Nerve tissue engineering using blends of Polyhydroxyalkanoates

PHAs are a family of linear polyesters consisting of 3, 4, 5 and 6-hydroxyacids, synthesized by a variety of bacterial species. They can be produced from renewable carbon sources, they are biodegradable, biocompatible and exhibit thermoplastic and elastomeric properties. Therefore, PHAs are a potential substitute for petroleum-derived plastics and have a wide range of biomedical applications, particularly in tissue engineering and drug delivery. The properties of PHA devices can be successfully modified through the fabrication of composites and blends. Such modifications allow for further tailoring of properties such as biocompatibility, mechanical properties and degradation times required for specific physiological conditions. PHAs have been used for the manufacture of a wide variety of surgical materials and implants in the areas of skin, nerve, dental, cardiac and bone tissue engineering. The latter two fields being where the PHAs have been the most extensively explored. These unique polymers possess significant advantages compared to their chemically-synthetized counterparts. Their structural diversity, adaptable properties, controllable surface degradation and biocompatibility with a wide range of cells, place PHAs as a biomaterial with immense potential for biomedical applications. The main aim of this project was to explore the use of Polyhydroxyalkanoates in nerve tissue engineering ultimately leading to the development of novel PHA-based nerve guidance conduits (NGCs). Production of the scl-PHA, P(3HB) and the mcl-PHA, P(3HO) through bacterial fermentation was performed to obtain appropriate amounts of polymer for chemical characterisation and further manufacturing of scaffolds for nerve tissue engineering applications. The average polymer yields achieved using a 20 L fermenter were 42.27 % dcw of P(3HO) using Pseudomonas mendocina CH50 and 49.22 % dcw of P(3HO) using Bacillus cereus SPV respectively. Novel PHA blends as resorbable biomaterials for use in the manufacture of NGCs were fabricated. PHA blend films with varying ratios of poly(3-hydroxyoctanoate)/poly(3-hydroxybutyrate), (P(3HO)/P(3HB), were produced using the solvent-casting method. Neat films of P(3HO) and P(3HB) along with 25:75, 50:50 and 75:25 blend films of P(3HO)/P(3HB) were characterised with respect to their chemical, material and biological properties in order to evaluate them as potential base materials for nerve tissue engineering. In the surface analysis the blends exhibited higher values of roughness compared with the neat films. The DSC characterisation of the blends confirmed that P(3HO) and P(3HB) formed immiscible blends. FTIR and XRD analysis of the blends showed a decrease in the crystallinity with increase in the proportion of P(3HO). An increase in the stiffness of the blends was observed when the proportion of P(3HB) increased. Although all of the blends were biocompatible with NG108-15 neuronal cells, the 25:75 P(3HO)/P(3HB) blend showed significantly better support for the growth and differentiation of these cells. Mechanical properties of PHA blends corresponded to the reported properties of peripheral nerves providing potential materials for their use as base material for the manufacture of NGCs. The 25:75 P(3HO)/P(3HB) blend was used for the manufacturing of electrospun fibres as resorbable scaffolds for their use in the manufacture of NGCs as lumen structure. The biocompatibility of these fibres with NG108-15 neuronal cells as well as the influence of RN22 Schwann cells on their growth and differentiation was studied. Highly aligned and uniform fibres with varying diameters were successfully fabricated by controlling electrospinning parameters. The resulting fibre diameters were 2.42 ± 0.34 μm, 3.68 ± 0.26 μm and 13.50 ± 2.33 μm for small, medium and large fibres respectively. The effect of the fibers on the growth of neuronal cells NG108-15 was investigated by live/dead cell test. Cell migration observed on the electrospun fibres showed directional alignment in accordance with the direction of the fibres. The correlation between 25:75 P(3HO)/P(3HB) micro-fibre diameter and neuronal growth under two conditions; individually and in co-culture with RN22 Schwann cells were evaluated. This was investigated using two types of cell staining; live/dead cell test and anti-beta tubulin immunolabelling. Results displayed from both assays revealed that all 25:75 P(3HO)/P(3HB) blend fibre groups were able to support growth and guide aligned distribution of neuronal cells when grown individually and in the presence of RN22 Schwann cells. Results also revealed a direct correlation between fiber diameter and neuronal growth and differentiation. Although neuronal cell viability was similar for all the substrates (approximately 99%) except on glass, large fibres supported the highest number of live neuronal cells grown individually compared to the rest of substrates. Biocompatibility and neuron regenerating properties of various Bioactive glasse (BG)/PHA blend composites were assessed in order to study their suitability for peripheral nerve tissue applications. BG/PHA blend composites were fabricated using Bioglass® 45S5 (BG1) and BG 1393 (BG2) along with 25:75 P(3HO)/P(3HB) blend. Different proportions of each BG (0.5, 1.0 and 2.5 % w/v) were used to determine the BG concentration that resulted in superior neuronal growth and differentiation of NG108-15 in single culture and in co-culture system with Schwannoma RN22 cells. NG108-15 cells displayed good growth and differentiation performance on all the PHA blend composites showing that both BGs (BG1 and BG2) have good biocompatibility at 2.5, 1.0 and 0.5 % w/v in the PHA blend solution. The Young’s modulus values displayed by all the PHA blend/BG composites ranged from 385.6 MPa to 1792.6 MPa, which are much higher than that of peripheral nerves. However, the tensile strength obtained in PHA blend/BG1 (1% w/v) (10.0 ± 0.6 MPa) was found to be similar to that of rabbit peroneal nerve measured in another study. Therefore, although PHA blend/BG1 (1% w/v) does not provide the adequate elasticity, it has the appropriate strength that NGCs require. PHA blend/BG1 (1% w/v) showed the best performance in supporting growth and neuronal differentiation of NG108-15 amongst all the substrates in all the cell culture experiments (Live/dead cell test, neurite outgrowth assessment on NG108-15 neuronal cell and on NG108-15/Schwann cell co-cultures). Moreover, neurite extension found on the PHA blend/BG1 (1% w/v) was remarkable as neurites formed a complex connection network. No correlation was found between the surface characteristics (roughness, hydrophilicity and pore size) of PHA blend/BGs at different concentrations of BG and cell growth and differentiation. Two prototypes of NGCs were fabricated using blends of PHAs as base materials. Since 75:25 P(3HO)/P(3HB) blend films have shown to possess the required flexibility to be implanted in peripheral nerves, this polymer blend was chosen for manufacturing of NGCs. The NGC prototype 1 consisted of a nerve guidance conduit made from 75:25 P(3HO)/P(3HB) blend with luminal electrospun aligned fibres fabricated with 25:75 P(3HO)/P(3HB) blend. The prototype 2 comprised of a hollow tube made from 75:25 P(3HO)/P(3HB) blend manufactured by dip-moulding using various dipping conditions. The NGC prototype 2 was implanted in rats for in vivo work, which was carried out in Neuroscience Institute Cavalieri Ottolenghi, Italy. The regenerated nerves were removed and processed for high-resolution light microscopy, transmission electron microscopy and immunohistochemistry analysis. Myelinated and unmyelinated fibres, Schwann cells, connective tissue and vessels were observed in the middle of NGCs through transmission electron microscopy. Although, quantitative estimation of myelinated and unmyelinated nerve fibres was not carried out, these preliminary in vivo results revealed the potential of 75:25 P(3HO)/P(3HB) NGCs to support regeneration. To summarize, the biocompatibility and mechanical properties of theP(3HO)/P(3HB) blends made these polymers excellent materials for nerve tissue engineering and for the manufacture of novel nerve guidance conduits

Urban development and transport disadvantage: Methodology to evaluate social transport needs in Latin American cities

This article examines the theoretical framework for accessibility, social exclusion and provision of public transport. The socio-economic and urban characteristics of Latin American cities require the creation of specific indices to determine social needs for public transport. In the article an index of social transport needs is drawn up. It can be used to highlight a problem which is severely affecting wide groups in Latin America who suffer social exclusion aggravated by a deficient provision of public transport, and that the planning of public transport systems must take into account mechanisms which include social needs in the decision making process.

Análisis y propuestas de acción sobre legislación referida al consumo eficiente del agua

El agua es un recurso limitado e indispensable para la supervivencia humana. Una política sustentable, garantiza la preservación de la misma en las fuentes evitando su agotamiento, reduce costos y contaminación, ya que produce una disminución en su consumo y la cantidad de fluentes a tratar. Toda estrategia de ahorro debe abarcar: una legislación acorde a nivel, provincial y nacional, métodos y técnicas de eficiencia, la tecnología disponible, recomendaciones y acciones. Nuestro país se ha caracterizado por carecer de una legislación y políticas referidas al consumo eficiente del agua. En este trabajo se plantea un análisis de la legislación referida al tema. Se comparó la legislación existente en Argentina y en diversos centros urbanos a nivel internacional, con el objetivo de poder determinar las carencias existentes en nuestro medio y definir así conclusiones que lleven a posibles líneas de acción al respecto.Fil: Rodríguez, Viviana L. Universidad Nacional de Cuyo. Facultad de Filosofía y LetrasFil: Lizarraga, Susana B. Universidad Nacional de Cuyo. Facultad de Filosofía y LetrasFil: Araujo, Héctor. Universidad Nacional de Cuyo. Facultad de Filosofía y Letra

Unidirectional Neuronal Cell growth and Differentiation on Aligned Polyhydroxyalkanoate Blend Microfibres with Varying Diameters

Polyhydroxyalkanoates (PHAs) are a family of prokaryotic-derived biodegradable and biocompatible natural polymers known to exhibit neuroregenerative properties. In this work, poly(3-hydroxybutyrate), P(3HB) and poly(3-hydroxyoctanoate), P(3HO), have been combined to form blend fibres for directional guidance of neuronal cell growth and differentiation. A 25:75 P(3HO)/P(3HB) blend (PHA blend) was used for the manufacturing of electrospun fibres as resorbable scaffolds to be used as internal guidance lumen structures in nerve conduits. The biocompatibility of these fibres was studied using neuronal and Schwann cells. Highly aligned and uniform fibres with varying diameters were fabricated by controlling electrospinning parameters. The resulting fibre diameters were 2.4 ± 0.3 µm, 3.7 ± 0.3 µm and 13.5 ± 2.3 µm for small, medium and large diameter fibres respectively. The cell response to these electrospun fibres was investigated with respect to growth and differentiation. Cell migration observed on the electrospun fibres showed topographical guidance in accordance with the direction of the fibres. The correlation between fibre diameter and neuronal growth under two conditions; individually and in co-culture with Schwann cells was evaluated. Results obtained from both assays revealed that all PHA blend fibre groups were able to support growth and guide aligned distribution of neuronal cells and there was a direct correlation between the fibre diameter and neuronal growth and differentiation. This work has led to the development of a family of unique biodegradable and highly biocompatible 3D substrates capable of guiding and facilitating the growth, proliferation and differentiation of neuronal cells as internal structures within nerve conduits

Nerve tissue engineering using blends of poly(3-hydroxyalkanoates) for peripheral nerve regeneration

The only types of polyhydroxyalkanoates (PHAs) that have been explored for use in nerve regeneration are poly(3‐hydroxybutyrate), P(3HB), and poly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate) (P(3HB‐co‐3HHx)). However, nerve regeneration induced by these PHAs is inferior to that of autologous nerve grafting. The aim of this work was to study novel PHA blends as resorbable biomaterials for the manufacture of nerve guidance conduits. PHA blend films with varying ratios of poly(3‐hydroxyoctanoate)/poly(3‐hydroxybutyrate) (P(3HO)/P(3HB)) were produced using the solvent‐casting method. Neat films of P(3HO) and P(3HB), along with 25:75, 50:50, and 75:25 blend films of P(3HO)/P(3HB), were characterized with respect to chemical, material, and biological properties. On surface analysis, the blends exhibited higher values of roughness compared with the neat films. The differential scanning calorimetry characterization of the blends confirmed that P(3HO) and P(3HB) formed immiscible blends. FTIR and XRD analysis of the blends showed a decrease in crystallinity along with an increase of the proportion of P(3HO) . However, an increase in the stiffness of the blends was observed when the proportion of P(3HB) increased. Although all of the blends were biocompatible with NG108‐15 neuronal cells, the 25:75 P(3HO)/P(3HB) blend showed significantly better support for growth and differentiation of these cells. The mechanical properties of PHA blends correspond to the reported properties of peripheral nerves. Therefore, they could serve as base material for the manufacture of nerve guidance conduits

Guest editorial: special issue on vision-based human activity recognition

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Preclinical study of peripheral nerve regeneration using nerve guidance conduits based on polyhydroxyalkanaotes

Nerve guidance conduits (NGCs) are used as an alternative to the “gold standard” nerve autografting, preventing the need for surgical intervention required to harvest autologous nerves. However, the regeneration outcomes achieved with the current NGCs are only comparable with autografting when the gap is short (less than 10 mm). In the present study, we have developed NGCs made from a blend of Polyhydroxyalkanoates, a family of natural resorbable polymers. Hollow NGCs made from a 75:25 Poly(3‐hydroxyoctanoate)/Poly(3‐hydroxybutyrate) blend (PHA‐NGCs) were manufactured using dip‐moulding. These PHA‐NGCs showed appropriate flexibility for peripheral nerve regeneration. In vitro cell studies performed using RT4‐D6P2T rat Schwann cell line confirmed that the material is capable of sustaining cell proliferation and adhesion. PHA‐NGCs were then implanted in vivo to repair 10 mm gaps of the median nerve of female Wistar rats for 12 weeks. Functional evaluation of the regenerated nerve using the grasping test showed that PHA‐NGCs displayed similar motor recovery as the autograft, starting from week 7. Additionally, nerve cross‐sectional area, density and number of myelinated cells, as well as axon diameter, fibre diameter, myelin thickness and g‐ratio obtained using the PHA‐NGCs were found comparable to an autograft. This preclinical data confirmed that the PHA‐NGCs are indeed highly promising candidates for peripheral nerve regeneration

Cáncer de mama durante el embarazo

As women in western countries delay childbearing, it has been hypothesized that the incidence of breast cancer diagnosed during pregnancy will increase. Breast carcinoma during pregnancy(BCP) put the health of the mother in conflict with that of the fetus. The aim is to give optimal treatment to the mother to maximise the chances of survival, whilst minimising the risk of harm of the fetus. Few breast surgeons or oncologist develop expertise in this area owing the rarity of the association. We report the epidemiology, pathology, clinical picture, therapeutic management and fetal outcome of pregnant women with breast cancer treated in our institutio

Cross-species complementation reveals conserved functions for EARLY FLOWERING 3 between monocots and dicots

Plant responses to the environment are shaped by external stimuli and internal signaling pathways. In both the model plant Arabidopsis thaliana (Arabidopsis) and crop species, circadian clock factors are critical for growth, flowering, and circadian rhythms. Outside of Arabidopsis, however, little is known about the molecular function of clock gene products. Therefore, we sought to compare the function of Brachypodium distachyon (Brachypodium) and Setaria viridis (Setaria) orthologs of EARLY FLOWERING 3, a key clock gene in Arabidopsis. To identify both cycling genes and putative ELF3 functional orthologs in Setaria, a circadian RNA-seq dataset and online query tool (Diel Explorer) were generated to explore expression profiles of Setaria genes under circadian conditions. The function of ELF3 orthologs from Arabidopsis, Brachypodium, and Setaria was tested for complementation of an elf3 mutation in Arabidopsis. We find that both monocot orthologs were capable of rescuing hypocotyl elongation, flowering time, and arrhythmic clock phenotypes. Using affinity purification and mass spectrometry, our data indicate that BdELF3 and SvELF3 could be integrated into similar complexes in vivo as AtELF3. Thus, we find that, despite 180 million years of separation, BdELF3 and SvELF3 can functionally complement loss of ELF3 at the molecular and physiological level