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.

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 Hyaluronic Acid Demilune Scaffold and Polypyrrole-Coated Fibers Carrying Embedded Human Neural Precursor Cells and Curcumin for Surface Capping of Spinal Cord Injuries.

Tissue engineering, including cell transplantation and the application of biomaterials and bioactive molecules, represents a promising approach for regeneration following spinal cord injury (SCI). We designed a combinatorial tissue-engineered approach for the minimally invasive treatment of SCI-a hyaluronic acid (HA)-based scaffold containing polypyrrole-coated fibers (PPY) combined with the RAD16-I self-assembling peptide hydrogel (Corning® PuraMatrix™ peptide hydrogel (PM)), human induced neural progenitor cells (iNPCs), and a nanoconjugated form of curcumin (CURC). In vitro cultures demonstrated that PM preserves iNPC viability and the addition of CURC reduces apoptosis and enhances the outgrowth of Nestin-positive neurites from iNPCs, compared to non-embedded iNPCs. The treatment of spinal cord organotypic cultures also demonstrated that CURC enhances cell migration and prompts a neuron-like morphology of embedded iNPCs implanted over the tissue slices. Following sub-acute SCI by traumatic contusion in rats, the implantation of PM-embedded iNPCs and CURC with PPY fibers supported a significant increase in neuro-preservation (as measured by greater ßIII-tubulin staining of neuronal fibers) and decrease in the injured area (as measured by the lack of GFAP staining). This combination therapy also restricted platelet-derived growth factor expression, indicating a reduction in fibrotic pericyte invasion. Overall, these findings support PM-embedded iNPCs with CURC placed within an HA demilune scaffold containing PPY fibers as a minimally invasive combination-based alternative to cell transplantation alone.

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.

New bioreactor for mechanical stimulation of cultured tendon-like constructs: design and validation.

OBJECTIVE: Although several different types of bioreactors are currently available with mechanical stimulation of constructs or prostheses for tendon regeneration, they are in many cases expensive and difficult to operate. This paper proposes a simple bioreactor to mechanically stimulate up to three constructs for tendon and ligament repair, composed of a stainless-steel frame and an electric motor. METHODS: The deformation is produced by a cam wheel, whose eccentricity defines the maximum deformation. The test samples, braids of PLA seeded in surface with mouse fibroblasts, are immersed in the culture medium during mechanical stimulation. RESULTS: Its advantages over existing similar bioreactor designs include: easy renewal of the culture medium and an external electric motor to avoid heating and contamination issues. After 14 days of stretching, the culture samples showed enhanced cellular proliferation and cell fiber alignment in addition to higher production of type I collagen. The cells initially seeded on the braid surface migrated to the inside of the braid. CONCLUSION: Although the results obtained have a poor statistical basis, they do suggest that the bioreactor could be usefully applied to stimulate constructs for tendon and ligament repair. Anyway, further experiments should be conducted in the future.

Biohybrids for spinal cord injury repair.

Spinal cord injuries (SCIs) result in the loss of sensory and motor function with massive cell death and axon degeneration. We have previously shown that transplantation of spinal cord-derived ependymal progenitor cells (epSPC) significantly improves functional recovery after acute and chronic SCI in experimental models, via neuronal differentiation and trophic glial cell support. Here, we propose an improved procedure based on transplantation of epSPC in a tubular conduit of hyaluronic acid containing poly (lactic acid) fibres creating a biohybrid scaffold. In vitro analysis showed that the poly (lactic acid) fibres included in the conduit induce a preferential neuronal fate of the epSPC rather than glial differentiation, favouring elongation of cellular processes. The safety and efficacy of the biohybrid implantation was evaluated in a complete SCI rat model. The conduits allowed efficient epSPC transfer into the spinal cord, improving the preservation of the neuronal tissue by increasing the presence of neuronal fibres at the injury site and by reducing cavities and cyst formation. The biohybrid-implanted animals presented diminished astrocytic reactivity surrounding the scar area, an increased number of preserved neuronal fibres with a horizontal directional pattern, and enhanced coexpression of the growth cone marker GAP43. The biohybrids offer an improved method for cell transplantation with potential capabilities for neuronal tissue regeneration, opening a promising avenue for cell therapies and SCI treatment.

Biohybrids of scaffolding hyaluronic acid biomaterials plus adipose stem cells home local neural stem and endothelial cells: Implications for reconstruction of brain lesions after stroke.

Endogenous neurogenesis in stroke is insufficient to replace the lost brain tissue, largely due to the lack of a proper biological structure to let new cells dwell in the damaged area. We hypothesized that scaffolds made of hyaluronic acid (HA) biomaterials (BM) could provide a suitable environment to home not only new neurons, but also vessels, glia and neurofilaments. Further, the addition of exogenous cells, such as adipose stem cells (ASC) could increase this effect. Athymic mice were randomly assigned to a one of four group: stroke alone, stroke and implantation of BM, stroke and implantation of BM with ASC, and sham operated animals. Stroke model consisted of middle cerebral artery thrombosis with FeCl3 . After 30 days, animals underwent magnetic resonance imaging (MRI) and were sacrificed. Proliferation and neurogenesis increased at the subventricular zone ipsilateral to the ventricle and neuroblasts, glial, and endothelial cells forming capillaries were seen inside the BM. Those effects increased when ASC were added, while there was less inflammatory reaction. Three-dimensional scaffolds made of HA are able to home newly formed neurons, glia, and endothelial cells permitting the growth neurofilaments inside them. The addition of ASC increase these effects and decrease the inflammatory reaction to the implant. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1598-1606, 2019.

Influence of synthesis parameters on hyaluronic acid hydrogels intended as nerve conduits.

Hydrogels have widely been proposed lately as strategies for neural tissue regeneration, but there are still some issues to be solved before their efficient use in tissue engineering of trauma, stroke or the idiopathic degeneration of the nervous system. In a previous work of the authors a novel Schwann-cell structure with the shape of a hollow cylinder was obtained using a three-dimensional conduit based in crosslinked hyaluronic acid as template. This original engineered tissue of tightly joined Schwann cells obtained in a conduit lumen having 400 µm in diameter is a consequence of specific cell-material interactions. In the present work we analyze the influence of the hydrogel concentration and of the drying process on the physicochemical and biological performance of the resulting tubular scaffolds, and prove that the cylinder-like cell sheath obtains also in scaffolds of a larger inner diameter. The diffusion of glucose and of the protein BSA through the scaffolds is studied and characterized, as well as the enzymatic degradation kinetics of the lyophilized conduits. This can be modulated from a couple of weeks to several months by varying the concentration of hyaluronic acid in the starting solution. These findings allow to improve the performance of hyaluronan intended for neural conduits, and open the way to scaffolds with tunable degradation rate adapted to the site and severity of the injury.

Combined application of polyacrylate scaffold and lipoic acid treatment promotes neural tissue reparation after brain injury.

PRIMARY OBJECTIVE: The aim of this study was to investigate the reparative potential of a polymeric scaffold designed for brain tissue repair in combination with lipoic acid. RESEARCH DESIGN: Histological, cytological and structural analysis of a combined treatment after a brain cryo-injury model in rats. METHODS AND PROCEDURES: Adult Wistar rats were subjected to cryogenic brain injury. A channelled-porous scaffold of ethyl acrylate and hydroxyethylacrylate, p(EA-co-HEA) was grafted into cerebral penumbra alone or combined with intraperitoneal LA administration. Histological and cytological evaluation was performed after 15 and 60 days and structural magnetic resonance (MRI) assessment was performed at 2 and 6 months after the surgery. MAIN OUTCOMES AND RESULTS: The scaffold was suitable for the establishment of different cellular types. The results obtained suggest that this strategy promotes blood vessels formation, decreased microglial response and neuron migration, particularly when LA was administrated. CONCLUSIONS: These evidences demonstrated that the combination of a channelled polymer scaffold with LA administration may represent a potential treatment for neural tissue repair after brain injury.

Peptide gel in a scaffold as a composite matrix for endothelial cells.

The performance of a composite environment with human umbilical vein endothelial cells (HUVECs) has been studied to provide an in vitro proof of concept of their potential of being easily vascularized. These cells were seeded in 1 mm thick scaffolds whose pores had been filled with a self-assembling peptide gel, seeking to improve cell adhesion, and viability of these very sensitive cells. The combination of the synthetic elastomer poly(ethyl acrylate), PEA, scaffold and the RAD16-I peptide gel provides cells with a friendly ECM-like environment inside a mechanically resistant structure. Immunocytochemistry, flow cytometry and scanning electron microscopy were used to evaluate the cell cultures. The presence of the self-assembling peptide filling the pores of the scaffolds resulted in a truly 3D nanoscale context mimicking the extracellular matrix environment, and led to increased cells survival, proliferation as well as developed cell-cell contacts. The combined system consisting of PEA scaffolds and RAD16-I, is a very interesting approach as seems to enhance endothelization, which is the first milestone to achieve vascularized constructs.

Engineered 3D bioimplants using elastomeric scaffold, self-assembling peptide hydrogel, and adipose tissue-derived progenitor cells for cardiac regeneration.

Contractile restoration of myocardial scars remains a challenge with important clinical implications. Here, a combination of porous elastomeric membrane, peptide hydrogel, and subcutaneous adipose tissue-derived progenitor cells (subATDPCs) was designed and evaluated as a bioimplant for cardiac regeneration in a mouse model of myocardial infarction. SubATDPCs were doubly transduced with lentiviral vectors to express bioluminescent-fluorescent reporters driven by constitutively active, cardiac tissue-specific promoters. Cells were seeded into an engineered bioimplant consisting of a scaffold (polycaprolactone methacryloyloxyethyl ester) filled with a peptide hydrogel (PuraMatrix™), and transplanted to cover injured myocardium. Bioluminescence and fluorescence quantifications showed de novo and progressive increases in promoter expression in bioactive implant-treated animals. The bioactive implant was well adapted to the heart, and fully functional vessels traversed the myocardium-bioactive implant interface. Treatment translated into a detectable positive effect on cardiac function, as revealed by echocardiography. Thus, this novel implant is a promising construct for supporting myocardial regeneration.

Creating the bioartificial myocardium for cardiac repair: challenges and clinical targets.

The association of stem cells with tissue-engineered scaffolds constitutes an attractive approach for the repair of myocardial tissue with positive effects to avoid ventricular chamber dilatation, which changes from a natural elliptical to spherical shape in heart failure patients. Biohybrid scaffolds using nanomaterials combined with stem cells emerge as new therapeutic tool for the creation of 'bioartificial myocardium' and 'cardiac wrap bioprostheses' for myocardial regeneration and ventricular support. Biohybrids are created introducing stem cells and self-assembling peptide nanofibers inside a porous elastomeric membrane, forming cell niches. Our studies lead to the creation of semi-degradable 'ventricular support bioprostheses' for adaptative LV and/or RV wrapping, designed with the concept of 'helical myocardial bands'. The goal is to restore LV elliptical shape, and contribute to systolic contraction and diastolic filling (suction mechanism). Cardiac wrapping with ventricular bioprostheses may reduce the risk of heart failure progression and the indication for heart transplantation.

New concept for a regenerative and resorbable prosthesis for tendon and ligament: physicochemical and biological characterization of PLA-braided biomaterial.

We present a concept for a new regenerative and resorbable prosthesis for tendon and ligament and characterize the physicomechanical and biological behavior of one of its components, a hollow braid made of poly-lactide acid (PLA) which is the load-bearing part of the prosthesis concept. The prosthesis consists of a braid, microparticles in its interior serving as cell carriers, and a surface non-adherent coating, all these parts being made of biodegradable materials. The PLA braid has a nonlinear convex stress-strain behavior with a Young modulus of 1370 ± 90 MPa in the linear, stretched state, and after 12 months of hydrolytic degradation the modulus shows a reduction by a factor of four. Different disinfection methods were tested as to their efficiency in cleansing the braid and preparing it for cell culture. Fibroblasts of L929 line were grown on the PLA braid for 14 days, showing good adherence and proliferation. These studies validate the PLA braid for the intended purpose in the regenerative prosthesis concept.

Channeled scaffolds implanted in adult rat brain.

Scaffolds with aligned channels based on acrylate copolymers, which had previously demonstrated good compatibility with neural progenitor cells were studied as colonizable structures both in vitro with neural progenitor cells and in vivo, implanted without cells in two different locations, in the cortical plate of adult rat brains and close to the subventricular zone. In vitro, neuroprogenitors colonize the scaffold and differentiate into neurons and glia within its channels. When implanted in vivo immunohistochemical analysis by confocal microscopy for neural and endothelial cells markers demonstrated that the scaffolds maintained continuity with the surrounding neural tissue and were colonized by GFAP-positive cells and, in the case of scaffolds implanted in contact with the subventricular zone, by neurons. Local angiogenesis was evidenced in the interior of the scaffolds' pores. New axons and neural cells from the adult neural niche abundantly colonized the biomaterial's inner structure after 2 months, and minimal scar formation was manifest around the implant. These findings indicate the biocompatibility of the polymeric material with the brain tissue and open possibilities to further studies on the relevance of factors such as scaffold structure, scaffold seeding and scaffold placement for their possible use in regenerative strategies in the central nervous system. The development of neural interfaces with minimized glial scar and improved tissue compatibility of the implants may also benefit from these results. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A:3276-3286, 2012.

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.

Mimicking natural dentin using bioactive nanohybrid scaffolds for dentinal tissue engineering.

Synthetic materials mimicking the internal porous structure of natural dentin were prepared as nanohybrid matrix scaffolds made of poly(ethyl methacrylate-co-hydroxyethyl acrylate), pure and with a sol-gel-derived interpenetrated silica nanophase, with aligned tubular pores in the micrometer range typical of dentinal tissue. Some of them were internally coated with a layer of hydroxyapatite by immersion in simulated body fluid. Their physicochemical and mechanical properties were investigated. The different types of scaffolds were implanted subcutaneously into immunocompromised nude mice for 4, 6, and 8 weeks and their biological response were analyzed. Optical microscopy was employed to study the scaffold structure and neovascularization. Cells origin, inflammation, and macrophagic responses were evaluated by optical microscopy, immunohistochemistry, and transmission electron microscopy. The scaffold ultrastructural pattern imitates dentinal histological structure. The materials allowed cell colonization and neoangiogenesis. These biomaterials were colonized by murine cells fenotypically different to those of dermal connective tissue, showing structural differentiations. Colonization and viability were improved by the use of mineralized interphases, which showed a cellular distribution resembling a neodentinal pattern. Invasion of the scaffold tubules by single odontoblast-like processes was ascertained both in the noncoated and coated scaffolds. Such materials thus seem promising in tissue engineering strategies for dentin regeneration.

In vivo evaluation of 3-dimensional polycaprolactone scaffolds for cartilage repair in rabbits.

BACKGROUND: Cartilage tissue engineering using synthetic scaffolds allows maintaining mechanical integrity and withstanding stress loads in the body, as well as providing a temporary substrate to which transplanted cells can adhere. PURPOSE: This study evaluates the use of polycaprolactone (PCL) scaffolds for the regeneration of articular cartilage in a rabbit model. STUDY DESIGN: Controlled laboratory study. METHODS: Five conditions were tested to attempt cartilage repair. To compare spontaneous healing (from subchondral plate bleeding) and healing due to tissue engineering, the experiment considered the use of osteochondral defects (to allow blood flow into the defect site) alone or filled with bare PCL scaffold and the use of PCL-chondrocytes constructs in chondral defects. For the latter condition, 1 series of PCL scaffolds was seeded in vitro with rabbit chondrocytes for 7 days and the cell/scaffold constructs were transplanted into rabbits' articular defects, avoiding compromising the subchondral bone. Cell pellets and bare scaffolds were implanted as controls in a chondral defect. RESULTS: After 3 months with PCL scaffolds or cells/PCL constructs, defects were filled with white cartilaginous tissue; integration into the surrounding native cartilage was much better than control (cell pellet). The engineered constructs showed histologically good integration to the subchondral bone and surrounding cartilage with accumulation of extracellular matrix including type II collagen and glycosaminoglycan. The elastic modulus measured in the zone of the defect with the PCL/cells constructs was very similar to that of native cartilage, while that of the pellet-repaired cartilage was much smaller than native cartilage. CONCLUSION: The results are quite promising with respect to the use of PCL scaffolds as aids for the regeneration of articular cartilage using tissue engineering techniques.

Microcomputed tomography and microfinite element modeling for evaluating polymer scaffolds architecture and their mechanical properties.

Detailed knowledge of the porous architecture of synthetic scaffolds for tissue engineering, their mechanical properties, and their interrelationship was obtained in a nondestructive manner. Image analysis of microcomputed tomography (microCT) sections of different scaffolds was done. The three-dimensional (3D) reconstruction of the scaffold allows one to quantify scaffold porosity, including pore size, pore distribution, and struts' thickness. The porous morphology and porosity as calculated from microCT by image analysis agrees with that obtained experimentally by scanning electron microscopy and physically measured porosity, respectively. Furthermore, the mechanical properties of the scaffold were evaluated by making use of finite element modeling (FEM) in which the compression stress-strain test is simulated on the 3D structure reconstructed from the microCT sections. Elastic modulus as calculated from FEM is in agreement with those obtained from the stress-strain experimental test. The method was applied on qualitatively different porous structures (interconnected channels and spheres) with different chemical compositions (that lead to different elastic modulus of the base material) suitable for tissue regeneration. The elastic properties of the constructs are explained on the basis of the FEM model that supports the main mechanical conclusion of the experimental results: the elastic modulus does not depend on the geometric characteristics of the pore (pore size, interconnection throat size) but only on the total porosity of the scaffold.

Three-dimensional nanocomposite scaffolds with ordered cylindrical orthogonal pores.

A silica reinforcement can improve the mechanical properties of hydrogels in the rubbery state. A method to prepare a scaffold with a well-ordered array of cylindrical pores is presented in this work, which yields a scaffold with a biphasic matrix of a hybrid nanocomposite: the hydrogel poly(2-hydroxyethyl acrylate) (PHEA) and a silica network obtained by an acid catalyzed sol-gel process of tetraethoxysilane (TEOS). As porogenic template of the scaffold stacked layers of commercial polyamide 6 fabrics were used, which were compressed and sintered. Porosity and dynamic mechanical response of the resulting scaffolds were measured and compared with the bulk properties. Removal of the organic polymer phase of the scaffold by pyrolysis revealed the overall continuity of the silica network; the residue maintained the original cylindrical pore structure of the scaffolds, though slightly shrunk. Atomic force microscopy topography measurements of these pyrolysed residues revealed a silica structure with particle aggregates having sizes around tens of nanometers. The silica distribution was assessed by X-ray microanalysis mapping, showing homogeneity at a micrometer scale.