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.

Low-Dose Gamma Radiation Sterilization for Decellularized Tracheal Grafts.

One of the main key aspects in ensuring that a transplant evolves correctly is the sterility of the medium. Decellularized tracheal transplantation involves implanting an organ that was originally in contact with the environment, thus not being sterile from the outset. While the decellularization protocol (through detergent exposition [2% sodium dodecyl sulfate], continuous stirring, and osmotic shocks) is conducted in line with aseptic measures, it does not provide sterilization. Therefore, one of the main challenges is ensuring sterility prior to in vivo implantation. Although there are established gamma radiation sterilization protocols for inorganic materials, there are no such measures for organic materials. Additionally, the protocols in place for inorganic materials cannot be applied to organic materials, as the established radiation dose (25 kGy) would completely destroy the implant. This paper studies the effect of an escalated radiation dose in a decellularized rabbit trachea. We maintained the dose range (kGy) and tested escalated doses until finding the minimal dose at which sterilization is achieved. After determining the dose, we studied effects of it on the organ, both histologically and biomechanically. We determined that while 0.5 kGy did not achieve sterility, doses of both 1 kGy and 2 kGy did, with 1 kGy, therefore, being the minimal dose necessary to achieve sterilization. Microscopic studies showed no relevant changes compared to non-sterilized organs. Axial biomechanical characteristics were not altered at all, and only a slight reduction in the force per unit of length that the organ can radially tolerate was observed. We can therefore conclude that 1 kGy achieves complete sterilization of decellularized rabbit trachea with a minimal, if any, effects on the organ.

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.

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.

Optimization of a decellularization protocol of porcine tracheas. Long-term effects of cryopreservation. A histological study.

OBJECTIVE: The aim of this study was to optimize a decellularization protocol in the trachea of Sus scrofa domestica (pig) as well as to study the effects of long-term cryopreservation on the extracellular matrix of decellularized tracheas. METHODS: Porcine tracheas were decellularized using Triton X-100, SDC, and SDS alone or in combination. The effect of these detergents on the extracellular matrix characteristics of decellularized porcine tracheas was evaluated at the histological, biomechanical, and biocompatibility level. Morphometric approaches were used to estimate the effect of detergents on the collagen and elastic fibers content as well as on the removal of chondrocytes from decellularized organs. Moreover, the long-term structural, ultrastructural, and biomechanical effect of cryopreservation of decellularized tracheas were also estimated. RESULTS: Two percent SDS was the most effective detergent tested concerning cell removal and preservation of the histological and biomechanical properties of the tracheal wall. However, long-term cryopreservation had no an appreciable effect on the structure, ultrastructure, and biomechanics of decellularized tracheal rings. CONCLUSION: The results presented here reinforce the use of SDS as a valuable decellularizing agent for porcine tracheas. Furthermore, a cryogenic preservation protocol is described, which has minimal impact on the histological and biomechanical properties of decellularized porcine tracheas.

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.

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.

[Mechanical behaviour of three types of surgical knots using 4/0 monofilament]. / Comportamiento mecánico de 3 tipos de anudados quirúrgicos usando monofilamentos de 4/0.

OBJECTIVE: To experimentally study the behaviour of 4 types of monofilament with 3 knotting techniques, very often used in surgery, employing mechanical tests. MATERIAL AND METHODS: Four 4/0 monofilaments were chosen, two of nylon, and the other two of polypropylene. Three types of knot designs were made with each thread. The first design (D-S-S) consisted of a double half-knot (D) followed by one single (S) in the opposite direction, a third S in turn in the opposite direction. The configuration of the second design was D-S-D, and the third, S-S-D. A mechanical fracture test was performed by stretching the thread at a rate of 4N/s, the force and deformation being recorded at intervals of 100 ms. RESULTS: There was a decrease in the force and deformation in the tests on threads with knots compared to threads without knots. In all cases the rupture of the thread occurred in the knot area, presumably due to damage caused to the thread during the knotting process. The D-S-D knot had the greatest resistance with polypropylene threads, and S-S-D provided the greatest resistance with nylon threads CONCLUSIONS: Polypropylene threads, with D-S-D knots, should be more indicated to suture tissues that have to support great forces and with little deformation of the suture (e.g., tendons). Nylon threads, with S-S-D knots, would be better indicated for tissues that have to support smaller forces and that require greater elasticity (e.g., skin).

Physical characterization of polycaprolactone scaffolds.

Films and sponges were prepared from a solution of Poly(epsilon-caprolactone) (PCL) in tetrahydrofuran (THF). The porosity, crystallinity, and mechanical properties of the samples were studied. Porosity of around 15% was obtained for the films produced by evaporation of THF at room temperature. A much more porous structure (50-70%) was found for the sponges obtained by cooling the solution at -30 degrees C and subsequently eliminating the solvent by freeze drying. The porosity of the samples was also observed by scanning electron microscopy (SEM). The crystallinity of the samples was studied by the calorimetric technique (DSC) before and after the compression scans. The mechanical properties of the different samples were determined by compression test, and were compared to those corresponding to the PCL in bulk. The compression scans did not affect the crystallinity of the samples. The variations observed in the results of the different scans were attributed to the differences in porosities and crystallinity.