Hydrogels with dual sensitivity to temperature and pH in physiologically relevant ranges as supports for versatile controlled cell detachment.

Thermosensitive hydrogels based on the N-vinyl caprolactam (VCL), capable of allowing for cell adhesion and proliferation, as well as non-aggressive detachment by controlled temperature drop, were functionalized with 23 % or lower molar percentages of the cationizable hydrophobic unit 2-(diisopropylamino) ethyl methacrylate (DPAEMA), to obtain networks with dual sensitivity to temperature and pH. The swelling analysis of the systems has shown a transition pK (pKb) close to physiological values, dependent on the temperature of the medium (pKb of 6.6 and 6.9 when the temperature of the medium is above and below the transition temperature VPTT, respectively) and little dependence on the degree of functionalization of DPAEMA. In addition, at temperatures below the transition temperature (VPTT), the systems have shown large swelling variations as a function of the pH (i.e. below and above the pKb), exhibiting greater absorption capacity at pHs below pKb, where the DPAEMA units are cationized. Cytocompatibility and transplant capacity have been evaluated using the C166-GFP endothelial cell line. None of the thermosensitive hydrogels with variable DPAEMA content showed a delay with respect to the control without DPAEMA neither in terms of adhesion nor in proliferation. However, by increasing the percentage of DPAEMA functionalization -and decreasing thermosensitivity-, a correlative decrease in mitochondrial activity was obtained in the transplant, with significant differences for the hydrogels with DPAEMA molar percentage of 3 % or higher. Taking advantage of the proximity of the pKb to the physiological value, we have evaluated the cellular response and the capacity for transplantation after lowering the pH to 6.5, below pKb. A direct relationship of the DPAEMA functionalization degree on the detachment efficiency was observed, since the hydrogels with the highest molar load of DPAEMA showed higher mitochondrial metabolic activity after cell detachment.

Vat Photopolymerization 3D Printing of Hydrogels with Re-Adjustable Swelling.

Vat photopolymerization typically prints highly crosslinked networks. Printing hydrogels, which are also networks but with a high swelling capacity in water and therefore with low crosslinking density, is a challenge for this technique. However, it may be of interest in medicine and in other areas, since it would allow for the preparation of this type of 3D-shaped material. In this work, an approach for printing hydrogels via vat photopolymerization that uses a mixture of stable and hydrolysable crosslinkers has been evaluated so that an initial highly crosslinked network can be printed, although after hydrolysis it becomes a network with low crosslinking. This approach has been studied with PEO/PEG-related formulations, that is, with a PEG-dimethacrylate as a stable crosslinker, a PEO-related derivative carrying ß-aminoesters as a degradable crosslinker, and PEG-methyl ether acrylate and hydroxyethyl acrylate as monofunctional monomers. A wide family of formulations has been studied, maintaining the weight percentage of the crosslinkers at 15%. Resins have been studied in terms of viscosity, and the printing process has been evaluated through the generation of Jacobs working curves. It has been shown that this approach allows for the printing of pieces of different shapes and sizes via vat photopolymerization, and that these pieces can re-ajust their water content in a tailored fashion through treatments in different media (PBS or pH 10 buffer).

Technological advances in fibrin for tissue engineering.

Fibrin is a promising natural polymer that is widely used for diverse applications, such as hemostatic glue, carrier for drug and cell delivery, and matrix for tissue engineering. Despite the significant advances in the use of fibrin for bioengineering and biomedical applications, some of its characteristics must be improved for suitability for general use. For example, fibrin hydrogels tend to shrink and degrade quickly after polymerization, particularly when they contain embedded cells. In addition, their poor mechanical properties and batch-to-batch variability affect their handling, long-term stability, standardization, and reliability. One of the most widely used approaches to improve their properties has been modification of the structure and composition of fibrin hydrogels. In this review, recent advances in composite fibrin scaffolds, chemically modified fibrin hydrogels, interpenetrated polymer network (IPN) hydrogels composed of fibrin and other synthetic or natural polymers are critically reviewed, focusing on their use for tissue engineering.

Evaluation of Poly(N-Ethyl Pyrrolidine Methacrylamide) (EPA) and Derivatives as Polymeric Vehicles for miRNA Delivery to Neural Cells.

MicroRNAs (miRNAs) are endogenous, short RNA oligonucleotides that regulate the expression of hundreds of proteins to control cells' function in physiological and pathological conditions. miRNA therapeutics are highly specific, reducing the toxicity associated with off-target effects, and require low doses to achieve therapeutic effects. Despite their potential, applying miRNA-based therapies is limited by difficulties in delivery due to their poor stability, fast clearance, poor efficiency, and off-target effects. To overcome these challenges, polymeric vehicles have attracted a lot of attention due to their ease of production with low costs, large payload, safety profiles, and minimal induction of the immune response. Poly(N-ethyl pyrrolidine methacrylamide) (EPA) copolymers have shown optimal DNA transfection efficiencies in fibroblasts. The present study aims to evaluate the potential of EPA polymers as miRNA carriers for neural cell lines and primary neuron cultures when they are copolymerized with different compounds. To achieve this aim, we synthesized and characterized different copolymers and evaluated their miRNA condensation ability, size, charge, cytotoxicity, cell binding and internalization ability, and endosomal escape capacity. Finally, we evaluated their miRNA transfection capability and efficacy in Neuro-2a cells and rat primary hippocampal neurons. The results indicate that EPA and its copolymers, incorporating ß-cyclodextrins with or without polyethylene glycol acrylate derivatives, can be promising vehicles for miRNA administration to neural cells when all experiments on Neuro-2a cells and primary hippocampal neurons are considered together.

Polycaprolactone with multiscale porosity and patterned surface topography prepared using sacrificial 3D printed moulds: Towards tailor-made scaffolds.

Biocompatible three-dimensional porous scaffolds are widely used in multiple biomedical applications. However, the fabrication of tailor-made 3D structures with controlled and combined multiscale macroscopic-microscopic, surface and inner porosities in a straightforward manner is still a current challenge. Herein, we use multimaterial fused deposition modeling (FDM) to generate poly (vinyl alcohol) (PVA) sacrificial moulds filled with poly (Ɛ-caprolactone) (PCL) to generate well defined PCL 3D objects. Further on, the supercritical CO2 (SCCO2) technique, as well as the breath figures mechanism (BFs), were additionally employed to fabricate specific porous structures at the core and surfaces of the 3D PCL object, respectively. The biocompatibility of the resulting multiporous 3D structures was tested in vitro and in vivo, and the versatility of the approach was assessed by generating a vertebra model fully tunable at multiple pore size levels. In sum, the combinatorial strategy to generate porous scaffolds offers unique possibilities to fabricate intricate structures by combining the advantages of additive manufacturing (AM), which provides flexibility and versatility to generate large sized 3D structures, with advantages of the SCCO2 and BFs techniques, which allow to finely tune the macro and micro porosity at material surface and material core levels.

Fabrication of 3D cylindrical thermosensitive hydrogels as supports for cell culture and detachment of tubular cell sheets.

Pseudo interpenetrating vinyl-caprolactam (VCL) based thermosensitive tubular hydrogels with a volume phase transition temperature, VPTT, around 35 °C, have been prepared by combining two different crosslinkers, a di-methacrylate (C1) and a di-vinyl urea (C2). The molar ratio between the two crosslinkers (for a global crosslinker molar percentage of 1.9) has shown to play a key role on the properties of the hydrogel. Increasing the amount of di-vinyl urea, leads to transparent but rather fragile materials and to a lower extent of thermosensitivity, that is, to a lower variation in the hydrogel swelling upon temperature change. However, tubes prepared with a selected crosslinker molar ratio C1/C2 of 65/35 provided a compromise between transparency, thermosensitivity and maneuverability and were, thus, evaluated as supports for cell culture using premyoblastic cells. These hydrogels, used as supports, allow for surface adhesion and cell proliferation until confluence, and eventually an efficient monolayer detachment (and transplant to a 3D-printed polylactic acid (PLA) support) through a controlled drop in temperature. As a result, this method permits to obtain tubular tissue constructs with potential applications in tissue engineering such as in the elaboration of vascular grafts.

Preparation and Characterization of Plasma-Derived Fibrin Hydrogels Modified by Alginate di-Aldehyde.

Fibrin hydrogels are one of the most popular scaffolds used in tissue engineering due to their excellent biological properties. Special attention should be paid to the use of human plasma-derived fibrin hydrogels as a 3D scaffold in the production of autologous skin grafts, skeletal muscle regeneration and bone tissue repair. However, mechanical weakness and rapid degradation, which causes plasma-derived fibrin matrices to shrink significantly, prompted us to improve their stability. In our study, plasma-derived fibrin was chemically bonded to oxidized alginate (alginate di-aldehyde, ADA) at 10%, 20%, 50% and 80% oxidation, by Schiff base formation, to produce natural hydrogels for tissue engineering applications. First, gelling time studies showed that the degree of ADA oxidation inhibits fibrin polymerization, which we associate with fiber increment and decreased fiber density; moreover, the storage modulus increased when increasing the final volume of CaCl2 (1% w/v) from 80 µL to 200 µL per milliliter of hydrogel. The contraction was similar in matrices with and without human primary fibroblasts (hFBs). In addition, proliferation studies with encapsulated hFBs showed an increment in cell viability in hydrogels with ADA at 10% oxidation at days 1 and 3 with 80 µL of CaCl2; by increasing this compound (CaCl2), the proliferation does not significantly increase until day 7. In the presence of 10% alginate oxidation, the proliferation results are similar to the control, in contrast to the sample with 20% oxidation whose proliferation decreases. Finally, the viability studies showed that the hFB morphology was maintained regardless of the degree of oxidation used; however, the quantity of CaCl2 influences the spread of the hFBs.

Cardiac Extracellular Matrix Hydrogel Enriched with Polyethylene Glycol Presents Improved Gelation Time and Increased On-Target Site Retention of Extracellular Vesicles.

Stem-cell-derived extracellular vesicles (EVs) have demonstrated multiple beneficial effects in preclinical models of cardiac diseases. However, poor retention at the target site may limit their therapeutic efficacy. Cardiac extracellular matrix hydrogels (cECMH) seem promising as drug-delivery materials and could improve the retention of EVs, but may be limited by their long gelation time and soft mechanical properties. Our objective was to develop and characterize an optimized product combining cECMH, polyethylene glycol (PEG), and EVs (EVs-PEG-cECMH) in an attempt to overcome their individual limitations: long gelation time of the cECMH and poor retention of the EVs. The new combined product presented improved physicochemical properties (60% reduction in half gelation time, p < 0.001, and threefold increase in storage modulus, p < 0.01, vs. cECMH alone), while preserving injectability and biodegradability. It also maintained in vitro bioactivity of its individual components (55% reduction in cellular senescence vs. serum-free medium, p < 0.001, similar to EVs and cECMH alone) and increased on-site retention in vivo (fourfold increase vs. EVs alone, p < 0.05). In conclusion, the combination of EVs-PEG-cECMH is a potential multipronged product with improved gelation time and mechanical properties, increased on-site retention, and maintained bioactivity that, all together, may translate into boosted therapeutic efficacy.

Hyaluronic acid-fibrin hydrogels show improved mechanical stability in dermo-epidermal skin substitutes.

Human plasma-derived bilayered skin substitutes have been successfully used by our group in different skin tissue engineering applications. However, several issues associated with their poor mechanical properties were observed, and they often resulted in rapid contraction and degradation. In this sense, hydrogels composed of plasma-derived fibrin and thiolated-hyaluronic acid (HA-SH, 0.05-0.2% w/v) crosslinked with poly(ethylene glycol) diacrylate (PEGDA, 2:1, 6:1, 10:1 and 14:1 mol of thiol to moles of acrylate) were developed to reduce the shrinking rates and enhance the mechanical properties of the plasma-derived matrices. Plasma/HA-SH-PEGDA hydrogels showed a decrease in the contraction behaviour ranging from 5% to 25% and an increase in Young's modulus. Furthermore, the results showed that a minimal amount of the added HA-SH was able to escape the plasma/HA-SH-PEGDA hydrogels after incubation in PBS. The results showed that the increase in rigidity of the matrices as well as the absence of adhesion cellular moieties in the second network of HA-SH/PEGDA, resulted in a decrease in contraction in the presence of the encapsulated primary human fibroblasts (hFBs), which may have been related to an overall decrease in proliferation of hFBs found for all hydrogels after 7 days with respect to the plasma control. The metabolic activity of hFB returned to the control levels at 14 days except for the 2:1 PEGDA crosslinking ratio. The metabolic activity of primary human keratinocytes (hKCs) seeded on the hydrogels showed a decrease when high amounts of HA-SH and PEGDA crosslinker were incorporated. Organotypic skins formed in vitro after 21 days with plasma/HA-SH-PEGDA hydrogels with an HA content of 0.05% w/v and a 2:1 crosslinking ratio were up to three times thicker than the plasma controls, evidencing a reduction in contraction, while they also showed better and more homogeneous keratin 10 (K10) expression in the supra-basal layer of the epidermis. Furthermore, filaggrin expression showed the formation of an enhanced stratum corneum for the constructs containing HA. These promising results indicate the potential of using these biomimetic hydrogels as in vitro skin models for pharmaceutical products and cosmetics and future work will elucidate their potential functionality for clinical treatment.

Contraction of fibrin-derived matrices and its implications for in vitro human skin bioengineering.

It is well-known that fibroblasts play a fundamental role in the contraction of collagen and fibrin hydrogels when used in the production of in vitro bilayered skin substitutes. However, little is known about the contribution of other factors, such as the hydrogel matrix itself, on this contraction. In this work, we studied the contraction of plasma-derived fibrin hydrogels at different temperatures (4, 23, and 37°C) in an isotonic buffer (phosphate-buffered saline). These types of hydrogels presented a contraction of approximately 30% during the first 24 hr, following a similar kinetics irrespectively of the temperature. This kinetics continued in a slowed down manner to reach a plateau value of 40% contraction after 10-15 days. Contraction of commercial fibrinogen hydrogels was studied under similar conditions and the kinetics was completed after 8 hr, reaching values between 20 and 70% depending on the temperature. We attribute these substantial differences to a modulatory effect on the contraction due to plasma proteins which are initially embedded in, and progressively released from, the plasma-based hydrogels. The elastic modulus of hydrogels measured at a constant frequency decreased with increasing temperature in 7-day gels. Rheological measurements showed the absence of a strain-hardening behavior in the plasma-derived fibrin hydrogels. Finally, plasma-derived fibrin hydrogels with and without human primary fibroblast and keratinocytes were prepared in transwell inserts and their height measured over time. Both cellular and acellular gels showed a height reduction of 30% during the first 24 hr likely due to the above-mentioned intrinsic fibrin matrix contraction.

Design of novel small molecule base-pair recognizers of toxic CUG RNA transcripts characteristics of DM1.

Myotonic Dystrophy type 1 (DM1) is an incurable neuromuscular disorder caused by toxic DMPK transcripts that carry CUG repeat expansions in the 3' untranslated region (3'UTR). The intrinsic complexity and lack of crystallographic data makes noncoding RNA regions challenging targets to study in the field of drug discovery. In DM1, toxic transcripts tend to stall in the nuclei forming complex inclusion bodies called foci and sequester many essential alternative splicing factors such as Muscleblind-like 1 (MBNL1). Most DM1 phenotypic features stem from the reduced availability of free MBNL1 and therefore many therapeutic efforts are focused on recovering its normal activity. For that purpose, herein we present pyrido[2,3-d]pyrimidin-7-(8H)-ones, a privileged scaffold showing remarkable biological activity against many targets involved in human disorders including cancer and viral diseases. Their combination with a flexible linker meets the requirements to stabilise DM1 toxic transcripts, and therefore, enabling the release of MBNL1. Therefore, a set of novel pyrido[2,3-d]pyrimidin-7-(8H)-ones derivatives (1a-e) were obtained using click chemistry. 1a exerted over 20% MBNL1 recovery on DM1 toxic RNA activity in primary cell biology studies using patient-derived myoblasts. 1a promising anti DM1 activity may lead to subsequent generations of ligands, highlighting a new affordable treatment against DM1.

Efficient and Low Cytotoxicity Gene Carriers Based on Amine-Functionalized Polyvinylpyrrolidone.

Non-viral vectors are a safety tool for gene therapy to deliver therapeutic genes. Among the different non-viral vectors, polyvinylpyrrolidone (PVP), a well-known hydrosoluble, neutral, and non-toxic polymer, satisfies the requirements and becomes a suitable candidate for gene delivery. In this study, we describe the preparation of polyvinylpyrrolidones decorated with pyrrolidine, piperidine, and piperazine groups, and evaluate them in vitro as non-viral gene carriers. The properties of these new systems are compared with those of hyperbranched polyethyleneimine (PEI) used as a positive control. Their ability to complex DNA at different N/P molar ratios, from 1:1 up to 10:1, was studied through agarose gel electrophoresis and dynamic light scattering. The resulting complexes (polyplexes) were characterized and evaluated in vitro with murine fibroblast (Swiss 3T3) as non-viral gene carriers, using luciferase as the reporter gene and a calcein cytocompatibility assay. All the copolymers condensed DNA to a particle average size between 100-400 nm when used at N/P ratios of 4:1 or higher. The copolymers with piperidine groups showed higher transfection efficiency than the pyrrolidine and piperazine modified copolymers, and even higher than the positive control of PEI at N/P ratios of 4:1 or higher. All the synthesized polyplexes from an aminated PVP displayed a general tendency of high cytocompatibility (75-95%) in comparison with the positive control PEI (55%).

Categorías diagnósticas y resultados a corto plazo en los pacientes con sospecha de COVID-19 atendidos en un servicio de urgencias / Diagnostic groups and short-term outcomes in suspected COVID-19 cases treated in an emergency department

OBJETIVO: EL objetivo principal fue describir el perfil clínico y la mortalidad a los 30 días de diferentes categorías diagnósticas en los casos de COVID-19 atendidos en un servicio de urgencias (SU). MÉTODO: Análisis secundario del registro COVID-19_URG-HCSC. Se seleccionaron los casos sospechosos de COVID-19 atendidos en un SU de Madrid desde el 28 de febrero hasta el 31 de marzo de 2020. La muestra se dividió: 1) sospecha con PCR no realizada (S/PCR NR); 2) sospecha con PCR negativa (S/PCR-); 3) sospecha con PCR positiva (S/PCR+); 4) alta sospecha con PCR negativa o no realizada (AS/PCR- o NR); y 5) alta sospecha con PCR positiva (AS/PCR+). Se recogieron variables clínicas, radiológicas y microbiológicas del episodio de urgencias. La variable de resultado principal fue la mortalidad por cualquier causa a los 30 días. Las variables secundarias fueron el ingreso y la gravedad del episodio. RESULTADOS: Se incluyeron 1.993 pacientes; 17,2% S/PCR NR, 11,4% S/PCR-, 22,1% S/PCR+, 11,7% AS/PCR- o NR y 37,6% AS/PCR+. Se hallaron diferencias estadísticamente significativas respecto a las variables demográficas, comorbilidad, clínicas, radiográficas, analíticas y terapéuticas y de resultados a corto plazo en función las categorías diagnósticas. La mortalidad global a los 30 días fue de un 11,5%, 56,5% casos fueron hospitalizados y 19,6% casos sufrieron un episodio grave. Las categorías de AS y de S/PCR+ tuvieron un incremento del riesgo ajustado de mortalidad a los 30 días y de sufrir un episodio grave durante el ingreso hospitalario respecto a S/PCR-. En relación al ingreso, solo las categorías de AS tuvieron un incremento del riesgo ajustado de hospitalización respecto a la categoría de S/PCR-. CONCLUSIONES: Existen diferentes categorías diagnósticas de la enfermedad COVID-19 en función del perfil clínico y microbiológico que tienen correlato con el pronóstico a 30 días

OBJECTIVE: The primary objective was to describe the clinical characteristics and 30-day mortality rates in emergency department patients with coronavirus disease 2019 (COVID-19) in different diagnostic groupings. METHODS: Secondary analysis of the COVID-19 registry compiled by the emergency department of Hospital Clínico San Carlos in Madrid, Spain. We selected suspected COVID-19 cases treated in the emergency department between February 28 and March 31, 2020. The cases were grouped as follows: 1) suspected, no polymerase chain reaction (PCR) test (S/no-PCR); 2) suspected, negative PCR (S/PCR-); 3) suspected, positive PCR (S/PCR+); 4) highly suspected, no PCR, or negative PCR (HS/no or PCR-); and 5) highly suspected, positive PCR (HS/PCR+). We collected clinical, radiologic, and microbiologic data related to the emergency visit. The main outcome was 30-day all-cause mortality. Secondary outcomes were hospitalization and clinical severity of the episode. RESULTS: A total of 1993 cases (90.9%) were included as follows: S/no-PCR, 17.2%; S/PCR-, 11.4%; S/PCR+, 22.1%; HS/no PCR or PCR-, 11.7%; and HS/PCR+, 37.6%. Short-term outcomes differed significantly in the different groups according to demographic characteristics; comorbidity and clinical, radiographic, analytical, and therapeutic variables. Thirty-day mortality was 11.5% (56.5% in hospitalized cases and 19.6% in cases classified as severe). The 2 HS categories and the S/PCR+ category had a greater adjusted risk for 30-day mortality and for having a clinically severe episode during hospitalization in comparison with S/PCR- cases. Only the 2 HS categories showed greater risk for hospitalization than the S/PCR- cases

Clinical presentation and outcome across age categories among patients with COVID-19 admitted to a Spanish Emergency Department.

PURPOSE: To determine the differences by age-dependent categories in the clinical profile, presentation, management, and short-term outcomes of patients with laboratory-confirmed COVID-19 admitted to a Spanish Emergency Department (ED). METHODS: Secondary analysis of COVID-19_URG-HCSC registry. We included all consecutive patients with laboratory-confirmed COVID-19 admitted to the ED of the University Hospital Clinico San Carlos (Madrid, Spain). The population was divided into six age groups. Demographic, baseline and acute clinical data, and in-hospital and 30-day outcomes were collected. RESULTS: 1379 confirmed COVID-19 cases (mean age 62 (SD 18) years old; 53.5% male) were included (18.1% < 45 years; 17.8% 45-54 years; 17.9% 55-64 years; 17.2% 65-74 years; 17.0% 75-84 years; and 11.9% ≥ 85 years). A statistically significant association was found between demographic, comorbidity, clinical, radiographic, analytical, and therapeutic variables and short-term results according to age-dependent categories. There were less COVID-specific symptoms and more atypical symptoms among older people. Age was a prognostic factor for hospital admission (aOR = 1.04; 95% CI 1.02-1.05) and in-hospital (aOR = 1.08; 95% CI 1.05-1.10) and 30-day mortality (aOR = 1.07; 95% CI 1.04-1.09), and was associated with not being admitted to intensive care (aOR = 0.95; 95% CI 0.93-0.98). CONCLUSIONS: Older age is associated with less COVID-specific symptoms and more atypical symptoms, and poor short-term outcomes. Age has independent prognostic value and may help in shared decision-making in patients with confirmed COVID-19 infection.

Diagnostic groups and short-term outcomes in suspected COVID-19 cases treated in an emergency department. / Categorías diagnósticas y resultados a corto plazo en los pacientes con sospecha de COVID-19 atendidos en un servicio de urgencias.

OBJECTIVES: The primary objective was to describe the clinical characteristics and 30-day mortality rates in emergency department patients with coronavirus disease 2019 (COVID-19) in different diagnostic groupings. MATERIAL AND METHODS: Secondary analysis of the COVID-19 registry compiled by the emergency department of Hospital Clínico San Carlos in Madrid, Spain. We selected suspected COVID-19 cases treated in the emergency department between February 28 and March 31, 2020. The cases were grouped as follows: 1) suspected, no polymerase chain reaction (PCR) test (S/no-PCR); 2) suspected, negative PCR (S/PCR-); 3) suspected, positive PCR (S/PCR+); 4) highly suspected, no PCR, or negative PCR (HS/no or PCR-); and 5) highly suspected, positive PCR (HS/PCR+). We collected clinical, radiologic, and microbiologic data related to the emergency visit. The main outcome was 30-day all-cause mortality. Secondary outcomes were hospitalization and clinical severity of the episode. RESULTS: A total of 1993 cases (90.9%) were included as follows: S/no-PCR, 17.2%; S/PCR-, 11.4%; S/PCR+, 22.1%; HS/no PCR or PCR-, 11.7%; and HS/PCR+, 37.6%. Short-term outcomes differed significantly in the different groups according to demographic characteristics; comorbidity and clinical, radiographic, analytical, and therapeutic variables. Thirty-day mortality was 11.5% (56.5% in hospitalized cases and 19.6% in cases classified as severe). The 2 HS categories and the S/PCR+ category had a greater adjusted risk for 30-day mortality and for having a clinically severe episode during hospitalization in comparison with S/PCR- cases. Only the 2 HS categories showed greater risk for hospitalization than the S/PCR- cases. CONCLUSION: COVID-19 diagnostic groups differ according to clinical and laboratory characteristics, and the differences are associated with the 30-day prognosis.

OBJETIVO: El objetivo principal fue describir el perfil clínico y la mortalidad a los 30 días de diferentes categorías diagnósticas en los casos de COVID-19 atendidos en un servicio de urgencias (SU). METODO: Análisis secundario del registro COVID-19_URG-HCSC. Se seleccionaron los casos sospechosos de COVID-19 atendidos en un SU de Madrid desde el 28 de febrero hasta el 31 de marzo de 2020. La muestra se dividió: 1) sospecha con PCR no realizada (S/PCR NR); 2) sospecha con PCR negativa (S/PCR­); 3) sospecha con PCR positiva (S/ PCR+); 4) alta sospecha con PCR negativa o no realizada (AS/PCR­ o NR); y 5) alta sospecha con PCR positiva (AS/ PCR+). Se recogieron variables clínicas, radiológicas y microbiológicas del episodio de urgencias. La variable de resultado principal fue la mortalidad por cualquier causa a los 30 días. Las variables secundarias fueron el ingreso y la gravedad del episodio. RESULTADOS: Se incluyeron 1.993 pacientes; 17,2% S/PCR NR, 11,4% S/PCR­, 22,1% S/PCR+, 11,7% AS/PCR­ o NR y 37,6% AS/PCR+. Se hallaron diferencias estadísticamente significativas respecto a las variables demográficas, comorbilidad, clínicas, radiográficas, analíticas y terapéuticas y de resultados a corto plazo en función las categorías diagnósticas. La mortalidad global a los 30 días fue de un 11,5%, 56,5% casos fueron hospitalizados y 19,6% casos sufrieron un episodio grave. Las categorías de AS y de S/PCR+ tuvieron un incremento del riesgo ajustado de mortalidad a los 30 días y de sufrir un episodio grave durante el ingreso hospitalario respecto a S/PCR­. En relación al ingreso, solo las categorías de AS tuvieron un incremento del riesgo ajustado de hospitalización respecto a la categoría de S/PCR­. CONCLUSIONES: Existen diferentes categorías diagnósticas de la enfermedad COVID-19 en función del perfil clínico y microbiológico que tienen correlato con el pronóstico a 30 días.

Fabrication of 3D-Printed Biodegradable Porous Scaffolds Combining Multi-Material Fused Deposition Modeling and Supercritical CO2 Techniques.

The fabrication of porous materials for tissue engineering applications in a straightforward manner is still a current challenge. Herein, by combining the advantages of two conventional methodologies with additive manufacturing, well-defined objects with internal and external porosity were produced. First of all, multi-material fused deposition modeling (FDM) allowed us to prepare structures combining poly (ε-caprolactone) (PCL) and poly (lactic acid) (PLA), thus enabling to finely tune the final mechanical properties of the printed part with modulus and strain at break varying from values observed for pure PCL (modulus 200 MPa, strain at break 1700%) and PLA (modulus 1.2 GPa and strain at break 5-7%). More interestingly, supercritical CO2 (SCCO2) as well as the breath figures mechanism (BFs) were additionally employed to produce internal (pore diameters 80-300 µm) and external pores (with sizes ranging between 2 and 12 µm) exclusively in those areas where PCL is present. This strategy will offer unique possibilities to fabricate intricate structures combining the advantages of additive manufacturing (AM) in terms of flexibility and versatility and those provided by the SCCO2 and BFs to finely tune the formation of porous structures.

Combining Breath Figures and Supercritical Fluids To Obtain Porous Polymer Scaffolds.

Supercritical fluids technology is a clean methodology to foam polymeric materials. However, this technique provides only the formation of inner porosity, whereas the so-called skin layer is commonly observed at the polymer surface. This article describes a new method for the preparation of outer and inner porous poly(ε-caprolactone) (PCL) scaffolds by combination of supercritical CO2 (SCCO2) foaming and the breath figures technique. In the first step, experiments with a SCCO2 reactor were performed at 35-45 °C, 100-250 bar, and 1-20 min depressurization time. The effect of these parameters in the formation of inner porosity was investigated for an adequate optimization. In a late stage, to provide also surface porosity to the polymeric samples and remove the skin layer, the breath figures technique was employed. The evaluation of porosity was determined by scanning electronic microscopy, mercury porosimetry, and micro X-ray computerized tomography scanning processing the images obtained with the ImageJ software. The results of this study using these two complementary techniques showed the existence of interconnectivity between inner and outer porosity of the samples. Furthermore, thermal transitions and crystallinity of the PCL samples have been analyzed by differential scanning calorimetry. Finally, a preliminary biological evaluation of the resulting scaffolds with mouse endothelial cells (C166-GFP) was performed to assess their biocompatibility and cellular viability.

Smart pH-Responsive Antimicrobial Hydrogel Scaffolds Prepared by Additive Manufacturing.

We report on the fabrication of 3D printed pH-responsive and antimicrobial hydrogels with a micrometer-scale resolution achieved by stereolithography (SLA) 3D printing. The preparation of the hydrogels was optimized by selecting the most appropriate difunctional polyethylene glycol dimethacrylates (testing cross-linking agents with chain lengths ranging from 2 up to 14 units ethylene glycol) and introducing acrylic acid (AA) as a monofunctional monomer. As a result of the incorporation of AA, the hydrogels described are able to reversibly swell and shrink upon environmental changes on the pH, and the swelling extent is directly related to the amount of AA and can be thus finely tuned. More interestingly, upon optimization of the UV penetration depth employing a photoabsorber (Sudan I), a reliable procedure for the fabrication of 3D objects with a high model accuracy is shown. Finally, the antimicrobial properties of all of the hydrogels were demonstrated using Staphylococcus aureus as a bacterial model. We found that even those hydrogels with a low amount of AA monomeric units presented excellent antimicrobial properties against S. aureus.

Stereoselectivity of Proline/Cyclobutane Amino Acid-Containing Peptide Organocatalysts for Asymmetric Aldol Additions: A Rationale.

Several α,ß,α- or α,γ,α-tripeptides, consisting of a central cyclobutane ß- or γ-amino acid being flanked by two d- or l-proline residues, have been synthesized and tested as organocatalysts in asymmetric aldol additions. High yields and enantioselectivities have been achieved with α,γ,α-tripeptides, being superior to peptides containing a cyclobutane ß-amino acid residue. This is probably due to their high rigidity, which hinders some of the peptide catalysts to adopt the proper active conformation. This reasoning correlates with the major conformation of the peptides in the ground state, as suggested by 1H NMR and computational calculations. The configuration of the aldol products is controlled by the proline chirality, and consequently, the R/S configuration of aldol products can be tuned by the use of either commercially available d- or l-proline. The enantioselectivity in the aldol reactions is reversed if the reactions are carried out in the presence of water or other protic solvents such as methanol. Spectroscopic and theoretical investigations revealed that this effect is not the consequence of conformational changes in the catalyst but rather caused by the participation of a water molecule in the rate determining transition state, in such a way that the preferential nucleophilic attack is oriented to the opposite enantiotopic aldehyde face.

Hydroxyl versus permethylated glycopolymers as gene carriers.

The main parameters that contribute to non-viral gene delivery are chemical structure and charge distribution. Indeed, saccharide units have been reported to have specific interactions with proteins located in the outer leaflet of the plasma cell membrane that facilitate the cellular internalization of plasmid-DNA vector complexes. In this work, glycopolymers based on statistical copolymers were synthesized through radical copolymerization of a cationic unit, N-ethyl pyrrolidine methacrylamide (EPA), with two styrenic monomers derived from the hydroxylated and permethylated forms of α-glucose. These copolymers were evaluated as possible non-viral gene carriers, and their ability to complex DNA was evaluated. The transfection efficiency and cytocompatibility of the polyplexes, in both fibroblastic and tumoral murine cell lines, was evaluated. Systems derived from α-glucose (GLCSt), over a monomer concentration range of 5-70mol%, exhibited high toxicity and low transfection efficiency, and were not able to significantly improve on results obtained from positive poly-EPA (PEPA) and polyethyleneimine (PEI) controls. However, systems derived from the permethylated form of α-glucose (MGLCSt), formed stable complexes with DNA or polyplexes, which showed improved transfection efficiency and cytocompatibility in comparison to positive controls. The high transfection efficiency can be clearly attributed to their cytocompatibility, which was notably found to be different for Swiss fibroblasts and B16 melanoma cells, high for Swiss and low for B16. As such, we present permethylated MCLCSt copolymers as good candidates for the possible development of therapies against melanoma.