A poly(lactic-co-glycolic acid) knitted scaffold for tendon tissue engineering: an in vitro and in vivo study

We have designed a composite scaffold for potential use in tendon or ligament tissue engineering. The composite scaffold was made of a cellularized alginate gel that encapsulated a knitted structure. Our hypothesis was that the alginate would act as a cell carrier and deliver cells to the injury site while the knitted structure would provide mechanical strength to the composite construct. The mechanical behaviour and the degradation profile of the poly(lactic-co-glycolic acid) knitted scaffolds were evaluated. We found that our scaffolds had an elastic modulus of 750 MPa and that they lost their physical integrity within 7 weeks of in vitro incubation. Autologous rabbit mesenchymal stem cell seeded composite scaffolds were implanted in a 1-cm-long defect created in the rabbit tendon, and the biomechanical properties and the morphology of the regenerated tissues were evaluated after 13 weeks. The regenerated tendons presented higher normalized elastic modulus of (60%) when compared with naturally healed tendons (40%). The histological study showed a higher cell density and vascularization in the regenerated tendons.

Preparation and characterization of poly(lactic-co-glycolic acid) microparticles containing DNA molecules encoding a malaria vaccine candidate

Background A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles of a malaria DNA vaccine is presented. A 40 kHz ultrasonic atomization device was used to create the microparticles from a feedstock containing 5 volumes of 0.5% w/v PLGA in acetone and 1 volume of condensed DNA which was fed at a flow rate of 18ml h-1. The plasmid DNA vectors encoding a malaria protein were condensed with a cationic polymer before atomization. Results High levels of gene expression in vitro were observed in COS-7 cells transfected with condensed DNA at a nitrogen to phosphate (N/P) ratio of 10. At this N/P ratio, the condensed DNA exhibited a monodispersed nanoparticle size (Z-average diameter of 60.8 nm) and a highly positive zeta potential of 38.8mV. The microparticle formulations of malaria DNA vaccine were quality assessed and it was shown that themicroparticles displayed high encapsulation efficiencies between 82-96% and a narrow size distribution in the range of 0.8-1.9 μm. In vitro release profile revealed that approximately 82% of the DNA was released within 30 days via a predominantly diffusion controlledmass transfer system. Conclusions This ultrasonic atomization technique showed excellent particle size reproducibility and displayed potential as an industrially viable approach for the formulation of controlled release particles.

Crystallization kinetics of poly(l-lactic acid)

The morphology and crystal growth of poly(l-lactic acid), PLLA have been studied from the melt as a function of undercooling and molecular weight using hot stage microscopy. Attention has been given to the application of growth rate equation on the growth rate data of PLLA and thus various nucleation parameters have been calculated. The criteria of Regime I and Regime II types of crystallization has been applied for the evaluation of substrate lengths.

Persistence of orally administered Megasphaera elsdenii and Ruminococcus bromii in the rumen of beef cattle fed a high grain (barley) diet

When cattle are fed grain, acidotic ruminal conditions and decreased efficiency in starch utilisation can result from the rapid production and accumulation of lactic acid in the rumen. The efficacy of drenching cattle with Megasphaera elsdenii and Ruminococcus bromii to improve animal performance was investigated. A feedlot trial was undertaken with 80 Bos indicus crossbred steers (initial liveweight 347.1 (s.d. 31.7) kg) in 10 pens in a randomised complete block design. An empty-pen-buffer was maintained between treated (inoculated) and untreated (control) groups to avoid transfer of inoculant bacteria to the control steers. Inoculated steers were orally drenched with M. elsdenii YE34 and R. bromii YE282, and populations increased rapidly over 3-14 days. The steers were fed for a total of 70 days with commercial, barley-based, feedlot rations. High growth rates (1.91 kg per day) were achieved throughout the experiment in both the inoculated and control steers. Intakes averaged 21.3 g dry matter (DM) per kg liveweight per day. There was probably no acidosis achieved in this trial following challenge (i.e. no change in pH occurred). There were no differences in any production or carcass measurements between the control and inoculated steers overall. However, the control group acquired dense ruminal populations of M. elsdenii by Day 14, while R. bromii populations established at high densities within the first 2 weeks but then declined and were undetectable by Day 50. R. bromii appears to be only transiently dominant, and once its dominance waned, it appeared that Ruminobacter spp. established in the rumen. Ruminobacter spp. became dominant between 14 and 28 days in all the steers examined and persisted through to the end of the study. These Ruminobacter spp. may be of future interest in the development of probiotics for grain-fed cattle.

Lactic acid bacteria and their antimicrobial peptides : Induction,detection,partial characterization, and potential applications

Bacteriocin-producing lactic acid bacteria and their isolated peptide bacteriocins are of value to control pathogens and spoiling microorganisms in foods and feed. Nisin is the only bacteriocin that is commonly accepted as a food preservative and has a broad spectrum of activity against Gram-positive organisms including spore forming bacteria. In this study nisin induction was studied from two perspectives, induction from inside of the cell and selection of nisin inducible strains with increased nisin induction sensitivity. The results showed that a mutation in the nisin precursor transporter NisT rendered L. lactis incapable of nisin secretion and lead to nisin accumulation inside the cells. Intracellular proteolytic activity could cleave the N-terminal leader peptide of nisin precursor, resulting in active nisin in the cells. Using a nisin sensitive GFP bioassay it could be shown, that the active intracellular nisin could function as an inducer without any detectable release from the cells. The results suggested that nisin can be inserted into the cytoplasmic membrane from inside the cell and activate NisK. This model of two-component regulation may be a general mechanism of how amphiphilic signals activate the histidine kinase sensor and would represent a novel way for a signal transduction pathway to recognize its signal. In addition, nisin induction was studied through the isolation of natural mutants of the GFPuv nisin bioassay strain L. lactis LAC275 using fl uorescence-activated cell sorting (FACS). The isolated mutant strains represent second generation of GFPuv bioassay strains which can allow the detection of nisin at lower levels. The applied aspect of this thesis was focused on the potential of bacteriocins in chicken farming. One aim was to study nisin as a potential growth promoter in chicken feed. Therefore, the lactic acid bacteria of chicken crop and the nisin sensitivity of the isolated strains were tested. It was found that in the crop Lactobacillus reuteri, L. salivarius and L. crispatus were the dominating bacteria and variation in nisin resistance level of these strains was found. This suggested that nisin may be used as growth promoter without wiping out the dominating bacterial species in the crop. As the isolated lactobacilli may serve as bacteria promoting chicken health or reducing zoonoosis and bacteriocin production is one property associated with probiotics, the isolated strains were screened for bacteriocin activity against the pathogen Campylobacter jejuni. The results showed that many of the isolated L. salivarius strains could inhibit the growth of C. jejuni. The bacteriocin of the L. salivarius LAB47 strain, with the strongest activity, was further characterized. Salivaricin 47 is heat-stable and active in pH range 3 to 8, and the molecular mass was estimated to be approximately 3.2 kDa based on tricine SDS-PAGE analysis.

Eutectic crystallization of poly(l-lactic acid) and pentaerythrityl tetrabromide

Diluents (either low molecular weight compounds orother polymers) are known to modify the morphology, the rates of nucleation and growth of polymers 1- 4. Recentlybinary systems in which both the components crystallize simultaneously to give a eutectic solid have been studied with great interest. Carbonnei et al.

Poly(lactic-co-glycolic acid): Carbon nanofiber composites for myocardial tissue engineering applications

The objective of the present in vitro research was to investigate cardiac tissue cell functions (specifically cardiomyocytes and neurons) on poly(lactic-co-glycolic acid) (PLGA) (50:50 wt.%)-carbon nanofiber (CNF) composites to ascertain their potential for myocardial tissue engineering applications. CNF were added to biodegradable PLGA to increase the conductivity and cytocompatibility of pure PLGA. For this reason, different PLGA:CNF ratios (100:0, 75:25, 50:50,25:75, and 0:100 wt.%) were used and the conductivity as well as cytocompatibility of cardiomyocytes and neurons were assessed. Scanning electron microscopy, X-ray diffraction and Raman spectroscopy analysis characterized the microstructure, chemistry, and crystallinity of the materials of interest to this study. The results show that PLGA:CNF materials are conductive and that the conductivity increases as greater amounts of CNF are added to PLGA, from OS m(-1) for pure PLGA (100:0 wt.%) to 5.5 x 10(-3) S m(-1) for pure CNF (0:100 wt.%). The results also indicate that cardiomyocyte density increases with greater amounts of CNF in PLGA (up to 25:75 wt.% PLGA:CNF) for up to 5 days. For neurons a similar trend to cardiomyocytes was observed, indicating that these conductive materials promoted the adhesion and proliferation of two cell types important for myocardial tissue engineering applications. This study thus provides, for the first time, an alternative conductive scaffold using nanotechnology which should be further explored for cardiovascular applications. (C) 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Molecular diagnosis of distal renal tubular acidosis in Tunisian patients: proposed algorithm for Northern Africa populations for the ATP6V1B1, ATP6V0A4 and SCL4A1 genes

Background: Primary distal renal tubular acidosis (dRTA) caused by mutations in the genes that codify for the H+ -ATPase pump subunits is a heterogeneous disease with a poor phenotype-genotype correlation. Up to now, large cohorts of dRTA Tunisian patients have not been analyzed, and molecular defects may differ from those described in other ethnicities. We aim to identify molecular defects present in the ATP6V1B1, ATP6V0A4 and SLC4A1 genes in a Tunisian cohort, according to the following algorithm: first, ATP6V1B1 gene analysis in dRTA patients with sensorineural hearing loss (SNHL) or unknown hearing status. Afterwards, ATP6V0A4 gene study in dRTA patients with normal hearing, and in those without any structural mutation in the ATP6V1B1 gene despite presenting SNHL. Finally, analysis of the SLC4A1 gene in those patients with a negative result for the previous studies. Methods: 25 children (19 boys) with dRTA from 20 families of Tunisian origin were studied. DNAs were extracted by the standard phenol/chloroform method. Molecular analysis was performed by PCR amplification and direct sequencing. Results: In the index cases, ATP6V1B1 gene screening resulted in a mutation detection rate of 81.25%, which increased up to 95% after ATP6V0A4 gene analysis. Three ATP6V1B1 mutations were observed: one frameshift mutation (c.1155dupC; p.Ile386fs), in exon 12; a G to C single nucleotide substitution, on the acceptor splicing site (c.175-1G > C; p.?) in intron 2, and one novel missense mutation (c. 1102G > A; p. Glu368Lys), in exon 11. We also report four mutations in the ATP6V0A4 gene: one single nucleotide deletion in exon 13 (c.1221delG; p. Met408Cysfs* 10); the nonsense c.16C > T; p.Arg6*, in exon 3; and the missense changes c.1739 T > C; p.Met580Thr, in exon 17 and c.2035G > T; p.Asp679Tyr, in exon 19. Conclusion: Molecular diagnosis of ATP6V1B1 and ATP6V0A4 genes was performed in a large Tunisian cohort with dRTA. We identified three different ATP6V1B1 and four different ATP6V0A4 mutations in 25 Tunisian children. One of them, c.1102G > A; p.Glu368Lys in the ATP6V1B1 gene, had not previously been described. Among deaf since childhood patients, 75% had the ATP6V1B1 gene c. 1155dupC mutation in homozygosis. Based on the results, we propose a new diagnostic strategy to facilitate the genetic testing in North Africans with dRTA and SNHL.

Poly(lactic acid)-based bio-blends and nanocomposites

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