Alginate Improves the Chondrogenic Capacity of 3D PCL Scaffolds In Vitro: A Histological Approach.

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.

Characterization of Electrospun BDMC-Loaded PLA Nanofibers with Drug Delivery Function and Anti-Inflammatory Activity.

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.

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.

Protein adsorption/desorption dynamics on Ca-enriched titanium surfaces: biological implications.

Calcium ions are used in the development of biomaterials for the promotion of coagulation, bone regeneration, and implant osseointegration. Upon implantation, the time-dependent release of calcium ions from titanium implant surfaces modifies the physicochemical characteristics at the implant-tissue interface and thus, the biological responses. The aim of this study is to examine how the dynamics of protein adsorption on these surfaces change over time. Titanium discs with and without Ca were incubated with human serum for 2 min, 180 min, and 960 min. The layer of proteins attached to the surface was characterised using nLC-MS/MS. The adsorption kinetics was different between materials, revealing an increased adsorption of proteins associated with coagulation and immune responses prior to Ca release. Implant-blood contact experiments confirmed the strong coagulatory effect for Ca surfaces. We employed primary human alveolar osteoblasts and THP-1 monocytes to study the osteogenic and inflammatory responses. In agreement with the proteomic results, Ca-enriched surfaces showed a significant initial inflammation that disappeared once the calcium was released. The distinct protein adsorption/desorption dynamics found in this work demonstrated to be useful to explain the differential biological responses between the titanium and Ca-ion modified implant surfaces.

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.

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.

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.

Synthesis and properties of caprolactone and ethylene glycol copolymers for neural regeneration.

Copolymer networks from poly(ethylene glycol) methacrylate (PEGMA) and caprolactone 2-(methacryloyloxy) ethyl ester were synthesized and the resulting structure of the copolymer network was characterized by differential scanning calorimetry, thermogravimetry, Fourier transform infrared spectroscopy, equilibrium water gain and dynamic mechanical analysis, results which were employed to conclude about the network structure of the resulting copolymers. The new material is a random copolymer with a good miscibility and increasing hydrophilicity as the PEGMA content increases in the composition. Physical data suggest an excess free volume and synergistic interactions between the lateral chains of both comonomers. Olfactory ensheathing cells were cultured on the different networks, and cell viability and proliferation were assessed by MTS assay. The copolymers with a 30 wt% of PEGMA showed the best results compared with the other compositions in this respect, indicating the relevance for biological performance of a balance of hydrophilic and hydrophobic functionalities in the polymer chain.

Multimodular Bio-Inspired Organized Structures Guiding Long-Distance Axonal Regeneration.

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.

Novel Tissue-Engineered Multimodular Hyaluronic Acid-Polylactic Acid Conduits for the Regeneration of Sciatic Nerve Defect.

The gold standard for the treatment of peripheral nerve injuries, the autograft, presents several drawbacks, and engineered constructs are currently suitable only for short gaps or small diameter nerves. Here, we study a novel tissue-engineered multimodular nerve guidance conduit for the treatment of large nerve damages based in a polylactic acid (PLA) microfibrillar structure inserted inside several co-linear hyaluronic acid (HA) conduits. The highly aligned PLA microfibers provide a topographical cue that guides axonal growth, and the HA conduits play the role of an epineurium and retain the pre-seeded auxiliary cells. The multimodular design increases the flexibility of the device. Its performance for the regeneration of a critical-size (15 mm) rabbit sciatic nerve defect was studied and, after six months, very good nerve regeneration was observed. The multimodular approach contributed to a better vascularization through the micrometrical gaps between HA conduits, and the pre-seeded Schwann cells increased axonal growth. Six months after surgery, a cross-sectional available area occupied by myelinated nerve fibers above 65% at the central and distal portions was obtained when the multimodular device with pre-seeded Schwann cells was employed. The results validate the multi-module approach for the regeneration of large nerve defects and open new possibilities for surgical solutions in this field.

A Strategy for Magnetic and Electric Stimulation to Enhance Proliferation and Differentiation of NPCs Seeded over PLA Electrospun Membranes.

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.

Engineered axon tracts within tubular biohybrid scaffolds.

Injuries to the nervous system that involve the disruption of axonal pathways are devastating to the individual and require specific tissue engineering strategies. Here we analyse a cells-biomaterials strategy to overcome the obstacles limiting axon regenerationin vivo, based on the combination of a hyaluronic acid (HA) single-channel tubular conduit filled with poly-L-lactide acid (PLA) fibres in its lumen, with pre-cultured Schwann cells (SCs) as cells supportive of axon extension. The HA conduit and PLA fibres sustain the proliferation of SC, which enhance axon growth acting as a feeder layer and growth factor pumps. The parallel unidirectional ensemble formed by PLA fibres and SC tries to recapitulate the directional features of axonal pathways in the nervous system. A dorsal root ganglion (DRG) explant is planted on one of the conduit's ends to follow axon outgrowth from the DRG. After a 21 d co-culture of the DRG + SC-seeded conduit ensemble, we analyse the axonal extension throughout the conduit by scanning, transmission electronic and confocal microscopy, in order to study the features of SC and the grown axons and their association. The separate effects of SC and PLA fibres on the axon growth are also experimentally addressed. The biohybrid thus produced may be considered a synthetic axonal pathway, and the results could be of use in strategies for the regeneration of axonal tracts.

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.

A Standardised Approach to the Biomechanical Evaluation of Tracheal Grafts.

The ideal tracheal substitute must have biomechanical properties comparable to the native trachea, but currently there is no standardised approach to evaluating these properties. Here we propose a novel method for evaluating and comparing the properties of tracheal substitutes, thus systematising both measurement and data curation. This system was tested by comparing native rabbit tracheas to frozen and decellularised specimens and determining the histological characteristics of those specimens. We performed radial compression tests on the anteroposterior tracheal axis and longitudinal axial tensile tests with the specimens anastomosed to the jaw connected to a measuring system. All calculations and results were adjusted according to tracheal size, always using variables relative to the tracheal dimensions, thus permitting comparison of different sized organs. The biomechanical properties of the decellularised specimens were only slightly reduced compared to controls and significant in regard to the maximum stress withstood in the longitudinal axis (-0.246 MPa CI [-0.248, -0.145] MPa) and the energy stored per volume unit (-0.124 mJ·mm-3 CI [-0.195, -0.055] mJ·mm-3). The proposed method is suitable for the systematic characterisation of the biomechanical properties of different tracheal substitutes, regardless of the size or nature of the substitute, thus allowing for direct comparisons.

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.

Influence of calcium ion-modified implant surfaces in protein adsorption and implant integration.

BACKGROUND: Calcium (Ca) is a well-known element in bone metabolism and blood coagulation. Here, we investigate the link between the protein adsorption pattern and the in vivo responses of surfaces modified with calcium ions (Ca-ion) as compared to standard titanium implant surfaces (control). We used LC-MS/MS to identify the proteins adhered to the surfaces after incubation with human serum and performed bilateral surgeries in the medial section of the femoral condyles of 18 New Zealand white rabbits to test osseointegration at 2 and 8 weeks post-implantation (n=9). RESULTS: Ca-ion surfaces adsorbed 181.42 times more FA10 and 3.85 times less FA12 (p<0.001), which are factors of the common and the intrinsic coagulation pathways respectively. We also detected differences in A1AT, PLMN, FA12, KNG1, HEP2, LYSC, PIP, SAMP, VTNC, SAA4, and CFAH (p<0.01). At 2 and 8 weeks post-implantation, the mean bone implant contact (BIC) with Ca-ion surfaces was respectively 1.52 and 1.25 times higher, and the mean bone volume density (BVD) was respectively 1.35 and 1.13 times higher. Differences were statistically significant for BIC at 2 and 8 weeks and for BVD at 2 weeks (p<0.05). CONCLUSIONS: The strong thrombogenic protein adsorption pattern at Ca-ion surfaces correlated with significantly higher levels of implant osseointegration. More effective implant surfaces combined with smaller implants enable less invasive surgeries, shorter healing times, and overall lower intervention costs, especially in cases of low quantity or quality of bone.

Evaluation of the inflammatory responses to sol-gel coatings with distinct biocompatibility levels.

The immune system plays a crucial role in determining the implantation outcome, and macrophages are in the frontline of the inflammatory processes. Further, cellular oxidative stress resulting from the material recognition can influence how cell responses develop. Considering this, the aim of this study was to study oxidative stress and macrophages phenotypes in response to sol-gel materials with distinct in vivo outcomes. Four materials were selected (70M30T and 35M35G30T, with high biocompatibility, and 50M50G and 50V50G, with low biocompatibility). Gene expression, immunocytochemistry and cytokine secretion profiles for M1 and M2 markers were determined. Moreover, oxidative stress markers were studied. Immunocytochemistry and ELISA showed that 50M50G and 50V50G lead to a higher differentiation to M1 phenotype, while 70M30T and 35M35G30T promoted M2 differentiation. In oxidative stress, no differences were found. These results show that the balance between M1 and M2, more than individual quantification of each phenotype, determines a biomaterial outcome.

Characterization of magnesium doped sol-gel biomaterial for bone tissue regeneration: The effect of Mg ion in protein adsorption.

Magnesium is the fourth most abundant element in the human body with a wide battery of functions in the maintenance of normal cell homeostasis. In the bone, this element incorporates in the hydroxyapatite structure and it takes part in mineral metabolism and regulates osteoclast functions. In this study, sol-gel materials with increasing concentrations of MgCl2 (0.5, 1, and 1.5%) were synthesized and applied onto Ti surfaces as coatings. The materials were first physicochemically characterized. In vitro responses were examined using the MC3T3-E1 osteoblastic cells and RAW264.7 macrophages. Human serum protein adsorption was evaluated employing nLC-MS/MS. The incorporation of Mg did not affect the crosslinking of the sol-gel network, and a controlled release of Mg was observed; it was not cytotoxic at any of the tested concentrations. The cytoskeleton arrangement of MC3T3-E1 cells cultured on the Mg-doped materials changed in comparison with controls; the cells became more elongated, with protruded lamellipodia and increased cell surface. The expression of integrins (ITGA5 and ITGB1) was boosted by Mg-coatings. The ALP activity and expression of TGF-ß, OSX and RUNX2 genes were also increased. In RAW264.7 cells, TNF-α secretion was reduced, while TGF-ß and IL-4 expression rose. These changes correlated with the altered protein adsorption patterns. The Mg-doped coatings showed increased adsorption of anti-inflammatory (CLUS, IC1, CFAH, and VTNC), cell adhesion (DSG1, FILA2, and DESP) and tissue regeneration (VTNC and CYTA) proteins. This integrated approach to biomaterial characterization revealed the potential of Mg in bone tissue regeneration.

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.