Polylactide, Processed by a Foaming Method Using Compressed Freon R134a, for Tissue Engineering

Fabricating polymeric scaffolds using cost-effective manufacturing processes is still challenging. Gas foaming techniques using supercritical carbon dioxide (scCO2) have attracted attention for producing synthetic polymer matrices; however, the high-pressure requirements are often a technological barrier for its widespread use. Compressed 1,1,1,2-tetrafluoroethane, known as Freon R134a, offers advantages over CO2 in manufacturing processes in terms of lower pressure and temperature conditions and the use of low-cost equipment. Here, we report for the first time the use of Freon R134a for generating porous polymer matrices, specifically polylactide (PLA). PLA scaffolds processed with Freon R134a exhibited larger pore sizes, and total porosity, and appropriate mechanical properties compared with those achieved by scCO2 processing. PLGA scaffolds processed with Freon R134a were highly porous and showed a relatively fragile structure. Human mesenchymal stem cells (MSCs) attached to PLA scaffolds processed with Freon R134a, and their metabolic activity increased during culturing. In addition, MSCs displayed spread morphology on the PLA scaffolds processed with Freon R134a, with a well-organized actin cytoskeleton and a dense matrix of fibronectin fibrils. Functionalization of Freon R134a-processed PLA scaffolds with protein nanoparticles, used as bioactive factors, enhanced the scaffolds' cytocompatibility. These findings indicate that gas foaming using compressed Freon R134a could represent a cost-effective and environmentally friendly fabrication technology to produce polymeric scaffolds for tissue engineering approaches.This work was supported by the Spanish Ministry of Economy and Competitiveness (MOTHER MAT2016-80826-R and Mol4Bio PID2019-105622RBI00); Spanish Ministry of Science, Innovation and Universities (RTI2018-095159-B-100); Instituto de Salud Carlos III (ISCIII)- European Regional Development Fund (ERDF); MINECO-AES (PI15/00752, PI15/01118 and PI18/00643); the Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN); CSIC through grant 2019AEP133; Comunidad Autónoma de Madrid (grant S2013/MIT-2862); Generalitat de Catalunya (grants 2017-SGR-918, 2017-SGR-229 and CERCA Programme); the Fundació Marató de TV3 (Nr. 201812); the COST Action CA15126 Between Atom and Cell; and the European Social Fund and EU to J.V., A.V. and I.R. (H2020-INFRAIA-2014-2015; NFFA-654360).With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).Peer reviewe

Lactic acid bacteria : reviewing the potential of a promising delivery live vector for biomedical purposes

Lactic acid bacteria (LAB) have a long history of safe exploitation by humans, being used for centuries in food production and preservation and as probiotic agents to promote human health. Interestingly, some species of these Gram-positive bacteria, which are generally recognized as safe organisms by the US Food and Drug Administration (FDA), are able to survive through the gastrointestinal tract (GIT), being capable to reach and colonize the intestine, where they play an important role. Besides, during the last decades, an important effort has been done for the development of tools to use LAB as microbial cell factories for the production of proteins of interest. Given the need to develop effective strategies for the delivery of prophylactic and therapeutic molecules, LAB have appeared as an appealing option for the oral, intranasal and vaginal delivery of such molecules. So far, these genetically modified organisms have been successfully used as vehicles for delivering functional proteins to mucosal tissues in the treatment of many different pathologies including GIT related pathologies, diabetes, cancer and viral infections, among others. Interestingly, the administration of such microorganisms would suppose a significant decrease in the production cost of the treatments agents since being live organisms, such vectors would be able to autonomously amplify and produce and deliver the protein of interest. In this context, this review aims to provide an overview of the use of LAB engineered as a promising alternative as well as a safety delivery platform of recombinant proteins for the treatment of a wide range of diseases

Producció d'amiloides bacterians lliures de toxines

Els cossos d'inclusió o amiloides són agregats de proteïnes nanoestructurades produïdes a l'interior d'una cèl·lula, freqüents en determinats bacteris, i amb interessants aplicacions biomèdiques, com ara l'alliberament de fàrmacs proteics. Fins ara, l'ús dels cossos d'inclusió estava limitat per la presència de toxines pròpies de les cèl·lules bacterianes que els produïen. Un estudi ha desenvolupat diverses soques d'Escherichia coli sense aquestes toxines a partir de les quals s'ha pogut produir amiloides lliures de contaminants potencialment perillososLos cuerpos de inclusión o amiloides son agregados de proteínas nanoestructuradas producidas en el interior de una célula, frecuentes en determinadas bacterias, y con interesantes aplicaciones biomédicas, tales como la liberación de fármacos proteicos. Hasta ahora, el uso de los cuerpos de inclusión estaba limitado por la presencia de toxinas propias de las células bacterianas que los producían. Un estudio ha desarrollado diversas cepas de Escherichia coli sin estas toxinas a partir de las cuales se ha podido producir amiloides libres de contaminantes potencialmente peligrosos.Inclusion bodies or amyloids are nanostructured protein aggregates produced inside a cell, frequent in certain bacteria, and with interesting biomedical applications such as drug protein delivery. Until now, the use of inclusion bodies was limited by the presence of own toxins from the producing bacterial cells. A study has developed several toxin-free Escherichia coli strains from which amyloids free from potentially hazardous cell contaminants have been produced

Producció d'amiloides bacterians lliures de toxines

Els cossos d'inclusió o amiloides són agregats de proteïnes nanoestructurades produïdes a l'interior d'una cèl·lula, freqüents en determinats bacteris, i amb interessants aplicacions biomèdiques, com ara l'alliberament de fàrmacs proteics. Fins ara, l'ús dels cossos d'inclusió estava limitat per la presència de toxines pròpies de les cèl·lules bacterianes que els produïen. Un estudi ha desenvolupat diverses soques d'Escherichia coli sense aquestes toxines a partir de les quals s'ha pogut produir amiloides lliures de contaminants potencialment perillosos.Los cuerpos de inclusión o amiloides son agregados de proteínas nanoestructuradas producidas en el interior de una célula, frecuentes en determinadas bacterias, y con interesantes aplicaciones biomédicas, tales como la liberación de fármacos proteicos. Hasta ahora, el uso de los cuerpos de inclusión estaba limitado por la presencia de toxinas propias de las células bacterianas que los producían. Un estudio ha desarrollado diversas cepas de Escherichia coli sin estas toxinas a partir de las cuales se ha podido producir amiloides libres de contaminantes potencialmente peligrosos.Inclusion bodies or amyloids are nanostructured protein aggregates produced inside a cell, frequent in certain bacteria, and with interesting biomedical applications such as drug protein delivery. Until now, the use of inclusion bodies was limited by the presence of own toxins from the producing bacterial cells. A study has developed several toxin-free Escherichia coli strains from which amyloids free from potentially hazardous cell contaminants have been produced

Improving protein delivery of fibroblast growth factor-2 from bacterial inclusion bodies used as cell culture substrates

Altres ajuts: We are indebted CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN, Spain) for funding our research on inclusion bodies.Bacterial inclusion bodies (IBs) have recently been used to generate biocompatible cell culture interfaces, with diverse effects on cultured cells such as cell adhesion enhancement, stimulation of cell growth or induction of mesenchymal stem cell differentiation. Additionally, novel applications of IBs as sustained protein delivery systems with potential applications in regenerative medicine have been successfully explored. In this scenario, with IBs gaining significance in the biomedical field, the fine tuning of this functional biomaterial is crucial. In this work, the effect of temperature on fibroblast growth factor-2 (FGF-2) IB production and performance has been evaluated. FGF-2 was overexpressed in Escherichia coli at 25 and 37 °C, producing IBs with differences in size, particle structure and biological activity. Cell culture topographies made with FGF-2 IBs biofabricated at 25 °C showed higher levels of biological activity as well as a looser supramolecular structure, enabling a higher protein release from the particles. In addition, the controlled use of FGF-2 protein particles enabled the generation of functional topographies with multiple biological activities being effective on diverse cell types

Isolation of cell-free bacterial inclusion bodies

Background: Bacterial inclusion bodies are submicron protein clusters usually found in recombinant bacteria that have been traditionally considered as undesirable products from protein production processes. However, being fully biocompatible, they have been recently characterized as nanoparticulate inert materials useful as scaffolds for tissue engineering, with potentially wider applicability in biomedicine and material sciences. Current protocols for inclusion body isolation from Escherichia coli usually offer between 95 to 99% of protein recovery, what in practical terms, might imply extensive bacterial cell contamination, not compatible with the use of inclusion bodies in biological interfaces. Results: Using an appropriate combination of chemical and mechanical cell disruption methods we have established a convenient procedure for the recovery of bacterial inclusion bodies with undetectable levels of viable cell contamination, below 10-1 cfu/ml, keeping the particulate organization of these aggregates regarding size and protein folding features. Conclusions: The application of the developed protocol allows obtaining bacterial free inclusion bodies suitable for use in mammalian cell cultures and other biological interfaces

Complex Particulate Biomaterials as Immunostimulant-Delivery Platforms

The control of infectious diseases is a major current challenge in intensive aquaculture. Most commercial vaccines are based on live attenuated or inactivated pathogens that are usually combined with adjuvants, oil emulsions being as the most widely used for vaccination in aquaculture. Although effective, the use of these oil emulsions is plagued with important side effects. Thus, the development of alternative safer and cost-effective immunostimulants and adjuvants is highly desirable. Here we have explored the capacity of inclusion bodies produced in bacteria to immunostimulate and protect fish against bacterial infections. Bacterial inclusion bodies are highly stable, non-toxic protein-based biomaterials produced through fully scalable and low-cost bio-production processes. The present study shows that the composition and structured organization of inclusion body components (protein, lipopolysaccharide, peptidoglycan, DNA and RNA) make these protein biomaterials excellent immunomodulators able to generically protect fish against otherwise lethal bacterial challenges. The results obtained in this work provide evidence that their inherent nature makes bacterial inclusion bodies exceptionally attractive as immunostimulants and this opens the door to the future exploration of this biomaterial as an alternative adjuvant for vaccination purposes in veterinary

Production of functional inclusion bodies in endotoxin-free Escherichia coli

Escherichia coli is the workhorse for gene cloning and production of soluble recombinant proteins in both biotechnological and biomedical industries. The bacterium is also a good producer of several classes of protein-based self-assembling materials such as inclusion bodies (IBs). Apart from being a relatively pure source of protein for in vitro refolding, IBs are under exploration as functional, protein-releasing materials in regenerative medicine and protein replacement therapies. Endotoxin removal is a critical step for downstream applications of therapeutic proteins. The same holds true for IBs as they are often highly contaminated with cell-wall components of the host cells. Here, we have investigated the production of IBs in a recently developed endotoxin-free E. coli strain. The characterization of IBs revealed this mutant as a very useful cell factory for the production of functional endotoxin-free IBs that are suitable for the use at biological interfaces without inducing endotoxic responses in human immune cell

Hyaluronic acid (HA)-coated naproxen-nanoparticles selectively target breast cancer stem cells through COX-independent pathways

Cytotoxic chemotherapy continues to be the main therapeutic option for patients with metastatic breast cancer. Several studies have reported a significant association between chronic inflammation, carcinogenesis and the presence of cancer stem cells (CSC). We hypothesized that the use of non-steroidal anti-inflammatory drugs targeted to the CSC population could help reducing tumor progression and dissemination in otherwise hard to treat metastatic breast cancer. Within this study cationic naproxen (NAP)-bearing polymeric nanoparticles (NPs) were obtained by self-assembly and they were coated with hyaluronic acid (HA) via electrostatic interaction. HA-coated and uncoated NAP-bearing NPs with different sizes were produced by changing the ionic strength of the aqueous preparation solutions (i.e. 300 and 350 nm or 100 and 130 nm in diameter, respectively). HA-NPs were fully characterized in terms of physicochemical parameters and biological response in cancer cells, macrophages and endothelial cells. Our results revealed that HA-coating of NPs provided a better control in NAP release and improved their hemocompatibility, while ensuring a strong CSC-targeting in MCF-7 breast cancer cells. Furthermore, the best polymeric NPs formulation significantly (p < 0.001) reduced MCF-7 cells viability when compared to free drug (i.e. 45 ± 6% for S-HA-NPs and 87 ± 10% for free NAP) by p53-dependent induction of apoptosis; and the migration of these cell line was also significantly (p < 0.01) reduced by the nano-formulated NAP (i.e. 76.4% of open wound for S-HA-NPs and 61.6% of open wound for NAP). This increased anti-cancer activity of HA-NAP-NPs might be related to the induction of apoptosis through alterations of the GSK-3β-related COX-independent pathway. Overall, these findings suggest that the HA-NAP-NPs have the potential to improve the treatment of advanced breast cancer by increasing the anti-proliferative effect of NAP within the CSC subpopulation.Authors would like to thank the Spanish Ministry of Science, Innovation and Universities (MAT2017-84277-R) and CIBER-BBN for the financial support of this project. CIBER-BBN is financed by the Instituto de Salud Carlos III (ISCIII) with assistance from the European Regional Development Fund (ERDF).The work was also partially funded by ISCIII (PI18_00871 co-founded by ERDF), and CIBER-BBN (EXPLORE) granted to I.A. ICTS “NANBIOSIS” has participated in this work, more specifically the U20/FVPR in the hemocompatibility assays (http://www. nanbiosis.es/portfolio/u20-in-vivo-experimental-platform/). E. Espinosa-Cano would like to thank the training program for Academic Staff (FPU15/06109) of the Spanish Ministry of Education Culture and Sport. The kind support by Alvaro González-Gomez Rosana Ramírez from the Biomaterials Group (ICTP-CSIC) and Rafael Nunez ˜ from the Center for Biological Research (CIB-CSIC), in the synthesis, cell culture and cryoTEM experiments, respectively, is greatly appreciated

Integrating mechanical and biological control of cell proliferation through bioinspired multi-effector materials

In nature, cells respond to complex mechanical and biological stimuli whose understanding is required for tissue construction in regenerative medicine. However, the full replication of such bimodal effector networks is far to be reached. Engineering substrate roughness and architecture allows regulating cell adhesion, positioning, proliferation, differentiation and survival, and the external supply of soluble protein factors (mainly growth factors and hormones) has been long applied to promote growth and differentiation. Further, bioinspired scaffolds are progressively engineered as reservoirs for the in situ sustained release of soluble protein factors from functional topographies. We review here how research progresses toward the design of integrative, holistic scaffold platforms based on the exploration of individual mechanical and biological effectors and their further combination