) or through ARID1A mutation, sensitizes cells to Ag5 mediated apoptosis. While cancers evolve to evade treatments designed to target pathways or genetic mutations that drive them, they cannot evade a treatment that takes advantage of aberrant redox homeostasis, which is essential for tumor progression and metastasis. Ag5 has antitumor activity in mice with orthotopic lung tumors reducing primary tumor size, and the burden of affected lymphatic nodes. The findings suggest the unique intracellular redox chemistry of Ag5 may lead to new redox-based approaches to cancer therapyThis research was partially supported by 1) “la Caixa” Foundation, Ref. LCF/PR/PR12/11070003 to F.D. and M.A.L.Q.; 2) Ministerio de Ciencia, Innovación y Universidades (MAT2017-89678-R, AEI/FEDER, UE) to F.D. and A.V.; 3) the Consellería de Educación (Xunta de Galicia), Grants No. Grupos Ref. Comp. ED431C 2017/22, ED431C 2019/13 and AEMAT-ED431E2018/08 to M.A.L.Q.; and ED431C 2019/13 to A.V. This project has received funding from the European Union's Horizon 2020 Research and Innovation Programme (Bac-To-Fuel) under Grant Agreement No. 825999 (M.A.L.Q.). J.C.H. acknowledge financial support from European Union's Horizon 2020 research and innovation programme under grant agreement no. 823717-ESTEEM3, and the MICIIN (projects PID2019-107578GA-100 and PID-110018GA-100). J.M.D, L.J.G., and F.G.R. thank to the ANPCyT (PICT 2015-2285 and 2017-3944), UNLP (Project 11/X790) and the partial support by the Laboratório Nacional de Luz Síncrotron (LNLS, Brazil) under proposal SXS-20180280. G.B. acknowledges the CINECA Award N. IsC51, year 2017, under the ISCRA initiative, for the availability of high-performance computing resources and support. D.B. expresses gratitude for a postdoctoral grant from Xunta de Galicia, Spain (POS-A/2013/018). B.D. expresses gratitude for a predoctoral grant from MICINN, Spain (BES-2016-076765). F.D. and A.V. also acknowledged Xunta de Galicia (Centro singular de investigación de Galicia accreditation 2019-2022 ref ED431G 2019/02) and the European Union (European Regional Development Fund – ERDF). Work in M.P.M.'s lab was supported by the Medical Research Council UK (MC_U105663142). T.G.C. gratefully acknowledges the technical assistance of María José Otero-Fraga (FIDIS)S

mRNA-activated matrices encoding transcription factors as primers of cell differentiation in tissue engineering

Gene-activated matrices (GAMs) encoding pivotal transcription factors (TFs) represent a powerful tool to direct stem cell specification for tissue engineering applications. However, current TF-based GAMs activated with pDNA, are challenged by their low transfection efficiency and delayed transgene expression. Here, we report a GAM technology activated with mRNAs encoding TFs SOX9 (cartilage) and MYOD (muscle). We find that these mRNA-GAMs induce a higher and faster TF expression compared to pDNA-GAMs, especially in the case of RNase resistant mRNA sequences. This potent TF expression was translated into a high synthesis of cartilage- and muscle-specific markers, and ultimately, into successful tissue specification in vitro. Additionally, we show that the expression of tissue-specific markers can be further modulated by altering the properties of the mRNA-GAM environment. These results highlight the value of this GAM technology for priming cell lineage specification, a key centerpiece for future tissue engineering devices.This work has been funded by Ministerio de Economía y Competitividad (MINECO-RETOS, Grant MAT2017-84361-R, Feder Funds), Fundación BBVA 2014-PO0110 and Xunta de Galicia (Grupos de Referencia Competitiva, Feder Funds; Convenio para fomentar a actividade investigadora do persoal investigador finalista nas convocatorias de axudas do ERC no marco da H2020). AML was a recipient of a FPU grant from Ministerio de Economía y Competitividad (FPU12/05528)S