Methodology for the production of human hair follicles

Hair follicles are a signature in mammals and cover almost their entire surface. They are the most important skin-derived organs, as they are involved in diverse biological functions such as: protection, thermal isolation and to comprise a reservoir of cells for skin regeneration and wound healing. With all this in mind, disorders associated with hair loss compromise the correct functioning of the human body. Additionally, hair follicles play a crucial role in social interactions, and their loss entails psychological consequences. During adulthood, hair follicle structure is not able to regenerate, reason why most hair follicle disorders imply permanent hair loss. From all of them, Androgenetic Alopecia is probably the most socially relevant as it has a prevalence of 70% in men older than 70 years. It is a non-scaring pathology in which hair is progressively lost, following a pattern distribution by genetically predisposed hair follicles that are sensitive to androgens. Nowadays there is not any hair follicle regenerative therapies available, being restoration surgeries the only available solution. High costs associated to the restoration treatments and the low availability of hair follicles in severe cases of hair loss underline the need to develop a hair regeneration therapy in adults. Nonetheless, hair follicle is a very complex and specialized structure difficult to replicate. In this scenario, a big effort has been made by the scientific community to understand and characterize human hair morphogenesis in the embryo. Among all the different structures present in the hair follicle, the dermal papilla (DP) focuses most of the attention for regenerative purposes. It is a spherical structure, located at the base of the hair follicle that contains highly specialized fibroblasts. This structure is crucial to induce the differentiation of the hair follicle in the embryo and its maintenance throughout life in adults. Furthermore, dissected murine DPs were shown to induce the generation of new follicles in vivo upon transplantation to host recipients. Later experiments demonstrated that this property was maintained by cultured DP cells, revealing the potential of these cells to induce hair follicles in adult humans. However, extrapolation of these results to human DP is not straightforward as, contrary to rodent cells, human dermal papilla cells lose their inductive capacity as soon as the DP structure is broken, and they are placed in 2D culture. Nonetheless, it has been demonstrated that human DP cells partially and inefficiently recover their in vivo inductive capacity if cultured as 3D aggregates (spheroids) emulating the in vivo microenvironment of a DP and transplanted to the dermo-epidermal interface of human skin transplanted to immunodeficient mice. On these bases, in this thesis we aimed at: 1) to find out the reasons of the poor efficiency of the spheroids to reprogram cultured DP cells and improve it; 2) to develop an in vitro system allowing to perform these studies in a quick and versatile way; 3) that this in vitro system may have clinical and / or industrial utility. To that, two different systems were proposed to culture DP cells and mimic human DP: dermal papilla spheroids and fibrin microgels with encapsulated DP cells. Both systems were analyzed in terms of the morphology, viability, and ability to promote stem cell fate recovery of DP cells. Later, these two systems were used, together with epidermal keratinocytes, to promote hair follicle differentiation in plasma-derived fibrin matrices. Preliminary results showed the formation of hair follicle-like structures similar to that present in the first stages of embryonic hair follicle morphogenesis for both DP cells culture systems. Furthermore, hair follicle differentiation was demonstrated by the positive expression of K14, K71, K75 and K15 present in the hair follicle outer root sheath, inner root sheath, companion layer and hair germ, respectively. The proposed in vitro DP culture system, together with the previous experience of our laboratory in skin bioengineering, opens the door to the generation of 3D organotypic skin cultures containing hair follicles.Programa de Doctorado en Ciencia y Tecnología Biomédica por la Universidad Carlos III de MadridPresidente: Álvaro Meana Infiesta.- Secretario: Carlos León Canseco.- Vocal: Joan Fontdevila Fon

Evaluation of different methodologies for primary human dermal fibroblast spheroid formation: automation through 3D bioprinting technology

Cell spheroids have recently emerged as an effective tool to recapitulate native microenvironments of living organisms in an in vitro scenario, increasing the reliability of the results obtained and broadening their applications in regenerative medicine, cancer research, disease modeling and drug screening. In this study the generation of spheroids containing primary human dermal fibroblasts was approached using the two-widely employed methods: hanging-drop and U-shape low adhesion plate (LA-plate). Moreover, extrusion-based three-dimensional (3D) bioprinting was introduced to achieve a standardized and scalable production of cell spheroids, decreasing considerably the possibilities of human error. This was ensured when U-shape LA-plates were used, showing an 85% formation efficiency, increasing up to a 98% when it was automatized using the 3D bioprinting technologies. However, sedimentation effect within the cartridge led to a reduction of 20% in size of the spheroid during the printing process. Hyaluronic acid (HA) was chosen as viscosity enhancer to supplement the bioink and overcome cell sedimentation within the cartridge due to the high viability values exhibited by the cells -around 80%- at the used conditions. Finally, (ANCOVA) of spheroid size over time for different printing conditions stand out HA 0.4% (w/v) 60 kDa as the viscosity-improved bioink that exhibit the highest cell viability and spheroid formation percentages. Besides, not only did it ensure cell spheroid homogeneity over time, reducing cell sedimentation effects, but also wider spheroid diameters over time with less variability, outperforming significantly manual loading.We kindly thank Daniel García for their guidance with the rheological experiments. This work was supported by Programa de Actividades de I + D entre Grupos de Investigación de la Comunidad de Madrid, S2018/ BAA-4480, Biopieltec-CM, Programa Estatal de I + D + i Orientada a los Retos de la Sociedad, RTI2018-101627-B-I00 and Cátedra Fundación Ramón Areces. The experimental techniques used during this study were performed in the CleanRooms of Bioengineering, Universidad Carlos III de Madrid, Madrid, Spain

Long-Term Real-World Effectiveness and Safety of Ustekinumab in Crohn’s Disease Patients: The SUSTAIN Study

Background Large real-world-evidence studies are required to confirm the durability of response, effectiveness, and safety of ustekinumab in Crohn’s disease (CD) patients in real-world clinical practice. Methods A retrospective, multicentre study was conducted in Spain in patients with active CD who had received ≥1 intravenous dose of ustekinumab for ≥6 months. Primary outcome was ustekinumab retention rate; secondary outcomes were to identify predictive factors for drug retention, short-term remission (week 16), loss of response and predictive factors for short-term efficacy and loss of response, and ustekinumab safety. Results A total of 463 patients were included. Mean baseline Harvey-Bradshaw Index was 8.4. A total of 447 (96.5%) patients had received prior biologic therapy, 141 (30.5%) of whom had received ≥3 agents. In addition, 35.2% received concomitant immunosuppressants, and 47.1% had ≥1 abdominal surgery. At week 16, 56% had remission, 70% had response, and 26.1% required dose escalation or intensification; of these, 24.8% did not subsequently reduce dose. After a median follow-up of 15 months, 356 (77%) patients continued treatment. The incidence rate of ustekinumab discontinuation was 18% per patient-year of follow-up. Previous intestinal surgery and concomitant steroid treatment were associated with higher risk of ustekinumab discontinuation, while a maintenance schedule every 12 weeks had a lower risk; neither concomitant immunosuppressants nor the number of previous biologics were associated with ustekinumab discontinuation risk. Fifty adverse events were reported in 39 (8.4%) patients; 4 of them were severe (2 infections, 1 malignancy, and 1 fever). Conclusions Ustekinumab is effective and safe as short- and long-term treatment in a refractory cohort of CD patients in real-world clinical practice

Using Interpretable Machine Learning to Identify Baseline Predictive Factors of Remission and Drug Durability in Crohn’s Disease Patients on Ustekinumab

Ustekinumab has shown efficacy in Crohn's Disease (CD) patients. To identify patient profiles of those who benefit the most from this treatment would help to position this drug in the therapeutic paradigm of CD and generate hypotheses for future trials. The objective of this analysis was to determine whether baseline patient characteristics are predictive of remission and the drug durability of ustekinumab, and whether its positioning with respect to prior use of biologics has a significant effect after correcting for disease severity and phenotype at baseline using interpretable machine learning. Patients' data from SUSTAIN, a retrospective multicenter single-arm cohort study, were used. Disease phenotype, baseline laboratory data, and prior treatment characteristics were documented. Clinical remission was defined as the Harvey Bradshaw Index <= 4 and was tracked longitudinally. Drug durability was defined as the time until a patient discontinued treatment. A total of 439 participants from 60 centers were included and a total of 20 baseline covariates considered. Less exposure to previous biologics had a positive effect on remission, even after controlling for baseline disease severity using a non-linear, additive, multivariable model. Additionally, age, body mass index, and fecal calprotectin at baseline were found to be statistically significant as independent negative risk factors for both remission and drug survival, with further risk factors identified for remission

Lidocaine-loaded solid lipid microparticles (SLMPs) produced from gas-saturated solutions for wound applications

This research was funded by Xunta de Galicia [ED431F 2016/010], MCIUN [RTI2018-094131-A-I00], Agrupación Estratégica de Materiales [AeMAT-BIOMEDCO2, ED431E 2018/08], Agencia Estatal de Investigación [AEI] and FEDER funds. C.A.G.-G. acknowledges to MINECO for a Ramón y Cajal Fellowship [RYC2014-15239]. This work was partially supported by Programa de Actividades de I+D entre Grupos de Investigación de la Comunidad de Madrid [S2018/BAA-4480, Biopieltec-CM], Programa Estatal de I+D+I Orientada a los Retos de la Sociedad [RTI2018-101627-B-I00] and Cátedra Fundación Ramón Areces.The delivery of bioactive agents using active wound dressings for the management of pain and infections offers improved performances in the treatment of wound complications. In this work, solid lipid microparticles (SLMPs) loaded with lidocaine hydrochloride (LID) were processed and the formulation was evaluated regarding its ability to deliver the drug at the wound site and through the skin barrier. The SLMPs of glyceryl monostearate (GMS) were prepared with different LID contents (0, 1, 2, 4, and 10 wt.%) using the solvent-free and one-step PGSS (Particles from Gas-Saturated Solutions) technique. PGSS exploits the use of supercritical CO2 (scCO2) as a plasticizer for lipids and as pressurizing agent for the atomization of particles. The SLMPs were characterized in terms of shape, size, and morphology (SEM), physicochemical properties (ATR-IR, XRD), and drug content and release behavior. An in vitro test for the evaluation of the influence of the wound environment on the LID release rate from SLMPs was studied using different bioengineered human skin substitutes obtained by 3D-bioprinting. Finally, the antimicrobial activity of the SLMPs was evaluated against three relevant bacteria in wound infections (Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa). SLMPs processed with 10 wt.% of LID showed a remarkable performance to provide effective doses for pain relief and preventive infection effects

Interaction of a 1,3-Dicarbonyl Toxin with Ru(II)-Biimidazole Complexes for Luminescence Sensing: A Spectroscopic and Photochemical Experimental Study Rationalized by Time-Dependent Density Functional Theory Calculations

A family of ruthenium(II) complexes containing one 2,2′-biimidazole (bim) ligand and two polypyridyl (NN) ligands has been prepared and their photophysical and photochemical features have been tested in the presence of tenuazonic acid (TeA), a widespread food and feed mycotoxin of current concern. While not tested in in vivo studies, TeA and other secondary metabolites of Alternaria fungi are suspected to exert adverse effects on the human health, so sensors and rapid analytical procedures are required. It is well-known that 1,3-dicarbonyl compounds such as TeA are relatively easy to deprotonate (the pKa of TeA is 3.5), yielding an enolate anion stabilized by resonance. The chelating and hydrogen-donor features of bim allow simultaneous binding to the metal core and to the target β-diketonate delocalized anion. Such a binding induces changes in the blue absorption (40 nm bathochromic shift), red luminescence intensity (>75% quenching), and triplet lifetime (0.2 μs decrease) of the Ru(NN)2(bim)2+ luminophore. Moreover, we have computationally rationalized, by time-dependent density functional theory, the structure of the different adducts of Ru–bim complexes with TeA and the electronic nature of the spectral absorption bands and their change upon the addition of TeA.Spanish Ministry of Science and Innovation (MICINN)Generalitat Valenciana and the European Social FundDepto. de Química OrgánicaFac. de Ciencias QuímicasTRUEpu

The role of versican in the skin ECM and its interaction with hyaluronic acid

Premio Accésit Congreso SIBB 2019The extracellular matrix (ECM) is a structural network that comprises the bulk of the tissues. It acts as a supporting scaffold for the cells to develop their function and plays an active role in many processes such as proliferation and migration. Therefore, the ECM is an interesting object of study for regenerative medicine. In this article we make an extensive review of two key components of the ECM: the glycosaminoglycan Hyaluronic Acid (HA) and the proteoglycan Versican (Ver). These two molecules are present in the skin ECM and play active roles in processes such as differentiation, wound healing, hair follicle cycle and development.Jorge González-Rico would like to acknowledge to the Convocatoria Ayudantes de Investigación de la Comunidad Autónoma de Madrid (CM 2018 - PEJ-2018-AI/IND-12079). This work was partially supported by Programa de Actividades de I+D entre Grupos de Investigación de la Comunidad de Madrid, S2018/BAA-4480, Biopieltec-CM, Programa Estatal de I+D+i Orientada a los Retos de la Sociedad, RTI2018-101627-B-I00, PID2019-109820RB-I00, Programa de Apoyo a la Realización de Proyectos Interdisciplinares de I+D para Jóvenes Investigadores de la Universidad Carlos III de Madrid (project: BIOMASKIN) and Cátedra Fundación Ramón Areces

Genetic Characterization of Extensively Drug-Resistant Shigella sonnei Infections, Spain, 2021-2022

In 2022, the United Kingdom reported an increase in drug resistance in Shigella sonnei isolates. We report 33 cases in Spain genetically related to the UK cases and 4 cases with similar antimicrobial resistance profiles infected with genetically distant strains. Our results suggest circulation of multiple genetic clusters of multidrug-resistant S. sonnei in Spain.Financial support for this work came from the National Institute of Health Carlos III with the project Acción Estratégica de Salud Intramural (AESI; PI21CIII/00029).S