Alternative tissue engineering scaffolds based on starch: processing methodologies, morphology, degradation and mechanical properties

http://www.sciencedirect.com/science/journal/09284931An ideal tissue engineering scaffold must be designed from a polymer with an adequate degradation rate. The processing technique must allow for the preparation of 3-D scaffolds with controlled porosity and adequate pore sizes, as well as tissue matching mechanical properties and an appropriate biological response. This communication revises recent work that has been developed in our laboratories with the aim of producing 3-D polymeric structures (from starch-based blends) with adequate properties to be used as scaffolds for bone tissue engineering applications. Several processing methodologies were originally developed and optimised. Some of these methodologies were based on conventional melt-based processing routes, such as extrusion using blowing agents (BA) and compression moulding (combined with particulate leaching). Other developed technologies included solvent casting and particle leaching and an innovative in situ polymerization method. By means of using the described methodologies, it is possible to tailor the properties of the different scaffolds, namely their degradation, morphology and mechanical properties, for several applications in tissue engineering. Furthermore, the processing methodologies (including the blowing agents used in the melt-based technologies) described above do not affect the biocompatible behaviour of starch-based polymers. Therefore, scaffolds obtained from these materials by means of using one of the described methodologies may constitute an important alternative to the materials currently used in tissue engineering

Cytocompatibility and response of osteoblastic-like cells to starch-based polymers : effect of several additives and processing conditions

This work reports on the biocompatibility evaluation of new biodegradable starch-based polymers that are under consideration for use in orthopaedic temporary applications and as tissue engineering scaffolds. It has been shown in previous works that by using these polymers it is both possible to produce polymer/hydroxyapatite (HA) composites (with or without the use of coupling agents) with mechanical properties matching those of the human bone, and to obtain 3D structures generated by solid blowing agents, that are suitable for tissue engineering applications. This study was focused on establishing the influence of several additives (ceramic fillers, blowing agents and coupling agents) and processing methods/conditions on the biocompatibility of the materials described above. The cytotoxicity of the materials was evaluated using cell culture methods, according to ISO/EN 109935 guidelines. A cell suspension of human osteosarcoma cells (HOS) was also seeded on a blend of corn starch with ethylene vinyl alcohol (SEVA-C) and on SEVA-C/HA composites, in order to have a preliminary indication on cell adhesion and proliferation on the materials surface. In general, the obtained results show that all the different materials based on SEVA-C, (which are being investigated for use in several biomedical applications), as well as all the additives (including the novel coupling agents) and different processing methods required to obtain the different properties/products, can be used without inducing a cytotoxic behaviour to the developed biomaterial

Conditional knockout of RAD51-related genes in Leishmania major reveals a critical role for homologous recombination during genome replication

Homologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome of Leishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genome-wide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins in Leishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal but in each case growth slows with time and leads to DNA damage and accumulation of cells with aberrant DNA content. Despite these similarities, we show that only loss of RAD51 or RAD51-3 impairs DNA synthesis and causes elevated levels of genome-wide mutation. Furthermore, we show that these two HR factors act in distinct ways, since ablation of RAD51, but not RAD51-3, has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival of Leishmania and reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote

Prediction of intracellular storage polymers using quantitative image analysis in enhanced biological phosphorus removal systems

Intracellular polymers, such as polyhydroxyalkanoates (PHA) synthesized by microorganisms for energy and carbon storage, can be commercially used as biodegradable plastics in a wide range of applications. The possibility of obtaining high PHA contents (where the most common monomers found are poly-hydroxybutyrate (PHB) and poly-hydroxyvalerate (PHV)) from inexpensive inocula and raw materials emerges as a promising and commercially interesting alternative. For this purpose, mixed cultures operated under feast/famine cycles are most frequently used. PHA is an important storage polymer in the metabolism of microorganisms involved in enhanced biological phosphorus removal (EBPR) systems. It is stored by polyphosphate accumulating organisms (PAO) and glycogen accumulating organisms (GAO), as described in previous publications [1,2]. Monitoring intracellular storage polymers in bacteria is usually performed through laborious and time consuming off-line chemical analyses. Thus, there is clearly a need to develop new techniques in order to promptly monitor these processes. Image analysis techniques have the potential to be a non-invasive and rapid means of assessing the amount of different storage polymers inside microbial cells, providing the evaluation of these important biotechnological processes. The present study focuses on predicting intracellular storage polymers in EBPR systems. For that purpose, quantitative image analysis techniques were developed and partial least squares (PLS) were used to model PHA results. An EBPR fed with synthetic wastewater containing volatile fatty acids (VFAs) and orthophosphate was used. Biomass samples were collected at the end of the anaerobic and aerobic phases. Analytical PHA quantification was performed by biomass digestion and gas chromatography analysis. In the concurring image analysis methodology Nile blue was used as a fluorescence staining method for PHA granules identification. The results from image analysis allowed establishing a PHB prediction ability presenting a regression value (R2) of 0.854, a PHA prediction regression value (R2) of 0.843 and a PHV prediction regression value (R2) of 0.779. The lower prediction ability for PHV could be explained since this parameter presented only a small contribution to the overall PHA. The analysis performed based on the variable importance in the projection (VIP) established a core of three image analysis parameters (granules total area, granules total intensity and image intensity) as the most important regarding PHA, PHB and PHV prediction

Development of image analysis methodologies to quantify intracellular PHA, polyphosphate and glycogen within wastewater treatment plants

In wastewater treatment plants (WWTP), enhanced biological phosphorus removal (EBPR) processes are performed by mixed cultures containing polyphosphate (PAO) and glycogen accumulating organisms (GAO). In these processes, it is of crucial importance to monitor the intracellular metabolism, namely glycogen, polyhydroxyalkanoates (PHA) and polyphosphate (polyP) inclusions, to determine its efficiency. However, traditional monitoring, carried out through off-line chemical analyses, is laborious and time-consuming. Therefore, there is a clear need to develop new techniques to promptly quantify these intracellular polymers, with image analysis emerging as a promising tool. The use of staining methodologies with specific fluorescent dyes is widespread in EBPR research, including Nile blue for PHA and DAPI for polyP. Although rarely applied in EBPR studies, Aniline blue is a fluorescent stain that can be used for glycogen determination. Furthermore, these fluorescent stains have generally been employed for qualitative rather than quantitative analysis. Therefore, this study aim focused on developing fluorescence-based staining methodologies for glycogen, and on acquisition, processing and image analysis procedures for PHA, polyP and glycogen. Image analysis data was then correlated with traditional analytical data by multivariable statistics. Regarding the determination of the glycogen intracellular concentration, results have been promising, presenting a good correlation (R2 of 0.915) between analytical and image analysis data. The staining and image analysis procedures for the determination of the intracellular concentration of PHA and polyP are currently being optimized. This study will provide a quantitative means to assess PAO and GAO metabolic activity in-situ in WWTP, facilitating the optimisation of these processes

Desenvolvimento de metodologias de análise de imagem para quantificar PHA, polifosfatos e glicogénio intracelular em estações de tratamento de águas residuais

O processo de remoção biológica de fósforo, em estações de tratamento de águas residuais, é um processo efetuado por culturas mistas contendo organismos acumuladores de polifosfatos (PAO) e de glicogénio (GAO). No decurso deste processo os microrganismos podem formar inclusões de glicogénio, polihidroxialcanoatos (PHA) e polifosfatos (poli-P). Neste processo, é fulcral monitorizar o metabolismo intracelular para determinar a sua eficiência. Contudo, a sua monitorização, realizada através de análise químicas em diferido, é laboriosa e morosa. Deste modo, existe uma clara necessidade do desenvolvimento de métodos mais expeditos, como metodologias de análise de imagens, para a monitorização destes polímeros intracelulares. Estas técnicas foram implementadas neste estudo, encontrando-se, no caso da determinação da concentração intracelular de poli-P, em fase de desenvolvimento dos protocolos de coloração e aquisição de imagens. Para a determinação da concentração intracelular de glicogénio, foi obtida uma boa correlação inicial. Na determinação da concentração intracelular de PHA, este estudo foca-se na otimização dos protocolos de coloração e no desenvolvimento do programa de análise de imagem

Novel method to quantify intracellular accumulation of polyphosphate in EBPR systems

A new method for intracellular storage polyphosphate (poly-P)identification and quantification in enhanced biological phosphorus removal (EBPR) systems is proposed based on image analysis. In EBPR systems, 4',6-diamidino-2-phenylindole (DAPI) is usually combined with fluororescent in situ hybridization (FISH) to evaluate the microbial community. The proposed technique is based on an image analysis procedure specifically developed for determining poly-P inclusions within biomass suspension using solely DAPI by epifluorescence microscopy. Due to contradictory literature DAPI concentrations used for poly-P detection, the present work assessed the optimal DAPI concentration for samples acquired at the end of the EBPR aerobic stage when the accumulation is performed. Digital images were then acquired and processed by means of image processing and analysis. Regarding image analysis results and considering the current operational conditions, a promising correlation could be found between average poly-P intensity values and the analytical determination, although presenting a correlation coefficient somewhat far from the ideal. The proposed methodology can be seen as a promising alternative procedure to quantify intracellular poly-P accumulation in a faster and less labor intensive way

Author Correction: iLoF: An intelligent Lab on Fiber Approach for Human Cancer Single-Cell Type Identification

An amendment to this paper has been published and can be accessed via a link at the top of the paper.This work was partially funded by the projects NanoSTIMA and NORTE-01-0145-FEDER-000029, both supported by the North Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, and through the European Regional Development Fund (ERDF); and by the Portuguese Foundation for Science and Technology, within the scope of the PhD grant PD/BD/135023/2017 and the projects: PTDC/BBB-EBI/0567/2014 (to CAR) and UID/BIM/04293/2013. It was also funded by FEDER funds through the Operational Programme for Competitiveness Factors-COMPETE (POCI-01-0145-FEDER-016585; POCI-01-0145-FEDER-007274; PPBI-POCI-01-0145-FEDER-022122). MB acknowledges the Marie Sklodowska-Curie grant agreement No. 748880

Biopolymer monitoring using quantitative image analysis techniques

Polyhydroxyalkanoates (PHAs) are intracellular biopolymers with many applications, particularly as substitutes of polypropylene and polyethylene, due to their thermoplastic properties and biocompatible nature. Furthermore, glycogen is a polysaccharide of glucose with high importance in the metabolism of microbial communities and polyphosphate is a microbial storage compound that should be recovered in order to offset the worldwide depletion of phosphorus sources. The determination of these biopolymers by chemical analysis is a laborious task, often involving digestion processes prior to gas and high-performance liquid chromatography, which are time consuming and difficult to apply in industry. Currently, it is important to develop new, rapid and simple techniques to monitor these polymers. Image analysis is a non-invasive and rapid technique that has the potential to be used to quantify these intracellular polymers quickly, in real-time. Mesquita et al. (2013) showed that it is possible to predict the concentration of glycogen and PHAs by quantitative image analysis, using aniline blue and nile blue staining, respectively. Polyphosphate can also be predicted by this technique through DAPI staining, which is currently under development. These biopolymers are produced by several different microorganisms, and combining their quantification with fluorescence in situ hybridization (FISH) techniques for microbial identification can enable the determination of organisms that store high quantities of each biopolymer. In this work, an advanced quantitative technique is developed to perform real time monitoring of these three biopolymers in a bioreactor performing biological phosphorus removal. Image analysis of the biopolymers was combined with FISH to determine the storage level of each compound within the different microbial populations. This technique will further enable the assessment of biopolymer levels within microbial communities, which can be applied in the biopolymer production industry

Comparison of post-exercise hypotension responses in Paralympic powerlifting athletes after completing two bench press training intensities.

Background and objective: Post-exercise hypotension, the reduction of blood pressure after a bout of exercise, is of great clinical relevance. Resistance exercise training is considered an important contribution to exercise training programs for hypertensive individuals and athletes. In this context, post-exercise hypotension could be clinically relevant because it would maintain blood pressure of hypertensive individuals transiently at lower levels during day-time intervals, when blood pressure is typically at its highest levels. The aim of this study was to compare the post-exercise cardiovascular effects on Paralympic powerlifting athletes of two typical high-intensity resistance-training sessions, using either five sets of five bench press repetitions at 90% 1 repetition maximum (1RM) or five sets of three bench press repetitions at 95% 1RM. Materials and Methods: Ten national-level Paralympic weightlifting athletes (age: 26.1 ± 6.9 years; body mass: 76.8 ± 17.4 kg) completed the two resistance-training sessions, one week apart, in a random order. Results: Compared with baseline values, a reduction of 5–9% in systolic blood pressure was observed after 90% and 95% of 1RM at 20–50 min post-exercise. Furthermore, myocardial oxygen volume and double product were only significantly increased immediately after and 5 min post-exercise, while the heart rate was significantly elevated after the resistance training but decreased to baseline level by 50 min after training for both training conditions. Conclusions: A hypotensive response can be expected in elite Paralympic powerlifting athletes after typical high-intensity type resistance-training sessions.publishedVersio