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The formulation of hydrogels that meet the necessary flow characteristics for their extrusion-based 3D printing while providing good printability, resolution, accuracy and stability, requires long development processes. This work presents the technological development of a hydrogel-based ink of Laponite and alginate and evaluates its printing capacity. As a novelty, this article reports a standardizable protocol to quantitatively define the best printing parameters for the development of novel inks, providing new printability evaluation parameters such as the Printing Accuracy Escalation Index. As a result, this research develops a printable Laponite-Alginate hydrogel that presents printability characteristics. This ink is employed for the reproducible manufacture of 3D printed scaffolds with versatile and complex straight or curved printing patterns for a better adaptation to different final applications. Obtained constructs prove to be stable over time thanks to the optimization of a curing process. In addition, the study of the swelling and degradation behavior of the Laponite and alginate 3D printed scaffolds in different culture media allows the prediction of their behavior in future in vitro or in vivo developments. Finally, this study demonstrates the absence of cytotoxicity of the printed formulations, hence, setting the stage for their use in the field of biomedicine.

Background: Physical activity’s established health benefits include pain control, yet there is no consensus on the required dosage according to the literature. Aim: To test the influence of exercise on perceived health in patients whose daily activities are limited due to pain. Besides, analyze the possible moderating effect of age and find a formula for exercise dosage based on the perceived level of health. Methods: Cross-sectional study based on the 2017 Spanish National Health Survey with 4,123 participants reporting activity limitations in their daily activities due to pain. The International Physical Activity Questionnaire (short version) was used to calculate energy expenditure. Data included age, sex, leisure-time physical activity, sitting time, pain-related impairments, pain medication, and perceived health. Mediation regression analysis was conducted using the SPSS PROCESS 4.0 macro. Results: Results indicate a decline in perceived health with age. The relationship between exercise (energy expenditure) and perceived health was highly significant (d=-0.224, p<0.001). Age moderated the association between pain and energy expenditure up to 75.61 years (0.1568, p=0.05) and between energy expenditure and perceived health up to 75.74 years (-0.289, p=0.05), but this effect diminished afterwards. A formula was developed to estimate energy expenditure based on the desired level of health. Conclusions: The perception of health in people who are limited in their daily activities due to pain (moderate to very much) is mediated by the amount of physical activity they perform. Besides, this perception is moderated by age, up to 76 years.

Epistaxis is one of the most common otorhinolaryngology emergencies worldwide. Although there are currently several treatments available, they present several disadvantages. This, in addition to the increasing social need of being environmentally respectful, led us to investigate whether a sponge-like scaffold (SP-CH) produced from natural by-products of the food industry - soy protein and beta-chitin - can be employed as a nasal pack for the treatment of epistaxis. To evaluate the potential of our material as a nasal pack, it was compared with two of the most commonly used nasal packs in the clinic: a basic gauze and the gold standard Merocel. Our SP-CH presented great physicochemical and mechanical properties, lost weight in aqueous medium, and could even partially degrade when incubated in blood. It was shown to be both biocompatible and hemocompatible in vitro, clearing up any doubt about its safety. It showed increased blood clotting capacity in vitro, as well as increased capacity to bind both red blood cells and platelets, compared to the standard gauze and Merocel. Finally, a rat-tail amputation model revealed that our SP-CH could even reduce bleeding time in vivo. This work, carried out from a circular economy approach, demonstrates that a green strategy can be followed to manufacture nasal packs using valorized by-products of the food industry, with equal or even better hemostatic properties than the gold standard in the clinic.

[EN] The ability of mesenchymal stromal cells (MSCs) to release a plethora of immunomodulatory factors makes them valuable candidates to overcome inflammatory bowel diseases (IBD). However, this cell therapy approach is still limited by major issues derived from nude MSC-administration, including a rapid loss of their immunomodulatory phenotype that impairs factor secretion, low persistence and impossibility to retrieve the cells in case of adverse effects. Here, we designed a licensing hydrogel system to address these limitations and thus, obtain a continuous delivery of bioactive factors. IFN gamma-loaded heparin-coated beads were included in injectable in situ crosslinking alginate hydrogels, providing a 3D microenvironment that ensured continuous inflammatory licensing, cell persistence and implant retrievability. Licensing-hydrogel encapsulated human MSCs (hMSCs) were subcutaneously xenotransplanted in an acute mouse model of ulcerative colitis. Results showed that encapsulated hMSCs exerted a delocalized systemic protection, not presenting significant differences to healthy mice in the disease activity index, colon weight/length ratio and histological score. At day 7, cells were easily retrieved and ex vivo assays showed fully viable hMSCs that retained an immunomodulatory phenotype, as they continued secreting factors including PGE2 and Gal-9. Our data demonstrate the capacity of licensing hydrogelencapsulated hMSCs to limit the in vivo progression of IBD.

[EN] In the present study we developed an injectable, bioactive and degradable hydrogel composed of alginate at 2.5% oxidation degree and calcium-activated platelet rich plasma (PRP) for wound healing applications (PRP-HG-2.5%). The alginate gives mechanical support to the hydrogel while the activated PRP provides growth factors that enhance wound healing and fibrin which creates an adequate microenvironment for cell migration and proliferation. The rheological and mechanical properties of the hydrogel were characterized. Further characterization revealed that PRPHG-2.5% showed a faster hydrolitic degradation rate than unmodified alginate and a similar platelet derived growth factor (PDGF-BB) release profile. In vitro efficacy studies, carried out in human fibroblasts and keratinocytes, showed that PRP-HG-2.5% was not cytotoxic and that it was able to promote cell adhesion and proliferation. Thereafter, in an in vivo full thickness wound healing study conducted in diabetic mice, no differences were found among PRP-HG-2.5% and its counterpart without PRP, likely due to the xenogeneic origin of the PRP. This hypothesis was validated in vitro, since a cytotoxic effect was observed after human PRP application to mouse fibroblasts. Therefore, PRP-HG-2.5% might be a promising strategy for chronic woundstreatment, although its effectiveness should be evaluated in a more reliable preclinical model.

Considering the high impact that severe Coronavirus disease 2019 (COVID-19) cases still pose on public health and their complex pharmacological management, the search for new therapeutic alternatives is essential. Mesenchymal stromal cells (MSCs) could be promising candidates as they present important immunomodulatory and anti-inflammatory properties that can combat the acute severe respiratory distress syndrome (ARDS) and the cytokine storm occurring in COVID-19, two processes that are mainly driven by an immunological misbalance. In this review, we provide a comprehensive overview of the intricate inflammatory process derived from the immune dysregulation that occurs in COVID-19, discussing the potential that the cytokines and growth factors that constitute the MSC-derived secretome present to treat the disease. Moreover, we revise the latest clinical progress made in the field, discussing the most important findings of the clinical trials conducted to date, which follow 2 different approaches: MSC-based cell therapy or the administration of the secretome by itself, as a cell-free therapy.

The development of cell microencapsulation systems began several decades ago. However, today few systems have been tested in clinical trials. For this reason, in the last years, researchers have directed efforts towards trying to solve some of the key aspects that still limit efficacy and biosafety, the two major criteria that must be satisfied to reach the clinical practice. Regarding the efficacy, which is closely related to biocompatibility, substantial improvements have been made, such as the purification or chemical modification of the alginates that normally form the microspheres. Each of the components that make up the microcapsules has been carefully selected to avoid toxicities that can damage the encapsulated cells or generate an immune response leading to pericapsular fibrosis. As for the biosafety, researchers have developed biological circuits capable of actively responding to the needs of the patients to precisely and accurately release the demanded drug dose. Furthermore, the structure of the devices has been subject of study to adequately protect the encapsulated cells and prevent their spread in the body. The objective of this review is to describe the latest advances made by scientist to improve the efficacy and biosafety of cell microencapsulation systems for sustained drug delivery, also highlighting those points that still need to be optimized.

Microencapsulation of pancreatic islets for the treatment of Type I Diabetes Mellitus (T1DM) generates a high quantity of empty microcapsules, resulting in high therapeutic graft volumes that can enhance the host’s immune response. We report a 3D printed microfluidic magnetic sorting device for microcapsules purification with the objective to reduce the number of empty microcapsules prior transplantation. In this study, INS1E pseudoislets were microencapsulated within alginate (A) and alginate-poly-L-lysine-alginate (APA) microcapsules and purified through the microfluidic device. APA microcapsules demonstrated higher mechanical integrity and stability than A microcapsules, showing better pseudoislets viability and biological function. Importantly, we obtained a reduction of the graft volume of 77.5% for A microcapsules and 78.6% for APA microcapsules. After subcutaneous implantation of induced diabetic Wistar rats with magnetically purified APA microencapsulated pseudoislets, blood glucose levels were restored into normoglycemia (< 200 mg/dL) for almost 17 weeks. In conclusion, our described microfluidic magnetic sorting device represents a great alternative approach for the graft volume reduction of microencapsulated pseudoislets and its application in T1DM disease.

[EN] Diabetic foot ulcers (DFUs) represent a major clinical challenge in the ageing population. To address this problem, rhEGF-loaded Poly-Lactic-co-Glycolic-Acid (PLGA)-Alginate microspheres (MS) were prepared by a modified w/o/w-doubleemulsion/ solvent evaporation method. Different formulations were evaluated with the aim of optimising MSs properties by adding NaCl to the surfactant solution and/or the solvent removal phase and adding alginate as a second polymer. The characterization of the developed MS showed that alginate incorporation increased the encapsulation efficiency (EE) and NaCl besides increasing the EE also became the particle surface smooth and regular. Once the MS were optimised, the target loading of rhEGF was increased to 1% (PLGA-Alginate MS), and particles were sterilised by gamma radiation to provide the correct dosage for in vivo studies. In vitro cell culture assays demonstrated that neither the microencapsulation nor the sterilisation process affected rhEGF bioactivity or rhEGF wound contraction. Finally, the MS were evaluated in vivo for treatment of the full-thickness wound model in diabetised Wistar rats. rhEGF MS treated animals showed a statistically significant decrease of the wound area by days 7 and 11, a complete re-epithelisation by day 11 and an earlier resolution of the inflammatory process. Overall, these findings demonstrate the promising potential of rhEGF-loaded MS (PLGA-Alginate MS) to promote faster and more effective wound healing, and suggest its possible application in DFU treatment.

[Background]: Previous reports on the prevalence of autosomal recessive congenital ichthyosis (ARCI) were based on single source data, such as lists of members in a patient association. These sources are likely to be incomplete. [Objectives]: We sought to describe the prevalence of ARCI. [Methods]: We obtained data from 3 incomplete sources (dermatology departments, a genetic testing laboratory, and the Spanish ichthyosis association) and combined them using the capture-recapture method. [Results]: We identified 144 living patients with ARCI. Of these, 62.5% had classic lamellar ichthyosis and 30.6% had congenital ichthyosiform erythroderma. The age distribution included fewer elderly patients than expected. The prevalence of ARCI in patients younger than 10 years, the best estimate as less subject to bias, was 16.2 cases per million inhabitants (95% confidence interval 13.3-23.0). According to the capture-recapture model, 71% of the patients were not being followed up in reference units, 92% did not have a genetic diagnosis, and 78% were not members of the ichthyosis association. [Limitations]: The prevalence of ARCI in Spain and findings related to the Spanish health care system might not be generalizable to other countries. [Conclusions]: The prevalence of ARCI is higher than previously reported. Many patients are not being followed up in reference units, do not have a genetic diagnosis, and are not members of a patient association, indicating room for improvement in their care. Data suggesting a reduced number of older patients might imply a shorter life expectancy and this requires further study.

The immobilization of cells within alginate-poly-l-lysine-alginate (APA) microcapsules has been demonstrated to be an effective technology design for long term delivery of therapeutic products. Despite promising advances, biosafety aspects still remain to be improved. Here, we describe a complete characterization of the strategy based on TGL triple-fusion reporter gene – which codifies for Herpes Simplex virus type 1 thymidine-kinase (HSV1-TK), green fluorescent protein (GFP) and Firefly Luciferase – (SFGNESTGL) to inactivate encapsulated cells and their therapeutic effects. Myoblasts genetically engineered to secrete erythropoietin (EPO) were retroviraly transduced with the SFGNESTGL plasmid to further characterize their ganciclovir (GCV)-mediated inactivation process. GCV sensitivity of encapsulated cells was 100-fold lower when compared to cells plated onto 2D surfaces. However, the number of cells per capsule and EPO secretion decayed to less than 15% at the same time that proliferation was arrested after 14 days of GCV treatment in vitro. In vivo, ten days of GCV treatment was enough to restore the increased hematocrit levels of mice implanted with encapsulated TGL-expressing and EPO-secreting cells. Altogether, these results show that TGL triple-fusion reporter gene may be a good starting point in the search of a suitable biosafety strategy to inactivate encapsulated cells and control their therapeutic effects.

Recreating the most critical aspects of the native extracellular matrix (ECM) is fundamental to understand and control the processes regulating cell fate and cell function. From the ill-defined complexity to the controlled simplicity, we discuss the different strategies that are being carried out by scientists worldwide to achieve the latest advances in the sophistication of three-dimensional (3D) scaffolds, stressing their impact on cell biology, tissue engineering and regenerative medicine. Synthetic and naturally derived polymers like polyethylene glycol, alginate, agarose, etc., together with micro- and nanofabrication techniques are allowing the creation of 3D models where biophysical and biochemical variables can be modified with high precision, orthogonality and even in real-time.

The thermally unstable adduct TpMe2Ir(C2H4)(DMAD), which was generated “in situ” by the reaction of DMAD with TpMe2Ir(C2H4)2 (1) at low temperature, reacted with different carboxylic acids to produce the following compounds: TpMe2Ir(E-C(CO2Me)double bond; length as m-dashCH(CO2Me))(H2O)(OC(O)C6H4R), (R = H, 2a; o-OH, 2b; o-Cl, 2c; m-Cl, 2d; o-NO2, 2e; m-NO2, 2f;o-Me, 2g;p-Me, 2h) and TpMe2Ir(E-C(CO2Me)double bond; length as m-dashCH(CO2Me))(H2O)(OC(O)Me) 3. In the reaction of derivative 2a with Lewis bases, TpMe2Ir(E-C(CO2Me)double bond; length as m-dashCH(CO2Me))(L)(OC(O)C6H5), (L = Py, 4a; m-Br–Py, 4b; m-Cl–Py, 4c; NCMe, 5) were obtained, of which 4b and 4c were isolated as a mixture of two isomers in which the substituted pyridine ring was present at different rotational orientations. All new compounds prepared were characterized by 1H and 13C{1H} NMR spectroscopy, the structure of compounds 2d, 2h and 4a being determined by X-ray diffraction analysis. DFT was used to analyze the relative stability and the structural orientation of the isomers.

Cell microencapsulation may represent a breakthrough to overcome problems associated with cell therapy. Advances in material biocompatibility and production protocols have put this field close to its clinical application. However, issues such as the possibility of tracking cell-containing microcapsules, monitoring cell viability, and discontinuation of the therapeutic activity when necessary, still remain unsolved. We demonstrate here simultaneous monitoring and pharmacological control of myoblasts-containing alginate microcapsules, injected in immunocompetent mice after transduction with the SFGNESTGL triple reporter retroviral vector, which contains green fluorescence protein (GFP), firefly luciferase and herpes simplex virus type 1 thymidine-kinase (HSV1-TK). Naked (as controls) or microencapsulated cells were subcutaneously injected in C57BL/6J mice and followed up by luminometry. Signal for naked cells disappeared 2 weeks after cell injection, whereas signal for microencapsulated cells remained strong for 8 months, thus demonstrating the presence of living cells. Treatment of mice with the thymidine-kinase substrate ganciclovir caused death of microencapsulated myoblasts, as seen by a drastic decay in the light emission and histological analysis. Hence, we conclude that incorporation of the SFGNESTGL vector into microencapsulated cells represents an accurate tool for controlling cell location and viability in a non-invasive way. Moreover, cell death can be induced by administration of ganciclovir, in case therapy needs to be interrupted. This system may represent a step forward in the control and biosafety of cell- and gene- therapy-based microencapsulation protocols.