Characterization of a Delivery System Based on a Hyaluronic Acid 3D Scaffold and Gelatin Microparticles.

The objective of this study was to develop and characterize a novel hyaluronic acid (HA) 3D scaffold integrated with gelatin microparticles for sustained-delivery applications. To achieve this goal, the delivery microparticles were synthesized and thoroughly characterized, focusing on their crosslinking mechanisms (vanillin and genipin), degradation profiles, and release kinetics. Additionally, the cytotoxicity of the system was assessed, and its impact on the cell adhesion and distribution using mouse fibroblasts was examined. The combination of both biomaterials offers a novel platform for the gradual release of various factors encapsulated within the microparticles while simultaneously providing cell protection, support, and controlled factor dispersion due to the HA 3D scaffold matrix. Hence, this system offers a platform for addressing injure repair by continuously releasing specific encapsulated factors for optimal tissue regeneration. Additionally, by leveraging the properties of HA conjugates with small drug molecules, we can enhance the solubility, targeting capabilities, and cellular absorption, as well as prolong the system stability and half-life. As a result, this integrated approach presents a versatile strategy for therapeutic interventions aimed at promoting tissue repair and regeneration.

Electric field bridging-effect in electrified microfibrils' scaffolds.

Introduction: The use of biocompatible scaffolds combined with the implantation of neural stem cells, is increasingly being investigated to promote the regeneration of damaged neural tissue, for instance, after a Spinal Cord Injury (SCI). In particular, aligned Polylactic Acid (PLA) microfibrils' scaffolds are capable of supporting cells, promoting their survival and guiding their differentiation in neural lineage to repair the lesion. Despite its biocompatible nature, PLA is an electrically insulating material and thus it could be detrimental for increasingly common scaffolds' electric functionalization, aimed at accelerating the cellular processes. In this context, the European RISEUP project aims to combine high intense microseconds pulses and DC stimulation with neurogenesis, supported by a PLA microfibrils' scaffold. Methods: In this paper a numerical study on the effect of microfibrils' scaffolds on the E-field distribution, in planar interdigitated electrodes, is presented. Realistic microfibrils' 3D CAD models have been built to carry out a numerical dosimetry study, through Comsol Multiphysics software. Results: Under a voltage of 10 V, microfibrils redistribute the E-field values focalizing the field streamlines in the spaces between the fibers, allowing the field to pass and reach maximum values up to 100 kV/m and values comparable with the bare electrodes' device (without fibers). Discussion: Globally the median E-field inside the scaffolded electrodes is the 90% of the nominal field, allowing an adequate cells' exposure.

Hybrid Endometrial-Derived Hydrogels: Human Organoid Culture Models and In Vivo Perspectives.

The endometrium plays a vital role in fertility, providing a receptive environment for embryo implantation and development. Understanding the endometrial physiology is essential for developing new strategies to improve reproductive healthcare. Human endometrial organoids (hEOs) are emerging as powerful models for translational research and personalized medicine. However, most hEOs are cultured in a 3D microenvironment that significantly differs from the human endometrium, limiting their applicability in bioengineering. This study presents a hybrid endometrial-derived hydrogel that combines the rigidity of PuraMatrix (PM) with the natural scaffold components and interactions of a porcine decellularized endometrial extracellular matrix (EndoECM) hydrogel. This hydrogel provides outstanding support for hEO culture, enhances hEO differentiation efficiency due to its biochemical similarity with the native tissue, exhibits superior in vivo stability, and demonstrates xenogeneic biocompatibility in mice over a 2-week period. Taken together, these attributes position this hybrid endometrial-derived hydrogel as a promising biomaterial for regenerative treatments in reproductive medicine.

Polylactic Acid Nanofiber Membranes Grafted with Carbon Nanotubes with Enhanced Mechanical and Electrical Properties.

Electroconductive materials based on poly(lactic acid) (PLA) electrospinning membranes grafted with carbon nanotubes (CNTs) functionalized with the carboxylic group R-COOH have been obtained. PLA electrospun membranes were modified with sulfuric acid (H2SO4) to oxidize its surface to subsequently graft the CNTs, the treatment time and drying of the membranes before grafting with CNTs being critical, influencing the final properties of the materials. SEM images showed that CNTs presented a uniform distribution on the surface of the PLA nanofibers, while FTIR spectra of PLA-CNTs materials revealed characteristic hydroxyl groups, as evidenced by absorption peaks of CNTs. Thanks to the grafting with CNTs, the resulting PLA-CNTs membranes present an improvement of the mechanical and conductive properties when compared with PLA membranes. On the one hand, grafting with CNTs causes the nanofibers to have greater rigidity, so they are more manipulable and can more easily preserve their conformation when stress is exerted. On the other hand, grafting with CNTs allows elimination of the insulating barrier of the PLA, reducing the resistivity and providing high electrical conductivity to the PLA-CNTs membranes. The incorporation of CNTs into PLA electrospun membranes is expected to offer greater functionalities to electrospun composite nanofibers for medical and industrial applications.

Electrical Stimulation Increases Axonal Growth from Dorsal Root Ganglia Co-Cultured with Schwann Cells in Highly Aligned PLA-PPy-Au Microfiber Substrates.

Nerve regeneration is a slow process that needs to be guided for distances greater than 5 mm. For this reason, different strategies are being studied to guide axonal growth and accelerate the axonal growth rate. In this study, we employ an electroconductive fibrillar substrate that is able to topographically guide axonal growth while accelerating the axonal growth rate when subjected to an exogenous electric field. Dorsal root ganglia were seeded in co-culture with Schwann cells on a substrate of polylactic acid microfibers coated with the electroconductive polymer polypyrrole, adding gold microfibers to increase its electrical conductivity. The substrate is capable of guiding axonal growth in a highly aligned manner and, when subjected to an electrical stimulation, an improvement in axonal growth is observed. As a result, an increase in the maximum length of the axons of 19.2% and an increase in the area occupied by the axons of 40% were obtained. In addition, an upregulation of the genes related to axon guidance, axogenesis, Schwann cells, proliferation and neurotrophins was observed for the electrically stimulated group. Therefore, our device is a good candidate for nerve regeneration therapies.

Alginate-Agarose Hydrogels Improve the In Vitro Differentiation of Human Dental Pulp Stem Cells in Chondrocytes. A Histological Study.

Matrix-assisted autologous chondrocyte implantation (MACI) has shown promising results for cartilage repair, combining cultured chondrocytes and hydrogels, including alginate. The ability of chondrocytes for MACI is limited by different factors including donor site morbidity, dedifferentiation, limited lifespan or poor proliferation in vitro. Mesenchymal stem cells could represent an alternative for cartilage regeneration. In this study, we propose a MACI scaffold consisting of a mixed alginate-agarose hydrogel in combination with human dental pulp stem cells (hDPSCs), suitable for cartilage regeneration. Scaffolds were characterized according to their rheological properties, and their histomorphometric and molecular biology results. Agarose significantly improved the biomechanical behavior of the alginate scaffolds. Large scaffolds were manufactured, and a homogeneous distribution of cells was observed within them. Although primary chondrocytes showed a greater capacity for chondrogenic differentiation, hDPSCs cultured in the scaffolds formed large aggregates of cells, acquired a rounded morphology and expressed high amounts of type II collagen and aggrecan. Cells cultured in the scaffolds expressed not only chondral matrix-related genes, but also remodeling proteins and chondrocyte differentiation factors. The degree of differentiation of cells was proportional to the number and size of the cell aggregates that were formed in the hydrogels.

BDNF-Gene Transfected Schwann Cell-Assisted Axonal Extension and Sprouting on New PLA-PPy Microfiber Substrates.

The work here reported analyzes the effect of increased efficiency of brain-derived neurotrophic factor (BDNF) production by electroporated Schwann cells (SCs) on the axonal extension in a coculture system on a biomaterial platform that can be of interest for the treatment of injuries of the nervous system, both central and peripheral. Rat SCs are electrotransfected with a plasmid coding for the BDNF protein in order to achieve an increased expression and release of this protein into the culture medium of the cells, performing the best balance between the level of transfection and the number of living cells. Gene-transfected SCs show an about 100-fold increase in the release of BDNF into the culture medium, compared to nonelectroporated SCs. Cocultivation of electroporated SCs with rat dorsal root ganglia (DRG) is performed on highly aligned substrates of polylactic acid (PLA) microfibers coated with the electroconductive polymer polypyrrol (PPy). The coculture of DRG with electrotransfected SCs increase both the axonal extension and the axonal sprouting from DRG neurons compared to the coculture of DRG with nonelectroporated SCs. Therefore, the use of PLA-PPy highly aligned microfiber substrates preseeded with electrotransfected SCs with an increased BDNF secretion is capable of both guiding and accelerating axonal growth.

Axonal extension from dorsal root ganglia on fibrillar and highly aligned poly(lactic acid)-polypyrrole substrates obtained by two different techniques: Electrospun nanofibres and extruded microfibres.

The biological behaviour of Schwann cells (SCs) and dorsal root ganglia (DRG) on fibrillar, highly aligned and electroconductive substrates obtained by two different techniques is studied. Mats formed by nanometer-sized fibres of poly(lactic acid) (PLA) are obtained by the electrospinning technique, while bundles formed by micrometer-sized extruded PLA fibres are obtained by grouping microfibres together. Both types of substrates are coated with the electrically conductive polymer polypyrrole (PPy) and their morphological, physical and electrical characterization is carried out. SCs on micrometer-sized substrates show a higher motility and cell-cell interaction, while a higher cell-material interaction with a lower cell motility is observed for nanometer-sized substrates. This higher motility and cell-cell interaction of SCs on the micrometer-sized substrates entails a higher axonal growth from DRG, since the migration of SCs from the DRG body is accelerated and, therefore, the SCs tapestry needed for the axonal growth is formed earlier on the substrate. A higher length and area of the axons is observed for these micrometer-sized substrates, as well as a higher level of axonal sprouting when compared with the nanometer-sized ones. These substrates offer the possibility of being electrically stimulated in different tissue engineering applications of the nervous system.

Conduits based on the combination of hyaluronic acid and silk fibroin: Characterization, in vitro studies and in vivo biocompatibility.

We address the production of structures intended as conduits made from natural biopolymers, capable of promoting the regeneration of axonal tracts. We combine hyaluronic acid (HA) and silk fibroin (SF) with the aim of improving mechanical and biological properties of HA. The results show that SF can be efficiently incorporated into the production process, obtaining conduits with tubular structure with a matrix of HA-SF blend. HA-SF has better mechanical properties than sole HA, which is a very soft hydrogel, facilitating manipulation. Culture of rat Schwann cells shows that cell adhesion and proliferation are higher than in pure HA, maybe due to the binding motifs contributed by the SF protein. This increased proliferation accelerates the formation of a tight cell layer, which covers the inner channel surface of the HA-SF tubes. Biocompatibility of the scaffolds was studied in immunocompetent mice. Both HA and HA-SF scaffolds were accepted by the host with no residual immune response at 8 weeks. New collagen extracellular matrix and new blood vessels were visible and they were present earlier when SF was present. The results show that incorporation of SF enhances the mechanical properties of the materials and results in promising biocompatible conduits for tubulization strategies.