Development and evaluation of hyaluronan nanocomposite conduits for neural tissue regeneration.

Hyaluronan-based hydrogels are among the most promising neural tissue engineering materials because of their biocompatibility and the immunomodulation capabilities of their degradation byproducts. Despite these features, the problems related to their handling and mechanical properties have not yet been solved. In the present work it is proposed to address these drawbacks through the development of nanohybrid materials in which different nanometric phases (carbon nanotubes, mesoporous silica nanoparticles) are embedded in a crosslinked hyaluronan matrix. These nanohybrid matrices were next processed in the shape of cylindrical conduits aimed at promoting and improving neural stem cell differentiation and regeneration in neural tracts. These constructs could be of use specifically for peripheral nerve regeneration. Results of the study show that the inclusion of the different phases improved physico-chemical features of the gel such as its relative electrical permittivity, water intake and elastic modulus, giving hints on how the nanometric phase interacts with hyaluronan in the composite as well as for their potential in combined therapeutic approaches. Regarding the in vitro biological behavior of the hybrid tubular scaffolds, an improved early cell adhesion and survival of Schwann cells in their lumen was found, as compared to conduits made of pure hyaluronan gels. Furthermore, the differentiation and survival of neural precursors was not compromised, despite alleged safety concerns.

Improved Mechanical, Thermal, and Hydrophobic Properties of PLA Modified with Alkoxysilanes by Reactive Extrusion Process.

An eco-friendly strategy for the modification of polylactic acid (PLA) surface properties, using a solvent-free process, is reported. Reactive extrusion (REX) allowed the formation of new covalent bonds between functional molecules and the PLA polymeric matrix, enhancing its mechanical properties and modifying surface hydrophobicity. To this end, the PLA backbone was modified using two alkoxysilanes, phenyltriethoxysilane and N-octyltriethoxysilane. The reactive extrusion process was carried out under mild conditions, using melting temperatures between 150 and 180 °C, 300 rpm as screw speed, and a feeding rate of 3 kg·h-1. To complete the study, flat tapes of neat and functionalized PLA were obtained through monofilament melt extrusion to quantify the enhancement of mechanical properties and hydrophobicity. The results verified that PLA modified with 3 wt% of N-octyltriethoxysilane improves mechanical and thermal properties, reaching Young's modulus values of 4.8 GPa, and PLA hydrophobic behavior, with values of water contact angle shifting from 68.6° to 82.2°.

Role of Electrospinning Parameters on Poly(Lactic-co-Glycolic Acid) and Poly(Caprolactone-co-Glycolic acid) Membranes.

Poly(lactic-co-glycolic acid) (PLGA) and poly(caprolactone-co-glycolic acid) (PCLGA) solutions were electrospun into membranes with tailored fiber diameter of 1.8 µm. This particular fiber diameter was tuned depending on the used co-polymer by adjusting the electrospinning parameters that mainly influence the fiber diameter. The greatest setting of the fiber diameter was achieved by varying the polymer solution parameters (polymer concentration, solvents and solvents ratio). PLGA was adequately electrospun with 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), whereas PCLGA required a polar solvent (such as chloroform) with a lower dielectric constant. Moreover, due to the amorphous morphology of PCLGA, pyridine as salt had to be added to the starting solution to increase its conductivity and make it electrospinnable. Indeed, the electrospinning of this co-polymer presents notable difficulties due to its amorphous structure. Interestingly, PCLGA, having a higher glycolic acid molar fraction than commonly electrospun co-polymers (caprolactone:glycolic acid ratio of 45:55 instead of 90:10), could be successfully electrospun, which has not been reported to date. To an accurate setting of fiber diameter, the voltage and the distance from needle to collector were varied. Finally, the study of the surface tension, conductivity and viscosity of the polymer solutions allowed to correlate these particular characteristics of the solutions with the electrospinning variables so that prior knowledge of them enables predicting the required processing conditions.

Role of Curing Temperature of Poly(Glycerol Sebacate) Substrates on Protein-Cell Interaction and Early Cell Adhesion.

A novel procedure to obtain smooth, continuous polymeric surfaces from poly(glycerol sebacate) (PGS) has been developed with the spin-coating technique. This method proves useful for separating the effect of the chemistry and morphology of the networks (that can be obtained by varying the synthesis parameters) on cell-protein-substrate interactions from that of structural variables. Solutions of the PGS pre-polymer can be spin-coated, to then be cured. Curing under variable temperatures has been shown to lead to PGS networks with different chemical properties and topographies, conditioning their use as a biomaterial. Particularly, higher synthesis temperatures yield denser networks with fewer polar terminal groups available on the surface. Material-protein interactions were characterised by using extracellular matrix proteins such as fibronectin (Fn) and collagen type I (Col I), to unveil the biological interface profile of PGS substrates. To that end, atomic force microscopy (AFM) images and quantification of protein adsorbed in single, sequential and competitive protein incubations were used. Results reveal that Fn is adsorbed in the form of clusters, while Col I forms a characteristic fibrillar network. Fn has an inhibitory effect when incubated prior to Col I. Human umbilical endothelial cells (HUVECs) were also cultured on PGS surfaces to reveal the effect of synthesis temperature on cell behaviour. To this effect, early focal adhesions (FAs) were analysed using immunofluorescence techniques. In light of the results, 130 °C seems to be the optimal curing temperature since a preliminary treatment with Col I or a Fn:Col I solution facilitates the formation of early focal adhesions and growth of HUVECs.

Influence of pre-polymerisation atmosphere on the properties of pre- and poly(glycerol sebacate).

Poly(glycerol sebacate) (PGS) is a versatile biodegradable biomaterial on account of its adjustable mechanical properties as an elastomeric polyester. Nevertheless, it has shown dissimilar results when synthesised by different research groups under equivalent synthesis conditions. This lack of reproducibility proves how crucial it is to understand the effect of the parameters involved on its manufacturing and characterize the polymer networks obtained. Several studies have been conducted in recent years to understand the role of temperature, time, and the molar ratio of its monomers, while the influence of the atmosphere applied during its pre-polymerisation remained unknown. The results obtained here allow for a better understanding about the effect of inert (Ar and N2) and oxidative (oxygen, dry air, and humid air) atmospheres on the extent of the reaction. The molecular pattern of intermediate pre-polymers and the gelation time and morphology of their corresponding cured PGS networks were studied as well. Overall, inert atmospheres promote a rather linear growth of macromers, with scarce branches, resulting in loose elastomers with long chains mainly crosslinked. Conversely, oxygen in the latter atmospheres promotes branching through secondary hydroxyl groups, leading to less-crosslinked 'defective' networks. In this way, the pre-polymerisation atmosphere could be used advantageously to adjust the reactivity of secondary hydroxyls, in order to modulate branching in the elastomeric PGS networks obtained to suit the properties required in a particular application.

Amphipathic Substrates Based on Crosslinker-Free Poly(ε-Caprolactone):Poly(2-Hydroxyethyl Methacrylate) Semi-Interpenetrated Networks Promote Serum Protein Adsorption.

A simple procedure has been developed to synthesize uncrosslinked soluble poly(hydroxyethyl methacrylate) (PHEMA) gels, ready for use in a subsequent fabrication stage. The presence of 75 wt % methanol (MetOH) or dimethylformamide (DMF) impedes lateral hydroxyl-hydroxyl hydrogen bonds between PHEMA macromers to form during their solution polymerization at 60 °C, up to 24 h. These gels remain soluble when properly stored in closed containers under cold conditions and, when needed, yield by solvent evaporation spontaneous physically-crosslinked PHEMA adapted to the mould used. Moreover, this two-step procedure allows obtaining multicomponent systems where a stable and water-affine PHEMA network would be of interest. In particular, amphiphilic polycaprolactone (PCL):PHEMA semi-interpenetrated (sIPN) substrates have been developed, from quaternary metastable solutions in chloroform (CHCl3):MetOH 3:1 wt. and PCL ranging from 50 to 90 wt % in the polymer fraction (thus determining the composition of the solution). The coexistence of these countered molecules, uniformly distributed at the nanoscale, has proven to enhance the number and interactions of serum protein adsorbed from the acellular medium as compared to the homopolymers, the sIPN containing 80 wt % PCL showing an outstanding development. In accordance to the quaternary diagram presented, this protocol can be adapted for the development of polymer substrates, coatings or scaffolds for biomedical applications, not relying upon phase separation, such as the electrospun mats here proposed herein (12 wt % polymer solutions were used for this purpose, with PCL ranging from 50% to 100% in the polymer fraction).

Candidate Polyurethanes Based on Castor Oil (Ricinus communis), with Polycaprolactone Diol and Chitosan Additions, for Use in Biomedical Applications.

Polyurethanes are widely used in the development of medical devices due to their biocompatibility, degradability, non-toxicity and chemical versatility. Polyurethanes were obtained from polyols derived from castor oil, and isophorone diisocyanate, with the incorporation of polycaprolactone-diol (15% w/w) and chitosan (3% w/w). The objective of this research was to evaluate the effect of the type of polyol and the incorporation of polycaprolactone-diol and chitosan on the mechanical and biological properties of the polyurethanes to identify the optimal ones for applications such as wound dressings or tissue engineering. Polyurethanes were characterized by stress-strain, contact angle by sessile drop method, thermogravimetric analysis, differential scanning calorimetry, water uptake and in vitro degradation by enzymatic processes. In vitro biological properties were evaluated by a 24 h cytotoxicity test using the colorimetric assay MTT and the LIVE/DEAD kit with cell line L-929 (mouse embryonic fibroblasts). In vitro evaluation of the possible inflammatory effect of polyurethane-based materials was evaluated by means of the expression of anti-inflammatory and proinflammatory cytokines expressed in a cellular model such as THP-1 cells by means of the MILLIPLEX® MAP kit. The modification of polyols derived from castor oil increases the mechanical properties of interest for a wide range of applications. The polyurethanes evaluated did not generate a cytotoxic effect on the evaluated cell line. The assessed polyurethanes are suggested as possible candidate biomaterials for wound dressings due to their improved mechanical properties and biocompatibility.

Wideband phantoms of different body tissues for heterogeneous models in body area networks.

One of the key issues about wireless technologies is their interaction with the human body. The so-called internet of things will comprise many devices that will transmit either around or through the human body. These devices must be tested either in their working medium, when possible, or in the most realistic one. For this purpose, tissue-like phantoms are the best alternative to carry out realistic analyses of the performance of body area networks. In addition, they are the conventional way to certify the compliance of commercial standards by these devices. However, the number of phantoms that work in large bandwidths is limited in literature. This work aims at presenting chemical solutions that will be useful to prepare a variety of wideband tissue phantoms. Besides, the colon was mimicked in two ways, the healthy tissue and the malignant one, taking into account studies that relate changes on the relative permittivity with cancer. They were designed on the basis of acetonitrile in aqueous solutions as described in a previous work. Thus, many scenarios could be developed such as multilayers which imitate parts of the heterogeneous body.

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.

Experimental Path Loss Models for In-Body Communications Within 2.36-2.5 GHz.

Biomedical implantable sensors transmitting a variety of physiological signals have been proven very useful in the management of chronic diseases. Currently, the vast majority of these in-body wireless sensors communicate in frequencies below 1 GHz. Although the radio propagation losses through biological tissues may be lower in such frequencies, e.g., the medical implant communication services band of 402 to 405 MHz, the maximal channel bandwidths allowed therein constrain the implantable devices to low data rate transmissions. Novel and more sophisticated wireless in-body sensors and actuators may require higher data rate communication interfaces. Therefore, the radio spectrum above 1 GHz for the use of wearable medical sensing applications should be considered for in-body applications too. Wider channel bandwidths and smaller antenna sizes may be obtained in frequency bands above 1 GHz at the expense of larger propagation losses. Therefore, in this paper, we present a phantom-based radio propagation study for the frequency bands of 2360 to 2400 MHz, which has been set aside for wearable body area network nodes, and the industrial, scientific, medical band of 2400 to 2483.5 MHz. Three different channel scenarios were considered for the propagation measurements: in-body to in-body, in-body to on-body, and in-body to off-body. We provide for the first time path loss formulas for all these cases.

in vitro development of bioimplants made up of elastomeric scaffolds with peptide gel filling seeded with human subcutaneous adipose tissue-derived progenitor cells.

Myocardial tissue lacks the ability to regenerate itself significantly following a myocardial infarction. Thus, new strategies that could compensate this lack are of high interest. Cardiac tissue engineering (CTE) strategies are a relatively new approach that aims to compensate the tissue loss using combination of biomaterials, cells and bioactive molecules. The goal of the present study was to evaluate cell survival and growth, seeding capacity and cellular phenotype maintenance of subcutaneous adipose tissue-derived progenitor cells in a new synthetic biomaterial scaffold platform. Specifically, here we tested the effect of the RAD16-I peptide gel in microporous poly(ethyl acrylate) polymers using two-dimensional PEA films as controls. Results showed optimal cell adhesion efficiency and growth in the polymers coated with the self-assembling peptide RAD16-I. Importantly, subATDPCs seeded into microporous PEA scaffolds coated with RAD16-I maintained its phenotype and were able to migrate outwards the bioactive patch, hopefully toward the infarcted area once implanted. These data suggest that this bioimplant (scaffold/RAD16-I/cells) can be suitable for further in vivo implantation with the aim to improve the function of affected tissue after myocardial infarction.

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.

Design and assembly procedures for large-sized biohybrid scaffolds as patches for myocardial infarct.

OBJECTIVE: To assemble a biohybrid cardiac patch consisting of a large (5 × 5 cm) elastomer scaffold whose pores are filled with a self-assembling peptide (SAP) gel entrapping adipose stem cells, to be used as a novel implant in a big animal model (sheep) of myocardial infarction. The study focuses on the way to determine optimal procedures for incorporating the SAP solution and the cells in the patch to ensure cell colonization and a homogeneous cell distribution in the construct before implantation. The problems associated with the scale-up of the different procedures raised by the large size of the construct are discussed. MATERIALS AND METHODS: Experiments were performed to choose between different assembling alternatives: incorporation of the SAP gel before cell seeding or simultaneous SAP and cell loading of the scaffold; surface seeding of cells or cell injection into the scaffold pores; dissemination of the cells throughout the scaffold before incubation by gentle shaking or by centrifugation. Immunocytochemistry techniques and confocal and scanning electron microscopies were employed to assess and quantify cell colonization of the material and early cell distribution. Cell concentrations and the uniformity of cellular distribution throughout the scaffold were taken as the main criteria to decide between the different alternative procedures. RESULTS: The combination of peptide preloading, cell injection, and shaking before incubation yielded the best results in terms of greater cell density and the most uniform distribution after 24 h of culture compared with the other methods. These techniques could be scaled-up to obtain large biohybrid cardiac patches with success. CONCLUSIONS: The results obtained after the different seeding methods allowed us to establish an effective protocol for the assembly of large biohybrid patches for their subsequent implantation in the heart of a big animal model of myocardial infarct in the context of a preclinical study.

Unsupervised segmentation of brain regions with similar microstructural properties: application to alcoholism.

In this work, a novel brain MRI segmentation approach evaluates microstructural differences between groups. Going further from the traditional segmentation of brain tissues (white matter -WM-, gray matter -GM- and cerebrospinal fluid -CSF- or a mixture of them), a new way to classify brain areas is proposed using their microstructural MR properties. Eight rats were studied using the proposed methodology identifying regions which present microstructural differences as a consequence on one month of hard alcohol consumption. Differences in relaxation times of the tissues have been found in different brain regions (p<0.05). Furthermore, these changes allowed the automatic classification of the animals based on their drinking history (hit rate of 93.75 % of the cases).

One-dimensional migration of olfactory ensheathing cells on synthetic materials: experimental and numerical characterization.

Olfactory ensheathing cells (OECs) are of great interest for regenerative purposes since they are believed to aid axonal growth. With the view set on the strategies to achieve reconnection between neuronal structures, it is of great importance to characterize the behaviour of these cells on long thread-like structures that may efficiently guide cell spread in a targeted way. Here, rat OECs were studied on polycaprolactone (PCL) long monofilaments, on long bars and on discs. PCL turns out to be an excellent substrate for OECs. The cells cover long distances along the monofilaments and colonize completely these structures. With the help of a one-dimensional (1D) analytical model, a migration coefficient, a net proliferation rate constant and the fraction of all cells which undergo migration were obtained. The separate effect of the three phenomena summarized by these parameters on the colonization patterns of the 1D path was qualitatively discussed. Other features of interest were also determined, such as the speed of the advance front of colonization and the order of the kinetics of net cell proliferation. Characterizing migration by means of these quantities may be useful for comparing and predicting features of the colonization process (such as times, patterns, advance fronts and proportion of motile cells) of different cell-substrate combinations.

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.

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.

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.

Volume mesh generation and finite element analysis of trabecular bone magnetic resonance images.

In order to help the assessment of trabecular bone diseases and complement Dual X-Ray Absorptiometry (DXA) in diagnosis process, it is needed an accurate mechanical characterization of trabecular bone structure to estimate the risk of fracture and evaluate micro-architecture deterioration. As Finite Element modeling has become a well-established method for analysis of complex structures, an algorithm has been developed to build a Finite Element mesh from three-dimensional reconstruction information in voxels. Generated mesh is loaded in a Finite Element analysis software in order to simulate micro-architecture mechanical behavior under compression conditions. Most part of related researches have been based on ex vivo micro-Computed Tomography (microCT) scans. This study uses three-dimensional trabecular bone reconstructions from high resolution Magnetic Resonance images acquired in vivo.