Cristina Martínez-Ramos

Introduction: Articular cartilage degeneration can lead to a progressive loss of joint function and, eventually, total joint failure. The use of mesenchymal stem cells (MSCs) for cartilage regeneration has proven effective; however, the difficulty of their clinical application has prompted research into other strategies, such as using MSC secretome instead of cells themselves. Our aim was to generate a culture system to obtain large amounts of MSC secretome with a controlled and known composition and the potential to induce articular cartilage regeneration. Methods: Human dental pulp stem cells (hDPSCs) embedded in alginate/agarose beads were cultured in a bioreactor with constant agitation and periodic culture medium exchange. The secretome was collected and characterized according to known chondral markers (TGF‐β1, SERPINE1, miR‐140, miR‐675, miR‐23a, miR‐204, miR‐211, and miR‐337‐5p). Metabolomic and proteomic analyses were carried out for detailed characterization of the collected secretome. The chondrogenic potential of the collected secretome was tested in an in vitro model developed using human primary chondrocytes cultured in 3D on alginate‐agarose scaffolds. Results: Our system allowed us to obtain 1200 mL of secretome from intensive cultures of hDPSCs. The cells cultured on the platform remained viable and in an active anabolic state, secreting large amounts of prochondrogenic mediators. Variability between batches of secretome was low, and the collected secretome induced primary chondrocyte differentiation in vitro. Conclusions: Genomic, metabolomic, and proteomic data indicate that hDPSCs on the platform proliferate and acquire a chondrocyte‐like phenotype. These cells secrete mediators that define a microenvironment favorable for chondral regeneration. This is further supported by evidence found in the in vitro differentiation model. Our platform allows the production of large volumes of secretome with controlled composition and chondrogenic induction potential. This is a necessary preclinical study for the subsequent analysis of the secretome obtained using in vivo experimental models.

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.

Polycaprolactone (PCL) scaffolds have demonstrated an effectiveness in articular cartilage regeneration due to their biomechanical properties. On the other hand, alginate hydrogels generate a 3D environment with great chondrogenic potential. Our aim is to generate a mixed PCL/alginate scaffold that combines the chondrogenic properties of the two biomaterials. Porous PCL scaffolds were manufactured using a modified salt-leaching method and embedded in a culture medium or alginate in the presence or absence of chondrocytes. The chondrogenic capacity was studied in vitro. Type II collagen and aggrecan were measured by immunofluorescence, cell morphology by F-actin fluorescence staining and gene expression of COL1A1, COL2A1, ACAN, COL10A1, VEGF, RUNX1 and SOX6 by reverse transcription polymerase chain reaction (RT-PCR). The biocompatibility of the scaffolds was determined in vivo using athymic nude mice and assessed by histopathological and morphometric analysis. Alginate improved the chondrogenic potential of PCL in vitro by increasing the expression of type II collagen and aggrecan, as well as other markers related to chondrogenesis. All scaffolds showed good biocompatibility in the in vivo model. The presence of cells in the scaffolds induced an increase in vascularization of the PCL/alginate scaffolds. The results presented here reinforce the benefits of the combined use of PCL and alginate for the regeneration of articular cartilage.

Effective spinal cord injury (SCI) treatment remains a significant challenge, given the complex nature of the primary injury and associated devastating loss of neural activity. Neural progenitor cell (NPC)-based therapy has emerged as a potent strategy for the treatment of SCI. However, the invasive nature of direct cell transplantation and the need to enhance graft integration into host tissue remain critical issues. We implemented an improved combinatorial approach to SCI treatment by functionalizing electrospun poly-lactic acid (PLA) membranes that support the sustained delivery of curcumin (PLA-curcumin) and act as a carrier for NPC for local transplantation. In vitro experiments demonstrate that curcumin prevents harmful oxidative and inflammatory stress by preventing death and inhibiting NF-κB activation (mimicked by treatment with hydrogen peroxide or lipopolysaccharide acid). Curcumin also enhances neurite-like outgrowth in NPC and cortical neurons in culture, which may enhance neural connectivity. In vivo transplantation of NPC on a PLA-curcumin electrospun membrane enables cell migration, reduces injured area size, and increases neuronal fiber preservation to induce a slowing of acute neural damage.

Controlled drug release systems are the subject of many investigations to achieve the therapeutic effect of drugs. They have numerous advantages, such as localized effects, lower side effects, and less onset of action. Among drug-delivery systems, electrospinning is a versatile and cost-effective method for biomedical applications. Furthermore, electrospun nanofibers are promising as drug carrier candidates due to their properties that mimic the extracellular matrix. In this work, electrospun fibers were made of Poly-L-lactic acid (PLA), one of the most widely tested materials, which has excellent biocompatible and biodegradable properties. A curcuminoid, bisdemethoxycurcumin (BDMC) was added in order to complete the drug delivery system. The PLA/BDMC membranes were characterized, and biological characteristics were examined in vitro. The results show that the average fiber diameter was reduced with the drug, which was mainly released during the first 24 h by a diffusion mechanism. It was seen that the use of our membranes loaded with BDMC enhanced the rate of proliferation in Schwann cells, the main peripheral neuroglial cells, and modulated inflammation by reducing NLRP3 inflammasome activation. Considering the results, the prepared PLA/BDMC membranes hold great potential for being used in tissue engineering applications.

Neural progenitor cells (NPCs) have been shown to serve as an efficient therapeutic strategy in different cell therapy approaches, including spinal cord injury treatment. Despite the reported beneficial effects of NPC transplantation, the low survival and differentiation rates constrain important limitations. Herein, a new methodology has been developed to overcome both limitations by applying a combination of wireless electrical and magnetic stimulation to NPCs seeded on aligned poly(lactic acid) nanofibrous scaffolds for in vitro cell conditioning prior transplantation. Two stimulation patterns were tested and compared, continuous (long stimulus applied once a day) and intermittent (short stimulus applied three times a day). The results show that applied continuous stimulation promotes NPC proliferation and preferential differentiation into oligodendrocytic and neuronal lineages. A neural-like phenotypic induction was observed when compared to unstimulated NPCs. In contrast, intermittent stimulation patterns did not affect NPC proliferation and differentiation to oligodendrocytes or astrocytes morphology with a detrimental effect on neuronal differentiation. This study provides a new approach of using a combination of electric and magnetic stimulation to induce proliferation and further neuronal differentiation, which would improve therapy outcomes in disorders such as spinal cord injury.

Axonal bundles or axonal tracts have an aligned and unidirectional architecture present in many neural structures with different lengths. When peripheral nerve injury (PNI), spinal cord injury (SCI), traumatic brain injury (TBI), or neurodegenerative disease occur, the intricate architecture undergoes alterations leading to growth inhibition and loss of guidance through large distance. In order to overcome the limitations of long-distance axonal regeneration, here we combine a poly-L-lactide acid (PLA) fiber bundle in the common lumen of a sequence of hyaluronic acid (HA) conduits or modules and pre-cultured Schwann cells (SC) as cells supportive of axon extension. This multimodular preseeded conduit is then used to induce axon growth from a dorsal root ganglion (DRG) explant placed at one of its ends and left for 21 days to follow axon outgrowth. The multimodular conduit proved effective in promoting directed axon growth, and the results may thus be of interest for the regeneration of long tissue defects in the nervous system. Furthermore, the hybrid structure grown within the HA modules consisting in the PLA fibers and the SC can be extracted from the conduit and cultured independently. This “neural cord” proved to be viable outside its scaffold and opens the door to the generation of ex vivo living nerve in vitro for transplantation.

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.