Designing neuronal networks with chemically modified substrates: an improved approach to conventional in vitro neural systems

Highly organised structures have been well-known to be part of the complex neuronal network presented in the nervous system, where thousands of neuronal connections are arranged to give rise to critical physiological functions. Conventional in vitro culture methods are useful to represent simplistic neuronal behaviour, however, the lack of such organisation results in random and uncontrolled neurite spreading, leading to a lack of cell directionality and in turn, resulting in inaccurate neuronal in vitro models. Neurons are highly specialised cells, known to be greatly dependent on interactions with their surroundings. Therefore, when surface material is modified, drastic changes in neuronal behaviour can be achieved. The use of chemically modified surfaces in vitro has opened new avenues in cell culture, where the chaotic environment found in conventional culture methods can be controlled by the combination of surface modification methods with surface engineering techniques. Polymer brushes and self-assembled monolayers (SAMs) display a wide range of advantages as a surface modification tool for cell culture applications, since their properties can be finely tuned to promote or inhibit cellular adhesion, differentiation and proliferation. Therefore, when precisely combined with patterning techniques, a control over neuronal behaviour can be achieved. Neuronal patterning presents a system with instructive cues that can be used to study neuron-neuron communication by directing single neurites in specific locations to initiate synapses. Furthermore, although this area has not been much explored, the use of these patterned brushes could also be used in co-culture systems as a platform to closely monitor cell heterotypical communication. This research demonstrates the behaviour of SH-SY5Y neurons on a variety of SAMs and polymer brushes, both in isolation and combination to promote cellular spatial control. APTES and BIBB coatings promoted the highest cell viability, proliferation, metabolic activity and neuronal maturation, whilst low cell adhesion was seen on PKSPMA and PMETAC surfaces. Thereafter, PKSPMA brushes were used as a potential cell repulsive coating and its combination with micro- patterning techniques (photolithography and soft lithography) resulted in a system with instructive cues for neuronal guidance, where neuronal directionality was obtained. In the final chapter of this thesis, a chimeric co-culture system was developed where the patterned SH-SY5Y cells were co-cultured with C2C12 myoblasts in an attempt to obtain an organised neuronal-muscle co-culture system. Whilst preliminary observations showed first stages of a patterned neuronal-muscle co-culture, future work is necessary to refine and improve the patterned co-culture process

Decoding the endometrial niche of Asherman’s Syndrome at single-cell resolution

Infertility; Molecular medicineInfertilidad; Medicina molecularInfertilitat; Medicina molecularAsherman’s Syndrome is characterized by intrauterine adhesions or scarring, which cause infertility, menstrual abnormalities, and recurrent pregnancy loss. The pathophysiology of this syndrome remains unknown, with treatment restricted to recurrent surgical removal of intrauterine scarring, which has limited success. Here, we decode the Asherman’s Syndrome endometrial cell niche by analyzing data from over 200,000 cells with single-cell RNA-sequencing in patients with this condition and through in vitro analyses of Asherman’s Syndrome patient-derived endometrial organoids. Our endometrial atlas highlights the loss of the endometrial epithelium, alterations to epithelial differentiation signaling pathways such as Wnt and Notch, and the appearance of characteristic epithelium expressing secretory leukocyte protease inhibitor during the window of implantation. We describe syndrome-associated alterations in cell-to-cell communication and gene expression profiles that support a dysfunctional pro-fibrotic, pro-inflammatory, and anti-angiogenic environment.This study was jointly supported by Human Uterus Cell Atlas Project from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 874867, PROMETEO/2018/161 from the Valencia Government, IDI-20201142 CDTI from the Spanish Government and Carlos Simon Foundation, Spain. X.S. and E.F. were partially supported by IDI-20201142 CDTI from the Spanish Government. B.R. was supported by the H2020-funded project Human Uterus Cell Atlas (HUTER) (2020/2021) (Grant Agreement 874867). R.P. was supported by an Industrial Doctorate grant (DIN2020-011069) from the Spanish Ministry of Science and Innovation (MICINN). N.V. was supported by PROMETEO/2018/161. J.G.F. was supported by a PFIS grant [FI19/00159]. J.L. was supported by INVEST/2022/478 program. A.S. was supported by Estonian Research Council (PRG1076) and Horizon 2020 innovation grant (ERIN, grant no. EU952516). I.M. was supported by an FIS project grant [PI21/00235]. F.V. was supported by an FIS project grant [PI21/00528]. Other data that support the findings of this study are available from Asherman Therapy SL. Restrictions apply to data access with data used under license for the current clinical study and are not publicly available. Data are, however, available from the authors upon reasonable request and with permission of the Vall Hebron Ethical Committee

Neural and aneural regions generated by the use of chemical surface coatings

The disordered environment found in conventional neural cultures impedes various applications where cell directionality is a key process for functionality. Neurons are highly specialized cells known to be greatly dependent on interactions with their surroundings. Therefore, when chemical cues are incorporated on the surface material, a precise control over neuronal behavior can be achieved. Here, the behavior of SH-SY5Y neurons on a variety of self-assembled monolayers (SAMs) and polymer brushes both in isolation and combination to promote cellular spatial control was determined. APTES and BIBB coatings promoted the highest cell viability, proliferation, metabolic activity, and neuronal maturation, while low cell survival was seen on PKSPMA and PMETAC surfaces. These cell-attractive and repulsive surfaces were combined to generate a binary BIBB-PKSPMA coating, whereby cellular growth was restricted to an exclusive neural region. The utility of these coatings when precisely combined could act as a bioactive/bioinert surface resulting in a biomimetic environment where control over neuronal growth and directionality can be achieved

High-oxygen-barrier multilayer films based on polyhydroxyalkanoates and cellulose nanocrystals

This study reports on the development and characterization of organic recyclable high-oxygen-barrier multilayer films based on different commercial polyhydroxyalkanoate (PHA) materials, including a blend with commercial poly(butylene adipate-co-terephthalate) (PBAT), which contained an inner layer of cellulose nanocrystals (CNCs) and an electrospun hot-tack adhesive layer of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) derived from cheese whey (CW). As a result, the full multilayer structures were made from bio-based and/or compostable materials. A characterization of the produced films was carried out in terms of morphological, optical, mechanical, and barrier properties with respect to water vapor, limonene, and oxygen. Results indicate that the multilayer films exhibited a good interlayer adhesion and contact transparency. The stiffness of the multilayers was generally improved upon incorporation of the CNC interlayer, whereas the enhanced elasticity of the blend was reduced to some extent in the multilayer with CNCs, but this was still much higher than for the neat PHAs. In terms of barrier properties, it was found that 1 µm of the CNC interlayer was able to reduce the oxygen permeance between 71% and 86%, while retaining the moisture and aroma barrier of the control materials.This research work was funded by the H2020 EU project YPACK (reference number 773872) and by the Spanish Ministry of Science and Innovation (MICI) project RTI2018-097249-B-C21.Beatriz Melendez-Rodriguez would like to acknowledge the MICI for her FPI fellowship (BES-2016-077972) and Sergio Torres-Giner for his MICI Juan de la Cierva–Incorporación contract (IJCI-2016-29675). The authors would also like to acknowledge the Unidad Asociada in Polymer Technology, joint unit IATA(CSIC)-UJI

Controlled arrangement of neuronal cells on surfaces functionalized with micro-patterned polymer brushes

Conventional in vitro cultures are useful to represent simplistic neuronal behaviour, however the lack of organisation results in random neurite spreading. To overcome this problem, control over the directionality of SH-SY5Y cells was attained, utilising photolithography to pattern the cell-repulsive anionic brush poly(potassium 3-sulfopropyl methacrylate) (PKSPMA) into tracks of 20, 40, 80 and 100 µm width. This data validates the use of PKSPMA brush coatings for long-term culture of SH-SY5Y cells, as well as providing a methodology by which the precise deposition of PKSPMA can be utilised to achieve targeted control over SH-SY5Y cells. Specifically, PKSPMA brush patterns prevented cell attachment, allowing SH-SY5Y cells to grow only on the non-coated glass (gaps of 20, 50, 75 and 100 µm width) at different cell densities (5000, 10000 and 15000 cells/cm2). This research demonstrates the importance of achieving cell directionality in vitro, whilst these simplistic models could provide new platforms to study complex neuron-neuron interactions

Development of Active Barrier Multilayer Films Based on Electrospun Antimicrobial Hot-Tack Food Waste Derived Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and Cellulose Nanocrystal Interlayers

[EN] Active multilayer films based on polyhydroxyalkanoates (PHAs) with and without high barrier coatings of cellulose nanocrystals (CNCs) were herein successfully developed. To this end, an electrospun antimicrobial hot-tack layer made of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) derived from cheese whey, a by-product from the dairy industry, was deposited on a previously manufactured blown film of commercial food contact PHA-based resin. A hybrid combination of oregano essential oil (OEO) and zinc oxide nanoparticles (ZnONPs) were incorporated during the electrospinning process into the PHBV nanofibers at 2.5 and 2.25 wt%, respectively, in order to provide antimicrobial properties. A barrier CNC coating was also applied by casting from an aqueous solution of nanocellulose at 2 wt% using a rod at 1m/min. The whole multilayer structure was thereafter assembled in a pilot roll-to-roll laminating system, where the blown PHA-based film was located as the outer layers while the electrospun antimicrobial hot-tack PHBV layer and the barrier CNC coating were placed as interlayers. The resultant multilayer films, having a final thickness in the 130-150 mu m range, were characterized to ascertain their potential in biodegradable food packaging. The multilayers showed contact transparency, interlayer adhesion, improved barrier to water and limonene vapors, and intermediate mechanical performance. Moreover, the films presented high antimicrobial and antioxidant activities in both open and closed systems for up to 15 days. Finally, the food safety of the multilayers was assessed by migration and cytotoxicity tests, demonstrating that the films are safe to use in both alcoholic and acid food simulants and they are also not cytotoxic for Caco-2 cells.The Spanish Ministry of Science and Innovation (MICI) through the RTI2018-097249-B-C21 program number and the EU H2020 YPACK (reference number 773872) projects funded this research.Figueroa-Lopez, KJ.; Torres-Giner, S.; Angulo, I.; Pardo-Figuerez, M.; Escuin, JM.; Bourbon, AI.; Cabedo, L.... (2020). Development of Active Barrier Multilayer Films Based on Electrospun Antimicrobial Hot-Tack Food Waste Derived Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and Cellulose Nanocrystal Interlayers. Nanomaterials. 10(12):1-24. https://doi.org/10.3390/nano10122356S124101

Development of an Electrospun Patch Platform Technology for the Delivery of Carvedilol in the Oral Mucosa

The work herein presented aims to develop and characterize carvedilol (CVD) releasable non-water-soluble monolayers and a multilayer patch made of ultrathin micron and submicron fibers for drug delivery into the sublingual mucosa. Firstly, the developed formulations containing CVD within different biopolymers (PDLA, PCL, and PHB) were characterized by scanning electron microscopy (SEM), attenuated total reflectance Fourier transformed infrared spectroscopy (ATR-FTIR), differential scanning calorimetry (DSC), wide-angle X-ray scattering (WAXS), and for their in vitro drug release. SEM micrographs assessed the fiber morphology attained by adding carvedilol. ATR-FTIR spectra revealed good chemical compatibility between CVD and the tested biopolymers, whereas DSC and WAXS confirmed that CVD was in an amorphous state within the biopolymeric fibers. In vitro release studies showed enhanced CVD release kinetics from the electrospun biopolymer monolayers compared to the dissolution rate of the commercial form of the pure drug, except for the slow-releasing PDLA fibers. Finally, the selected CVD-loaded layer, i.e., electrospun PHB, was built into a three-layer patch to tackle mucosa adhesion and unidirectional release, while retaining the enhanced release kinetics. The patch design proposed here further demonstrates the potential of the electro-hydrodynamic processing technology to render unique mucoadhesive controlled delivery platforms for poorly water-soluble drugs

Super-Repellent Paper Coated with Electrospun Biopolymers and Electrosprayed Silica of Interest in Food Packaging Applications

In the current work, a super-repellent biopaper suitable for food contact applications was developed. To do this, three different kinds of biopolymers, namely polylactide (PLA), poly(ε-caprolactone) (PCL), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and hydrophobic silica microparticles (SiO2), were sequentially processed by electrohydrodynamic processing (EDHP). As a first step, the ultrathin biopolymer fibers were deposited onto a commercial food contact cellulose paper by electrospinning and, thereafter, the nanostructured silica was sequentially electrosprayed. The multilayer coated papers were annealed at different temperatures to promote adhesion between the layers and enhance the super-repellent properties. The developed coatings were characterized in terms of morphology, permeance to water vapor, adhesion, mechanical resistance, and contact and sliding angle. The resultant multilayer biopapers presented a hierarchical micro/nanostructured surface with an apparent water contact angle (WCA) higher than 155° and sliding angle (SA) lower than 10° for all the tested biopolymers used. Among the different multilayer approaches, it was observed that the paper/PHBV/SiO2 showed the best performance, in terms of water vapor permeance; resistance after the tape peeling-off test; and food super-repelling properties to water, yogurt, and custard. Overall, this study presents the successful generation of super-repellent biopapers coated with PLA, PCL, or PHBV along with hydrophobic silica microparticles and its effectiveness for easy emptying food packaging applications to reduce food waste

Development of Ciprofloxacin-Loaded Electrospun Yarns of Application Interest as Antimicrobial Surgical Suture Materials

Surgical site infections (SSI) occur very frequently during post-operative procedures and are often treated with oral antibiotics, which may cause some side effects. This type of infection could be avoided by encapsulating antimicrobial/anti-inflammatory drugs within the surgical suture materials so that they can more efficiently act on the site of action during wound closure, avoiding post-operative bacterial infection and spreading. This work was aimed at developing novel electrospun bio-based anti-infective fibre-based yarns as novel suture materials for preventing surgical site infections. For this, yarns based on flying intertwined microfibres (1.95 ± 0.22 µm) were fabricated in situ during the electrospinning process using a specially designed yarn collector. The electrospun yarn sutures (diameter 300–500 µm) were made of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with different contents of 3HV units and contained ciprofloxacin hydrochloride (CPX) as the antimicrobial active pharmaceutical ingredient (API). The yarns were then analysed by scanning electron microscopy, Fourier transform infrared spectroscopy, wide-angle X-ray scattering, differential scanning calorimetry, and in vitro drug release. The yarns were also analysed in terms of antimicrobial and mechanical properties. The material characterization indicated that the varying polymer molecular architecture affected the attained polymer crystallinity, which was correlated with the different drug-eluting profiles. Moreover, the materials exhibited the inherent stiff behaviour of PHBV, which was further enhanced by the API. Lastly, all the yarn sutures presented antimicrobial properties for a time release of 5 days against both Gram-positive and Gram-negative pathogenic bacteria. The results highlight the potential of the developed antimicrobial electrospun yarns in this study as potential innovative suture materials to prevent surgical infections

Preliminary Studies on an Innovative Bioactive Skin Soluble Beauty Mask Made by Combining Electrospinning and Dry Powder Impregnation

The world of cosmetics is now aiming at biobased materials which are skin-compatible and can be used to generate more sustainable beauty masks with enhanced bioactivity. This work presents, in this line of interest, the combination of two innovative technologies, namely electrospinning and dry powder impregnation, to generate biobased skin soluble electrospun pullulan carriers dry impregnated with chitin nanofibrils-nanolignin-glycyrrethinic acid (CLA) complexes, as effective biobased and skin compatible beauty masks. The scalability of the pullulan electrospun carrier and bioactive complexes impregnation were optimized and the morphology evaluated. Subsequently, skin compatibility and mask effectiveness were investigated in vitro and in vivo. The results showed that cell viability was optimal for both impregnated and neat pullulan fibers. Additionally, the CLA impregnated pullulan fibers were able to upregulate the endogenous antimicrobial molecule HBD-2. Preliminary studies in vivo indicated that the beauty mask containing the CLA complexes significantly decreased area, length and depth of forehead and crow’s feet wrinkles, and significantly increased moisturizing levels in the skin. The developed beauty mask was also seen to increase skin firmness, while it did not show skin irritation after the test. The work demonstrates that the combination of these two technologies may open new alternatives to more sustainable bioactive cosmetic products for the skin.This research was funded by the Bio-Based Industries Joint Undertaking under the European Union Horizon 2020 research program (BBI-H2020), PolyBioSkin project, grant number G.A. 745839.Peer reviewe