Biodegradable stents (BRS) offer enormous potential but first they must meet five specific requirements: (i) their manufacturing process must be precise; (ii) degradation should have minimal toxicity; (iii) the rate of degradation should match the recovery rate of vascular tissue; (iv) ideally, they should induce rapid endothelialization to restore the functions of vascular tissue, but at the same time reduce the risk of restenosis; and (v) their mechanical behavior should comply with medical requirements, namely, the flexibility required to facilitate placement but also sufficient radial rigidity to support the vessel. Although the first three requirements have been comprehensively studied, the last two have been overlooked. One possible way of addressing these issues would be to fabricate composite stents using materials that have different mechanical, biological, or medical properties, for instance, Polylactide Acid (PLA) or Polycaprolactone (PCL). However, fashioning such stents using the traditional stent manufacturing process known as laser cutting would be impossible. Our work, therefore, aims to produce PCL/PLA composite stents using a novel 3D tubular printer based on Fused Deposition Modelling (FDM). The cell geometry (shape and area) and the materials (PCL and PLA) of the stents were analyzed and correlated with 3T3 cell proliferation, degradation rates, dynamic mechanical and radial expansion tests to determine the best parameters for a stent that will satisfy the five strict BRS requirements. Results proved that the 3D-printing process was highly suitable for producing composite stents (approximately 85–95% accuracy). Both PCL and PLA demonstrated their biocompatibility with PCL stents presenting an average cell proliferation of 12.46% and PLA 8.28% after only 3 days. Furthermore, the PCL/PLA composite stents demonstrated their potential in degradation, dynamic mechanical and expansion tests. Moreover, and regardless of the order of the layers, the composite stents showed  medium levels of degradation rates and mechanical modulus. Radially, they exhibited the virtues of PCL in the expansion step (elasticity) and those of PLA in the recoil step (rigidity). Results have clearly demonstrated that composite PCL/PLA stents are a highly promising solution to fulfilling the rigorous BRS requirements. Biodegradable stents (BRS) were introduced to overcome the limitations of permanent stents and to offer significant advantages such as, among others, complete bioresorption and/or mechanical flexibility. . BRS hav2 laser cutting and sterilization have on Poly-L-Lactide (PLLA). Their results revealed the enormous influence sterilization has on the mechanical properties of PLLA . Using additive manufacturing, Park et al.  were tested. Two-dimensional (2D) cell culture was also performed to normalize the cell proliferation rates on the stents. Because of their rapid and stable growth kinetic, fibroblast cells were selected to carry out the first proof of concept for the PCL/PLA composite stents. However, further studies with other kinds of cells, such as endothelial human cell, are required and should be performed.Murine 3T3 fibroblasts cells were obtained from the American Type Culture Collection . The 3T3 cells were cultured in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% sodium pyruvate, 50 U/mL penicillin and 50 μg/mL . Cells were kept at 37 °C and 5% CO2 atmosphere for 3 days. Because our aims were to determine whether PCL and PLA are biologically valid and to define the stent geometry that produces the best cell proliferation, cell culture was performed in static conditions. However, further work using dynamic conditions to simulate a real environment would be beneficial because under dynamic conditions stents would be subjected to pressure and flow conditions that can modify the kinetics of cell proliferation.The stents were placed in 24-well non-adherent microplates , soaked with DMEM and incubated at 37 °C prior to cell seeding. Finally, due to cell growth kinetics and the culture period, the stents were seeded with a 20,000-cell-per-stent concentration and kept at 37 °C in a 5% CO®  was used to process the images and collect the data.A Nikon SMZ-745T optical microscope  attached to a ProgRes CT3 digital camera  was used to determine the dimensional features  of the samples. Image JG, NC, FR, and Ma) had on cell proliferation. The subsequent results allowed us to then select the best parameters with which to manufacture the final PCL/PLA composite stents with the best biological response. An MTT (3-4.5-dimethyl-2-thiazolyl)-2.5-diphenyltetrazolium bromide) assay was selected to test the cell proliferation on the stents. MTT is a yellow tetrazolium salt which can be reduced by the mitochondria of viable cells. This metabolic transformation results in water-insoluble purple crystals of formazan being produced, which can then be solubilized with dimethyl sulfoxide (DMSO) into a colored solution. The absorbance of DMSO solution is related to the number of cells. After incubation, the culture medium was removed, and the stents were put into new wells where they were incubated with 3 mL of DMEM medium and 300 µL of MTT  for 2.5 h. After this time, the MTT supplemented culture medium was removed, DMSO was added and the stents were shacked to ensure the complete dissolution of the resulting formazan crystals. Four 100 µL aliquots from each sample were pipetted into a 96-well plate and absorbance was measured at 570 nm using a microplate reader . Adherent 2D controls were likewise processed with the same number of seeded cells.MTT tests were performed to check the effect the process parameters  wash samples with phosphate buffered solution ; then (ii) fix samples with 4% paraformaldehyde at room temperature and wash samples; next (iii) permeabilize cells with 0.2% Triton X-100; and (iv) add blocking buffer at room temperature; then (v) dye cytoskeleton actin with rhodamine-phalloidin (1:200) in blocking buffer; and finally (vi) dye the cells nuclei with  (DAPI) (1/1000) in blocking buffer. The adhesion assay was performed simply to obtain qualitative data about cell adhesion.E’) was analyzed. The loss tangent is the ratio of loss modulus and storage modulus and this indicates the material’s viscosity and elastic properties, respectively. The loss tangent curve peak appears and can be defined as the material’s glass transition temperature . The dynamic storage modulus (W0), and its residual weight after degradation and once it had been completely dried (rW) in an oven at 30 °C for 24 h. Weight loss percentage, LW%, was estimated using the following equation:The PCL, PLA, and PCL/PLA samples were submerged in phosphate buffered solution  at 37 °C for 6 weeks. Samples were recovered after 2, 4, and 6 weeks under the same conditions (25 °C). Using a METTLER TOLEDO Sartorius 2MP Scale , weight loss was evaluated by measuring the original weight of the sample after the printing process (The PBS was changed frequently to keep the pH as constant as possible.Ø: 4 mm) until a maximum of 175% of their original diameter (MØ: 7 mm) was reached. Then, the radial force was removed, and the diameter was measured to determine the recoil ratio (RØ).The PCL, PLA, and PCL/PLA samples were expanded radially from their original diameter . The analysis was carried out at a 95% confidence level (α = 0.05). All observations were confirmed by at least three independent experiments. All data are expressed as mean ± standard error (SE).Results were subjected to regression analysis. The analysis of variance (ANOVA) method was applied to test the statistical significance of the process parameters , regression analysis showed the considerable statistical influence that flow rate, number of stent radial cells, and material (p < 0.05) have on cell proliferation. Stent cell geometry did not show any significant statistical influence on cell proliferation .Finally, in terms of the decisive effects materials have, lower molecular weights are known to produce poorer cell proliferation . Here, PConfocal Laser Microscopy was performed on the PCL and PLA samples to qualitatively determine how 3T3 cells stick to the stents . The objThe results prove the materials’ biological compatibility and encourage us to believe that PCL/PLA composite stents would comply with the fourth requirement, i.e., rapid endothelization without risk of restenosis. PCL’s better cell proliferation may be useful to increase the proliferation of endothelial vessel cells in the external wall of the stents, while an internal PLA wall may help to reduce the proliferation of cells that produce restenosis. However, further studies with other kinds of cells or substances need to be performed to confirm this. The results here show low cell proliferation because of the small amount of material that the stents have. Additional studies that use longer culture times may be beneficial to obtain better proliferation results.Ø: 4 mm), stent thickness (ST: 0.3 mm [0.15 mm/layer]), total length (L: 19 mm), printer nozzle Tª (220 °C), printer bed Tª (25 °C), printer infill (100%), and printer speed  were kept constant. All the layer sequences were tested to identify if they influenced the properties or not.As the properties of the stents are interrelated and sometimes contradictory, they require a careful compromise. For example, stents should be flexible enough to facilitate their placement but rigid enough to support the vessel. Sometimes higher tensile strength materials, desirable for bolstering radial strength, have higher yield strengths that promote undesired acute recoil. Biological aspects are also contradictory. For example, scaffolds (stents) with smaller pores (cell areas) will give better cell proliferation ,25; howeThe manufacturing process successfully produced PCL/PLA stents , requiriThe intrinsic properties of 3D-printing based on FDM, make producing an object with pointed corners (geometries A and C) impossible. However, the rounded corners obtained could be useful when stents are being implanted and/or might reduce any potential damage to the endothelial in the vessel wall.The mechanisms causing PCL and PLA degradation are well known: degradation occurs when water penetrates the bulk of the material and causes hydrolysis throughout the entire polymer matrix. From the literature it can be concluded that PCL and PLA undergo a two-stage degradation process: first, the non-enzymatic hydrolytic cleavage of ester groups and, second, when the polymer is more highly crystalline and of low molecular weight it experiences intracellular degradation and may be completely resorbed and degraded via an intracellular mechanism .G, NC, and FR) would have). Results have evidenced the variations in degradation rates between PCL and PLA ). Results  and the composition of the layers have on the radial behavior were analyzed.The radial behavior of stents is one of the most important properties because this is what provides the vessel with the appropriate support it needs. The effects the process parameters  and materials (PCL and PLA) exert were also analyzed. Samples were subjected to 3T3 cell proliferation, degradation, dynamic mechanical and radial expansion tests to determine the parameters that best meet the rigorous requirements for BRS.The results have demonstrated the considerable influence the cell area and material of a stent have on 3T3 proliferation. That said, the cell shape of the stent did not show any significant influence at all. Our initial hypothesis was confirmed, i.e., the smaller the cell area of a stent, the better the cell proliferation rate. Meanwhile, as a result of their different molecular weights, PCL demonstrated better cell proliferation than PLA.The degradation rate results revealed the limitations of PLA for BRS purposes as a consequence of its fast degradation rate, whereas PCL showed a better degradation rate. The composite PCL/PLA stents showed an almost medium degradation for all the layer configurations which was mainly due to PLA degradation. The faster PLA degradation rate would eventually leave a BRS made only of PCL. The differences in their degradation rates are produced by their different molecular weights, so employing PCL and PLA with similar molecular weights is a must to obtain a homogenous degradation of all the layers.E’ modulus in the dynamic mechanical results which, in turn, hinders its sole applicability for stent purposes. On the other hand, PLA showed a high E’ modulus and good properties for supporting the artery vessel once in place but its properties hinder its placement. Conversely, composite stents showed a middle E’ modulus regardless of the order the layers were made up of.PCL showed a very low Finally, the radial behavior results have proved that composite PCL/PLA stents could be used to improve each material’s separate limitations. For instance, PCL stents presented overly high recoil ratios but excellent expansion behavior, whereas PLA stents presented inadequate radial expansion, due to their rigidity, but excellent recoil ratio. Composite stents, either with PCL or PLA as the inner layer, demonstrated the virtues of PCL stents  and PLA stents  that could be combined to provide a good solution for BRS. Furthermore, their good radial behavior (regardless of the order of the layers) make composite stents a promising concept for cardiovascular problems.Based on the results presented here, we believe that polymer composite stents manufactured with 3D-printing processes could be a highly effective solution to the current problems that stents made of polymers have. However, FDA rules currently limit the use of 3D-printed stents in real clinical applications and, although PCL and PLA are FDA-approved materials, there are still open challenges to be met before approval for 3D-printed implantable medical devices can be obtained. This manuscript has presented a potential approach for future applications for stents.


Fog harvesting of the Namib desert beetles has inspired many researchers to design artificial fog harvesting hybrid surfaces, which commonly involve flat hydrophilic patterns on hydrophobic surfaces. However, relatively less interest has been shown in the bumpy topography of the Namib desert beetle’s dorsal surface as well as its curved body shape when designing artificial hybrid surfaces. In this work, we explore a fog harvesting flexible hybrid surface that has a superhydrophilic 3D copper oxide pattern on a hydrophobic rough elastomer background surface enabled by transferring a copper layer from a prepared donor substrate to a receiving elastomer substrate. The water collection rates of the hybrid surface and control samples are measured, and the results reveal the advantages of 3D bumpy structures on a curved shape surface to facilitate fog harvesting, particularly in more unfavorable fog stream conditions. The curved 3D bumpy hybrid surface exhibits an over 16 times higher water collection rate than the flat 2D hybrid surface in the fog stream in parallel to the hybrid surface. This work provides an improved understanding of the role of the Namib desert beetle’s bumpy dorsal surface and curved body shape, and offers an insight into the design of novel surfaces with enhanced fog harvesting performance. Water is the most abundant resource in nature. However, the portable water available for living organisms is rather confined, and the amount is constantly diminishing. On top of that, accessible fresh water is unevenly distributed over the surface of the earth. Water scarcity is more severe in arid areas, and living creatures in such areas must adopt special abilities to collect water from atmospheric air for survival ,2,3. ForWhile many efforts have been made on designing artificial hybrid surfaces with a large spatial wettability difference, there has been relatively little work done on 3D structured and curved fog harvesting surfaces mimicking the actual hydrophilic bumps and the curved body of Namib desert beetles. For example, there have been only a few previously reported flat hybrid surfaces with 3D bumpy structures although those are not morphologically comparable to the actual bumps of Namib desert beetles ,14. TherIn this work, we explore a flexible hybrid surface with a 3D superhydrophilic copper oxide (CuO) pattern on a hydrophobic rough polydimethylsiloxane (PDMS) background, which is inspired by the bumps on the curved dorsal surface of Namib desert beetles c–e. To e2, NaOH, and Na3PO4·12H2O were purchased from Sigma Aldrich, St. Louis, MI, USA. Cu etchant (CE-100) was purchased from Transene, Danvers, MA, USA. Hydrochloric acid (HCl) was purchased from Macron Fine Chemicals, Allentown, PA, USA.PDMS (Sylgard 184), Cu electroplating kits, including Microfill EVF Carrier, Brightner, and Leveler, were purchased from Dow Corning, Midland, MI, USA. Potassium hydroxide pellet (KOH) was purchased from Fisher Scientific, Waltham, MA, USA. AZ 5214 photoresist was purchased from MicroChemicals, Ulm, Germany. Trichlorosilane, NaClOThe scanning electron microscope (SEM) images of the hybrid surfaces were taken using an SEM . The CA of water droplets (7 μL) on spiky CuO and rough PDMS surfaces were measured using a goniometer . The sequential images of fog harvesting surfaces were taken by a digital camera  at the sample tilting angle of 90°.A commercially available ultrasonic humidifier  was used to examine the WCRs of those samples. The temperature and relative humidity of the surrounding were set to be 20 °C and 58%, respectively. The humidifier generating a fog stream with a 20° departure angle was placed 10 cm away from the sample stage and the collected water droplets were gathered in a bucket placed under the sample stage a. The saIn the FEA model, the fog stream was assumed to be in parallel to the 3D pattern sample surface d. The di3N4) etch mask. Then, the Si3N4 layer is removed and a silicon oxide (SiO2) layer is grown  layer , the 3D Cu pattern is achieved on a PDMS surface layer b. Additiubstrate c. Over tubstrate d. It is S surfacee. The PDS surfacec,d as shS surfacef. The faTo quantitatively characterize the fog harvesting abilities of the hybrid surface samples as a function of the superhydrophillic CuO pattern shapes, the water collection rate (WCR) is defined as the mass of collected water per hour divided by the sample surface area. Not only three samples with 2D square, 5-point star, and 7-point star CuO patterns, but also two control samples, including CuO and rough PDMS surfaces, are fabricated. DF2 associated with the 5-pointed and 7-pointed star pattern samples since a sharper corner results in smaller difference between innerθ and outerθ assuming that the droplet size is comparable to the pattern corner size. There is a secondary driving force to move surrounding water droplets into the inner area of the CuO region, which is caused by the shape gradient when the CuO pattern shape has sharp corners. A droplet on the sharp corner of the CuO region experiences asymmetric wetting because the contact area between a water droplet and the hybrid surface includes a larger rough PDMS portion when a water droplet covers the outer area. Thus, the CA of a water droplet on the inner area of the CuO region is lower than the CA on the outer area, which causes the asymmetric wetting. The secondary driving force by this shape gradient arising from the sharp corner is also estimated as below :(2)FD2≈γIn comparison to the 2D samples, which represent conventional fog harvesting surfaces with planar patterns, the hybrid surface with a 3D pattern is fabricated using the process depicted in The effect of 3D pattern becomes more obvious when the WCR of each sample is measured with lower sample tilting angles as exhibited in the relative WCR plot in The other unique feature of the hybrid surface in this work is its mechanical flexibility, such that the hybrid surface can be wrapped over a curved object as shown in In summary, Namib desert beetle-inspired hybrid surfaces composed of superhydrophilic CuO 2D or 3D patterns on flexible hydrophobic rough PDMS background surfaces were designed, fabricated, and characterized. The systematic experiments validate that 3D pattern hybrid surfaces exhibit superior water collection performance to 2D pattern hybrid surfaces. In addition, the flexible nature of the hybrid surface enables the study of the contribution of the curved surface shape to the WCR in unfavorable fog stream directions. The fabrication processes in this work involved no use of toxic chemicals, such as silane or fluoropolymers, coated on the water collecting surfaces for practical safer fog harvesting. This work provides an improved understanding of the role of bumpy structures and the rounded shape of the Namib desert beetle’s dorsal surface for fog harvesting, and thus, offers a guide to design 3D flexible fog harvesting surfaces.


In field conditions that favour high yields, final grain weight of wheat is not limited by sugar availability or activities of key enzymes of starch synthesis during grain filling. Since starch is by far the major component of the mature wheat grain, it has been assumed that variation in the capacity for starch synthesis during grain filling can influence final grain weight. We investigated this assumption by studying a total of 54 wheat genotypes including elite varieties and landraces that were grown in two successive years in fields in the east of England. The weight, water content, sugars, starch, and maximum catalytic activities of two enzymes of starch biosynthesis, ADP-glucose pyrophosphorylase and soluble starch synthase, were measured during grain filling. The relationships between these variables and the weights and starch contents of mature grains were analysed. Final grain weight showed few or no significant correlations with enzyme activities, sugar levels, or starch content during grain filling, or with starch content at maturity. We conclude that neither sugar availability nor enzymatic capacity for starch synthesis during grain filling significantly influenced final grain weight in our field conditions. We suggest that final grain weight may be largely determined by developmental processes prior to grain filling. Starch accumulation then fills the grain to a physical limit set by developmental processes. This conclusion is in accord with those from previous studies in which source or sink strength has been artificially manipulated. Triticum aestivum). Wheat yield per unit land area is a function of the weight of individual grains and the numbers of grains per unit land area (influenced by numbers of ears per plant and number of filled grains per ear). Numbers of grains per unit land area is a complex, weakly inherited trait, but individual grain weight is more stably inherited  in field-grown wheat , the first committed enzyme of starch synthesis, is reported to increase final grain weight under some conditions. Specifically, transgenic wheat plants with elevated AGPase activity during grain filling have been reported to have both more grains and higher yields , may be important for grain filling. First, SS activities are barely sufficient to support the rate of starch synthesis throughout wheat grain filling, and the rate of starch synthesis and SS activity decline in parallel as grains mature  of AGPase and SS, and the levels of carbohydrate substrates for starch synthesis during grain filling in a large panel of diverse genotypes of hexaploid wheat, and examined the relationship between these values, the accumulation of starch, and the weight of individual grains at maturity. To provide insights relevant to the wheat crop, samples were taken from field-grown plants, in two successive years. Plants were grown in Norfolk, UK, an area of high wheat yields with a moderate climate.Triticum aestivum) were from the Earliness & Resilience for Yield in a Changed Climate (ERYCC) panel generated with funding from the Department for Environment, Food and Rural Affairs (UK Government) and the UK Home-Grown Cereals Authority (Project LK0992: Elite cultivars of wheat (Winter wheat was grown in plots (one per genotype) of 1.5 × 1.5 m at Church Farm, Bawburgh, Norfolk, UK, and Morley Farm, Wymondham, Norfolk, UK (locations 10 km apart). In 2013, elite cultivars were grown at Morley and landraces at Bawburgh. In 2014, a subset of 21 elite cultivars and landraces was grown on 1.5 × 1.5 m plots at Bawburgh.Individual primary ears from plants inside the plot were tagged on the day on which they underwent anthesis. At time-points during development, ears were harvested from six plants per genotype, sealed in plastic bags and frozen in liquid nitrogen, then stored on dry ice for transport to a –80 °C freezer. Samples of grains for enzyme and metabolite assays were dissected into pre-weighed tubes, on dry ice. Each sample contained 10 grains from the outer  florets (positions F1 and F2) of spikelets in the central region (from 25–75% of the base-to-tip distance) of a single ear. Following weighing, sample tubes were stored at –80 °C.Grain was harvested after whole-plant senescence . This occurred after physiological grain maturity for all samples in both years. Mature grain samples were harvested and conditioned over at least 4 weeks in a common environment to ensure a common moisture content and comparability regardless of the exact date at which physiological maturity was reached. In 2013, mature grain was bulked for each genotype, then three subsamples were taken for weight and starch assays. In 2014, mature grain was harvested from the centre of a single primary ear each of six randomly selected plants per genotype.www.buxton-weather.co.uk/; accessed 05/09/18).From anthesis to harvest in both 2013 and 2014 the average maximum daily temperature was 22 °C. The temperature was over 25 °C on six days in 2013 and on three days in 2014. The highest temperatures were 29 °C in 2013 and 27 °C in 2014 .For developing grains, fresh weight was measured and then samples were dried at 60 °C for 72 h and reweighed to obtain dry weight. The two values were used to calculate water content. Final grain weight was measured using a Marvin seed analyser  in a tube containing a 7-mm steel ball and ~0.5 ml of 100 mM MOPS/NaOH, pH 7.2, 5 mM MgCl2, 5% v/v glycerol, 5 mM DTT, 10 mg ml–1 bovine serum albumin, 1% (w/v) polyvinylpolypyrrolidone, to give an extract concentration of 350 mg tissue ml–1. Insoluble material was pelleted by centrifugation and the supernatant used for assays.Samples of ~175 mg frozen grain were homogenized at 28 vibrations sAssay components were optimised to give maximum activities on extracts of developing grains. We checked that reaction rates were constant over time, and dependent on appropriate substrates and on extract concentration.2, 2 mM ADP-glucose, 1 unit glucose 6-phosphate dehydrogenase , 1 unit phosphoglucomutase , 1.5 mM Na pyrophosphate (used to initiate the assay), and 40 µl extract.AGPase was assayed at 25 °C and 340 nm in a microtitre plate reader. Assays were conducted in duplicate and contained, in 200 µl, 100 mM MOPS/NaOH, 2.5 mM MgCl14C glucose method of Starch synthase was assayed at 25 °C by the ADPSamples of ~350 mg frozen grain were homogenized on a Retsch ball mill  in 0.5 ml of 0.77 M perchloric acid. After 45 min at 0 °C, debris was pelleted by centrifugation and the supernatant removed. The pellet was washed by resuspension in water and centrifugation, and the washings added to the supernatant. The combined supernatant was neutralized with 2 M KOH, 0.4 M MES, 0.4 M KCl and used for sugar assays following removal of the precipitate by centrifugation. The pellet was washed three times by resuspension in 80% v/v ethanol, and used for starch assays.Sugars and starch were assayed enzymatically according to JXB online. Pearson’s correlation coefficient, r, was calculated between pairs of variables of interest using the means of data per cultivar to ensure independent observations given that the field trials used single plots. Correlations were tested for statistical significance using the F-test and a significance level of P<0.05 was used. The multivariate techniques Principal Coordinates (PCo) analysis and cluster analysis were used to assess the similarity between cultivars. Euclidean distance measures of similarity were used to reduce the 21 (genotypes) × 68 responses  statistical package was used for all analyses.To assess the extent of variation for maximum catalytic activities of AGPase and SS and sugar and starch contents between genotypes, and to look for correlations between these variables and final grain weight, we harvested field-grown developing and mature grain from 18 UK elite cultivars and 36 landraces in 2013, and from a subset of these lines  in 2014. Detailed comparisons between landraces and the elite cultivars in 2013, and between 2013 and 2014 datasets, are precluded by differences between these three trials in field locations and in mature grain sampling strategies (see Methods). The three datasets (in full in P=0.202 for grains at 20 d after anthesis (DAA), P=0.312 for 30 DAA]. Maximum catalytic activities of AGPase and sucrose content on a fresh-weight basis in developing grains also showed little correlation between 2013 and 2014  correlations between final grain weight and SS activity on a FW basis in both years: for activity at 10 DAA and 30 DAA in landraces in 2013, and for activity at 30 DAA and 44 DAA in 2014  correlations of final grain weight with sucrose content at 30 DAA on a FW basis , the year of their release, and the number of years in which they were on the UK Recommended List for winter wheats. None of these measures provided an explanation for the clusters derived from the similarity matrix . Our conclusions are subject to the limitations of our experimental set-up. The technical demands of assaying enzymes and metabolites on numerous samples meant that it was not possible to replicate the genotypes in independent plots, as is usual practice for field experimentation. Instead, we repeated a large part of the experiment in two growing seasons. Our conclusions are based on consideration of these two, independent sets of analyses.In general, our results indicated that final grain weight was not strongly influenced by the availability of sugars or the activities of AGPase or starch synthase in the genotypes and under the environmental conditions we studied. We first discuss the roles of sugar availability, starch synthetic capacity, and starch accumulation in determining final grain weight, and compare our data in these individual respects with the outcomes of previous studies. We then evaluate the idea that developmental processes rather than grain filling determine final grain weight, and discuss whether this is likely to be the case for genotypes and environments different from those in our experiments.Our data revealed no consistent, positive correlations between final grain weight and levels of sucrose, glucose, and fructose in the grain on a weight basis from 10 DAA onwards . It is thus unlikely that grain filling and hence final grain weight were limited by AGPase activity during the main part of grain filling.We found no significant correlations between final grain weight and AGPase activity on a weight basis from 10 DAA onwards , was investigated by over-expression of ndosperm .Our conclusion that that final grain weight was not limited by the maximum catalytic activities of AGPase or starch synthase or by substrate availability during grain filling is seemingly at odds with the demonstration that application of T6P analogues during grain filling increases starch content and final grain size  and rice  or starch-synthesising enzymes during grain filling. We emphasise that these conclusions apply to the main period of grain filling. As discussed above, the capacity for starch synthesis and the supply of substrates from photosynthesis may be crucial determinants of various yield parameters in the period around anthesis and early in grain development see also . The avoAlthough our results were obtained with winter wheat grown under near-optimal conditions for high yield, our conclusions may be more widely applicable. Many experiments in which assimilate supply to the developing grain has been manipulated—through reduction of photosynthesis (defoliation or shading) or through removal of grains—have led to the conclusion that the supply of assimilates from photosynthesis does not limit the growth of grains. The failure of grain weight to respond to either a limitation or an increase in assimilate supply has been observed in both near-optimal and stress conditions  classification, and Recommended List data for elite cultivars.Text S1. Evaluation of two-dimensional plots of Principal Coordinates.Supplementary Table S1Click here for additional data file.Supplementary Table S2Click here for additional data file.Supplementary Tables and FiguresClick here for additional data file.


Mesocestoides vogae larvae represent a suitable model for evaluating the larvicidal potential of various compounds. In this study we investigated the in vitro effects of three natural flavonolignans—silybin (SB), 2,3-dehydrosilybin (DHSB) and silychristin (SCH)—on M. vogae larvae at concentrations of 5 and 50 μM under aerobic and hypoxic conditions for 72 h. With both kinds of treatment, the viability and motility of larvae remained unchanged, metabolic activity, neutral red uptake and concentrations of neutral lipids were reduced, in contrast with a significantly elevated glucose content. Incubation conditions modified the effects of individual FLs depending on their concentration. Under both sets of conditions, SB and SCH suppressed metabolic activity, the concentration of glucose, lipids and partially motility more at 50 μM, but neutral red uptake was elevated. DHSB exerted larvicidal activity and affected motility and neutral lipid concentrations differently depending on the cultivation conditions, whereas it decreased glucose concentration. DHSB at the 50 μM concentration caused irreversible morphological alterations along with damage to the microvillus surface of larvae, which was accompanied by unregulated neutral red uptake. In conclusion, SB and SCH suppressed mitochondrial functions and energy stores, inducing a physiological misbalance, whereas DHSB exhibited a direct larvicidal effect due to damage to the tegument and complete disruption of larval physiology and metabolism. Raillietina echinobothrida in vitro by causing flaccid paralysis accompanied by alterations in tegumental architecture [Echinococcus multilocullaris and Echinococcus granulosus [Schistosoma mansoni worms in vitro at concentrations of 50 and 100 μM. All pairs of coupled adult worms were separated by the action of curcumin at doses of 20–100 μM, and it also reduced egg production by 50% [Natural products are an important source of structural motifs in drug discovery. About half of the small molecules approved as new drugs by FDA each year come directly, or are semi-synthesized, from natural products . Secondaitecture ,7. Metacanulosus . The poln by 50% .Silybum marianum L. (Asteraceae) and the major components (about or more than 10% relative abundance in HPLC) are the flavonolignans silybin A, silybin B, silychristin A, silydianin and isosilybin A  and the major biological activities of silymarin and silybin include antioxidant/prooxidant, hepatoprotective and chemoprotective . It is considered to be a suitable laboratory model for studying cestode biology, host-parasite interactions and the antiparasitic potential of various compounds. In our previous studies on mice with M. vogae infection, silymarin in combination with praziquantel, an increased efficacy of the drug praziquantel compared to monotherapy was found, along with a decrease in liver fibrosis and liver damage due to oxidative stress [M. corti can generate energy under both aerobic and anaerobic conditions [Silymarin is a mixture of phenolic molecules isolated from the fruits of ilybin A  Silybum iew, see . Among oiew, see ,13. Howeiew, see ,15,16. Sfibrosis . Fibrosie stress ,19. In tnditions , which snditions . Whereasnditions , values nditions . TherefoWe recorded the motility of larvae in all the tested groups and the data expressed as motility scores are summarized in The MTT test is widely used to study the cytotoxic effect of compounds on cells, and is often used to study the effects of helminth parasites on viability/respiration. Here we tested whether the mitochondrial respiration of larvae cultivated under aerobic and hypoxic conditions can influence the effects of individual FLs on metabolic activity. We evaluated the time-dependent effect of SB, SCH and DHSB at 5 and 50 µM concentrations under aerobic A–C and hFifty µM DHSB exhibited the opposite effect on metabolic activity to SB and SCH at each incubation interval. The increased metabolic activity in larvae cultured under hypoxia with 50 µM DHSB was in contrast with the effects of DHSB during aerobic cultivation. This might be a result of the overstimulation of enzymatic systems of complex II in mitochondria, leading to their collapse and resulting in an uncontrolled reduction of MTT in dead larvae. A high accumulation of formazan in dead larvae was observed under the microscope (not shown).It was shown that viable eukaryotic cells will uptake neutral red dye by active transport, and incorporate it into lysosomes . In thisp < 0.01), but had no significant effect at 50 µM. SCH did not modulate the transport of dye significantly. After treatment with 50 µM DHSB, the concentration of neutral red in larvae was significantly elevated (139.58 ± 12.29%), but to a lower extent than was found in larvae after aerobic cultivation (p < 0.05). Representative images of larvae from hypoxic cultivations exposed to FLs and the distribution of neutral red are shown in panel C of After the cultivation of larvae under hypoxia, SB at a low concentration increased the uptake of dye  under aerobic conditions (16.59 ± 4.19), whereas it elevated its concentration after hypoxic cultivation (49.57 ± 10.21), correlated with a markedly reduced larval motility.Neutral lipids, comprised mainly of triacylglycerol, cholesterol and free fatty acids, can also serve as energy stores for helminths . Here weThe localization of lipid droplets and nuclei on whole-mount preparations of larvae was examined by confocal scanning laser microscopy after histochemical staining. Very similar staining patterns were observed in control larvae and larvae treated with SB and SCH (50 μM) under aerobic and hypoxic conditions, which is shown in the selected images (upper panel). Representative images (lower panel) of larvae after DHSB treatment (50 μM) exhibited different staining patterns for individual cultivations. In contrast to glycogen, lipid droplets were localized mainly in the central parenchymal part of larval bodies and below the suckers in larvae from the control cultivation. Changes in the concentrations of lipids after exposure to FLs correlated with the distribution and fluorescence intensity of lipid droplets, being the most affected in the neck regions.The shape of the body and effect on the tegument of larvae after three days of incubation with 50 µM of FLs were analyzed by scanning electron microscopy, and representative images are shown in Mesocestoides vogae are characterized by asexual growth in rodents and lizards [Mesocestoides spp. were found to be responsible for serious infections in dogs and cats with poor prognosis [Larvae of  lizards , and prorognosis ,29,30. Trognosis . From thM. vogae larvae are metabolically active in both an aerobic and anaerobic/hypoxic microenvironment. This and our findings indicate that they can utilize the NADH-fumarate reductase system and mitochondrial respiration involving NADH-oxidase localized in complex I, an enzyme involved in aerobic respiration described in mammals. Therefore, in this in vitro study we evaluated the activities of SB, SCH and DHSB under aerobic cultivation conditions and in a cultivation system with a reduced external supply of oxygen/CO2, partially resembling hypoxic environments in hosts. Firstly, we found that larvae transferred from mice to in vitro cultivation systems adapted to the new microenvironments by gradually decreasing metabolic activity, neutral red uptake and concentrations of neutral lipids in contrast with significantly elevated glucose content, without visibly affecting their viability and motility. These changes in larval physiology due to in vitro cultivation conditions, which differ from those in their natural habitats in the hosts, have to be considered in the evaluation of the effects of the compounds.Helminths have exploited a variety of energy transducing systems in their adaptations to the specific habitats in their hosts, and the most important factors are the nutrient and oxygen supply . SeveralM. vogae larvae is a process coordinated by multiple neurotransmitters and neuropeptides and other molecules  and moderately reduced the frequency of body contractions at high concentration (50 μM) . The effa et al. ), which a et al. . They sha et al. ,37. We sM. corti cultured under anaerobic conditions produce more succinate, suggesting the presence and high activity of an NADH-fumarate reductase system. In this study, with aerobic cultivation, the metabolic activity of larvae was suppressed by the low concentration of each of the FLs, already within 4 h of exposure, but the most after 72 h of cultivation with the 50 μM concentration. In accordance with the previous results, the effects of DHSB under hypoxic conditions differed from that of SB and SCH, and a high amount of formazan was extracted from larvae after exposure to the 50 μM concentration. Using the enriched mitochondrial fraction, Matsumoto et al. [E. multilocullaris, indicating that parasite mitochondria are highly adapted to anaerobic environments. So far, no such study on the enzyme activities that form mitochondrial respiratory systems in M. vogae larvae has been conducted. The decreased metabolic activity caused by SB and SCH, regardless of the cultivation conditions, could imply that these FLs interfere with the functions of the enzymes, probably dehydrogenases, involved in both aerobic and anaerobic respiration. The significant increase in reduced formazan after DHSB treatment over the control might be due to the overstimulation of dehydrogenases or uncontrolled release of reducing substrates in damaged/dying larvae. The different effects of individual FLs on larval motility, physiology and respiration could be linked to their chemical properties, which determine the interactions with proteins, nucleic acids and lipids. As in other polyphenols, all three FLs are lipophilic in the order DHSB > SB> SCH [We further tested the activities of FLs on these processes by means of the MTT test, in which the reduction of MTT to formazan during mitochondrial respiration is catalyzed by succinate dehydrogenase using succinate as an electron donor or by the activity of NADH dehydrogenase ,37. Kӧhlo et al.  demonstr SB> SCH , which e SB> SCH . Using m SB> SCH  identifi SB> SCH .Raillientina echinobothrida [Echinocossus spp. [The internalization of neutral red dye in eukaryotic cells is an active process mediated via pinocytosis  and/or pbothrida  and Echisus spp. . By usinsus spp. .Glycogen is a carbohydrate complex formed predominantly of glucose units, and serves as the most important energy reserve in both the larval and adult stages of cestodes . After PTaenia taeniaeformis, the isolated lipid droplets mainly consisted of neutral lipids with triglycerides, sterol esters, sterols and free fatty acids being the major components [Schistosoma mansoni, which was confirmed by Huang et al. [Results from various experiments suggest that helminths are unable to synthesize sterols (cholesterol) and fatty acids de novo  and depemponents . In thismponents . Informamponents . We founmponents , and enzg et al. , though Silymarin was purchased from Liaoning Senrong Pharmaceutical Co. . Silybin  and silychristin were isolated from silymarin as described previously . Brieflyd-glucose, Nile Red powder, glyceryl trioleate (Triolein), DABCO octane, amphotericin B, paraformaldehyde (PFA). RPMI-1640 with stable glutamine and HEPES, PenStrep (penicillin/streptomycin), bovine foetal serum, Hoechst 33433, anthrone, glutaraldehyde (10% solution) and DMSO were purchased from Merck .The producers of chemicals and kits are indicated in the text, except for those purchased from Sigma Aldrich, Darmstadt, Germany): MTT -2,5-diphenyltetrazolium bromide), Neutral Red powder, M. vogae tetrathyridia was maintained in an outbred ICR strain of mice by the intraperitoneal passage of larvae isolated from chronically infected mice (3–5 months of age). Maintaining the infection in ICR mice was approved by the ethics committee of the State Veterinary Administration of the Slovak Republic with reference number Ro-3871/15-221c. For each in vitro experiment, larvae were aseptically isolated from the peritoneal cavity of an infected mouse, washed several times with sterile PBS and larval inoculation samples were prepared in a volume of 0.4 mL for each cultivation, instead of counting the larvae. This volume of settled larvae contained approximately 3.0–3.5 × 103 larvae. The larvae were then transferred into 50 mL cell culture flasks  with a plug seal cap for hypoxic conditions or with a ventilated cap for the cultivations in aerobic conditions (5% CO2 and access to atmospheric oxygen) at 37 °C. Individual experimental flasks were then filled with 10 mL of RPMI 1640 medium supplemented with 5% bovine foetal serum, 1% PenStrep antibiotic solution, 0.1% gentamycin  and 0.1% amphotericin B. Stock solutions of silybin (SB), silychristin (SCH) and dehydrosilybin (DHSB) were prepared in 100% DMSO. At the concentration of 50 μM, the culture medium contained 0.1% DMSO, which had no effect on larval viability and motility. One control flask (medium with corresponding DMSO concentration) and cultures of larvae treated with SB, SCH and DHSB, respectively, were set in each in vitro experiment. Medium in flasks was replaced with fresh medium containing the corresponding concentration of individual FLs once after the first 36 h.Infection with A simple procedure for the quantitative assessment of the effects on a given amount of larvae in all assays was established. As larvae are of various sizes and multiplication stages, throughout this study wet weight of larval mass was determined instead of counting them, and was done in pre-weighed 1.5 mL test tubes. Briefly, at the selected time of incubation, larvae were aseptically transferred from the culture flask to pre-weighed tubes in a volume of approximately 50 μL and left to settle. After removing the liquid, tubes were weighed and the wet weight of larval sample was calculated, being in the range of 20 to 35 mg. For each assay, individual parameters were first calculated for the precise weight of larvae in each sample tube and then re-calculated for the selected amount of wet larval tissue (50 mg or 1 g). In each biochemical assay and under each experimental set of conditions, triplicate larval samples were used for each treatment.Metabolic activity of larvae was evaluated by the MTT test, which we initially used to assess the time-dependent effects of SB, SCH and DHSB at concentrations of 5 μM and 50 μM after 4 h, 24 h and 72 h of cultivation. Larvae exposed to FLs were transferred to pre-weighed tubes for hypoxic conditions and to 24-well plates for aerobic cultivation in 1 mL of RPMI medium. 4 h before the selected period of exposure, 40 μL of 0.5 % MTT was added to the tubes/plates. The supernatant was then removed, larvae were washed with PBS and homogenized with a pestle and mortar in 200 μL of DMSO. After centrifugation at 3000 rpm for 10 min, DMSO containing formazan was transferred to a 96-well plate and absorbance was measured at 550 nm with the reference filter at 630 nm using a Multiscan FC Plate Reader . The absorbance obtained for the real weight of larval tissue in tubes was re-calculated for 50 mg of tissue/sample, and these values were used to express changes in metabolic activity relative to untreated control samples (100%) for each time point and concentration.Neutral red uptake is a common quantitative assay to determine cell viability and toxic effects of the compounds in vitro. This method was used to examine larval viability in freshly isolated larvae and in larvae after 72 h of incubation with 5 μM and 50 μM of SB, SCH, DHSB as well as in untreated controls under both types of conditions. In the assay we used the In vitro Toxicology Assay Kit Neutral Red Based  with some modifications. Briefly, larvae were transferred from flasks into 1.5 mL tubes (in triplicates/group) and filled with 1.0 mL of fresh RPMI medium containing 20 μg/mL of neutral red. Incubation involved mild shaking at 37 °C and was terminated after 60 min. After washing the larvae, they were homogenized in 1.5 mL of solubilization solution. The supernatant containing neutral red was collected into another tube after centrifugation. The optical density (OD) of the samples was measured at 540 nm in a Specord S600 spectrophotometer . Concentrations of neutral red expressed as OD for the actual amount of larval tissue were re-calculated for 50 mg of tissue in each sample, and these values were used to calculate the viability of larval cells (in %) relative to untreated control samples (100%) at each time point.Mesocestoides spp. (tetrathyridium) is an organism with a size between 0.1 to 2 mm, which is morphologically formed of unsegmented parenchymal tissue, scolex and well developed muscle and nervous system [The larval stage of s system ,35. Unde2SO4. The concentration of glucose was measured in larvae isolated from mice  and in larval samples exposed to FLs (5 μM and 50 μM of each FLs) for 72 h. Larval samples, with pre-determined weight as indicated above, were transferred into glass tubes and hydrolyzed in 4 mL of 10% KOH in a boiling water bath for 15–20 min. 0.5 mL of the hydrolysate was slowly mixed with 2 mL of anthrone reagent in an ice bath. The reaction mixture was boiled for exactly 10 min, cooled rapidly and used for the spectrophotometric determination of glucose at 630 nm. Simultaneously, a standard curve using d-glucose as standard in 10% KOH was prepared over the range of 0.2 to 1.0 mg/mL. Glucose in experimental samples was then calculated for 1 g of larval tissue/sample.The concentration of free glucose and glycogen-derived glucose in the larval tissue was quantified by the anthrone method with some modifications. Anthrone reagent was prepared freshly each time by dissolving 20 mg of anthrone in 10 mL of 96% HTM (Thermo Scientific) fluid for frozen tissue and 12 μm-thick cryosections were cut in a Cryocut 1800 (Leica). Sections were then mounted on slides coated with 2% gelatin solution, fixed with 4% PFA for 10 min, washed in PBS, stained with periodic acid-Schiff (PAS) staining system  and observed under a light microscope .The localization of glycogen and molecules containing saccharides, for example glycoproteins, was examined in cryostat sections of larvae after treatment with 50 μM of FLs. Larvae were immediately embedded into NEG 50Nile red is a fluorescent stain for the detection of cytoplasmic lipid droplets in eukaryotic cells of animal as well as plant origin . We deveThe affinity of nile red to neutral lipids was employed to study the localization of lipid droplets in larvae after 72 h of cultivation with 50 µM FLs. Specimens were fixed in 4% PFA in PBS (pH 7.2) for 4 h and processed for whole-mount histochemistry. After washing in PBS, larvae were immersed in the staining mixture containing 10 µg/mL nile red in PBS (stock solution was 1 mg/mL DMSO) and also the fluorescent dye Hoechst 33433 at a concentration of 5 µg/mL of PBS, which allowed the visualization of nuclei in blue. Larvae were stained for 5 h at 8 °C in the dark and after washing, they were mounted on slides in glycerol solution containing 2.5% DABCO. The distribution of neutral lipid droplets was visualized using a LSCM Olympus Fv1000 confocal scanning laser microscope  and analyzed with a FV10-ASW 2.0 Viewer .2 in a K 850 unit . The dried samples were mounted onto Die-Cut Carbon Conductive Double-Sided Adhesive Discs  and sputter-coated with 20 nm of gold in a Polaron Sputter-Coater (E5100) (Quorum Technologies Ltd.). The final samples were examined in a FEI Nova NanoSem 450 scanning electron microscope  at 5 kV using secondary electron detectors (SE or TLD) or the back-scattered electron detector (CBS).Tegumental changes in the tetrathyridia following in vitro incubation with 50 µM were examined by scanning electron microscopy (SEM). Specimens were washed in warm PBS and immediately fixed with 2.5% glutaraldehyde in PBS buffer overnight at 4 °C and stored at the same temperature before further processing. Samples were washed three times in PBS and dehydrated through an alcohol series . The samples were then transferred into porous specimen pots , treated twice in 100% ethanol for 20 min and critical point dried with liquid CO® Software, Inc., San Diego, CA, USA). Results were expressed as mean values ± standard deviation (SD) and were calculated from triplicate samples/group obtained from three independent in vitro experiments. For analysis, one way ANOVA, followed by Tukey’s post-hoc test was used and statistical differences were evaluated between mean values in the control and treated groups and in indicated assays also between basal and control groups. p < 0.05 were considered statistically significant.Statistical analysis of data was performed using GraphPad Prism version 7.00 for Windows (GraphPad PrismIn summary, we demonstrated that SB and SCH significantly suppressed the functions of mitochondrial enzymes, reduced glycogen/glucose and neutral lipid stores, thereby inducing a physiological misbalance, which however did not lead to the death of larvae. In contrast, DHSB exhibited a concentration- and time-dependent larvicidal effect due to marked damage to the tegument and probably protein transporters, as well as the complete disruption of larval respiration, physiology and metabolism.


At 60 days, BCPG showed a higher percentage area of new bone formation (p < 0.05). (4) Conclusion: BCP promoted a new bone formation by osteoconduction and might be considered a valid alternative in bone regeneration procedures.(1) Background: Evaluate the osteoconduction capability of a biphasic calcium phosphate (BCP) ceramic composed of hydroxyapatite and β-tricalcium phosphate 60%/40% in a rat model. (2) Methods: In the calvarial bone of 54 adult male rats, 7-mm diameter critical size defects were performed. The animals were randomly allocated to three experimental groups according to the type of material: blood clot (BCG), blood clot covered with a bovine-derived collagen membrane (MBCG), and BCP ceramic covered with a bovine-derived collagen membrane (BCPG). In each group, 6 animals were euthanatized at post-operative days 7, 30, and 60 for histological and histometric analysis. (3) Results: The qualitative analysis revealed the persistence of the collagen membrane at seven days, with no relevant newly bone formation in all groups. At 30 days, centripetal bone formation was observed residual particles of the biomaterial surrounded by fibroblasts noted in the BCPG. At 60 days, while BCG and MBCG showed a partial maturation with the central part of the defect populated by a fibrous connective tissue, in the BCPG the critical area was entirely occupied by newly formed bone. In the intra groups analysis was noted a significant increase in new bone formation during the experimental period ( Replacement of missing teeth with osseointegrated dental implants is a well-established treatment option associated with high long-term survival rates . The proIn this respect, a lack of bone is mainly due to the physiologic bone remodeling occurring in response to the loss of function as a consequence of tooth extraction. This may be exacerbated by other conditions, including dento-alveolar trauma, traumatic dental extractions, dental congenital absence, tumor resections, chronic/acute infections or the consequence of severe periodontal disease. The final result is a recipient site that is unsuitable to properly receive a dental implant, as an inadequate bone volume is a factor that negatively affects the primary stability and the long-term outcome .The bone tissue has an intrinsic self-repairing potential that involves a tightly regulated sequential process, capable of restoring structure and functions . HoweverDue to their bioactive properties and chemical similarity to the mineral phase of bone, calcium phosphate-based biomaterials (CaP) are widely used for bone regeneration . In partIn view of the aforesaid characteristics, encouraging results emerged when BCPs were used alone ,15 or as®, Latin American Solutions—LAS, Brazil) marketed in the forms of granules, sticks, cylinders, and wedges, has been developed as an alternative to autogenous bone grafts in case of bone regeneration procedures. Graftys BCP® is a microporous, mesoporous, and macroporous two-phase calcium phosphate ceramic made of 60% HA and 40% β-TCP that resorbs and is gradually replaced by newly formed bone during the healing process. These characteristics facilitate long-term volume stability by decelerating the overall resorption capabilities, promoting a more stable and uniform bone growth.A new BCP biomaterial , and was submitted to and approved by the Ethics Committee on Animal Experimentation of the School of Dentistry, Sao Paulo State University, Araçatuba, Brazil, under the Process FOA-01245-2012.®, Genius Biomaterials—Baumer SA, São Paulo, Brazil); in the biphasic calcium phosphate ceramic group (BCPG), the critical size defect was filled with BCP ceramic particles  covered with a bovine-derived cortical membrane.In total, 54 adult male rats , weighing from 450 to 500 g, were used during the experimental procedures. The animals were provided by the animal facility of the same University , where they were kept in individual cages in air-conditioned environment, and were fed with standard solid food and water ad libitum throughout the experiment. The animals were randomly divided into three groups according to the type of material grafted in the critical size defect that was created. In the clot group (BCG), the critical size defect was filled with blood clot; in the membrane clot group (MBCG), the critical size defect was filled with blood clot and covered with a type 1 bovine-derived collagen membrane , at a dose of 10 mg/kg and xylazine hydrochloride 2% , at a dose of 5 mg/kg.Before the surgical procedures, all acclimatized animals were fasted for 12 h. General anesthesia was induced through intramuscular administration of ketamine hydrochloride 1% .Manual shaving was then performed in the frontoparietal region. Subsequently, each animal was placed in a prone position in a sterile field, and a meticulous antisepsis of the surgical area was accomplished with topic polyvinylpyrrolidone solution . The musculocutaneous flap was raised and retracted with surgical retractors to expose the parietal bone on both sides. A 7-mm trephine drill  mounted on a 20:1 reduction contra-angle  connected to a straight surgical handpiece  at a speed of 1500 rpm was used to prepare a bicortical osteotomy in the middle region between the parietal bones. The osteotomized parietal bone was carefully removed to preserve the dura mater [The calvarial surgical access consisted of a median linear incision of 2 cm performed with a scalpel blade number 15C  mounted on a number 3 scalpel handle  simple interrupted stitches.The critical size defect was then grafted according to the group allocation . In grouEuthanasia and specimen retrieval were performed on post-operative days 7, 30, and 60, upon six animals per group for each time period. The animals were sacrificed with an overdose of anesthetic.® Reagents, Dental-Hospital Dynamics Ltd., Catanduva, SP, Brazil) and decalcified in 20% ethylenediaminetetraacetic acid  dissolved in Milli-Q water , changed weekly over a period of six weeks at room temperature. The samples were then dehydrated in an ascending series of ethanol concentrations , with the solution being replaced every 1 h, in an orbital shaker . The samples were successively cleared in xylol, paraffin-embedded and prepared using a precision saw to obtain 6-μm thick sections. The sections were mounted onto slides and stained with hematoxylin eosin . The slides were finally coded to perform a blind histometric analysis.Osteotomies were performed on each calvaria to obtain bone samples with at least 3 cm of margins circumferentially around the grafted area. The pieces were immersion-fixed in 10% neutral buffered formalin , version 3.1, and the area of newly formed bone was expressed in micrometers and converted in percentage as the ratio of newly formed bone area/total area × 100. Five sections from each sample were prepared in a plane parallel to the sagittal suture and through the center of the augmented area and stained with HE. At the surface of the calvaria bone, we determined the newly formed bone inside the calvarial defect without difference between the groups. The slices were scanned using an optical microscope  paired with an image capture camera  and connected to a microcomputer Intel core i5 . The slides were photomicrographs magnified from the originals by 4× and 40×. The scanned images were recorded in JPEG-format files and analyzed using the “hands free” tools in the Image J® , and then the area of newly formed bone was selected inside all area of the calvarial defect, even in the area of connective tissue or biomaterial, and blood clot.A single investigator performed the histological analysis, blinded with respect to the group allocation. A qualitative histological description was performed for all specimens. The histometric evaluation were performed using ImageJp < 0.05). Hence, an ANOVA test and, when necessary, post hoc Tukey’s test was applied to compare the experimental times  and types of graft within groups . A linear regression model was used to assess the time effect (in days) with respect to the daily percentage increase of new bone formation in each group. A significance level of 0.05 was adopted for all tests.The VPR independent statistician performed the statistical analysis. Initially, data were submitted to normality Shapiro-Wilk test, which showed normal distribution . The MBCG have not presented statistically significant differences when compared to the other two groups. At 30 days the area of new bone formation has not showed differences statistically significant between groups (p = 0.065). At 60 days BCPG showed a higher area of new bone formation when compared to MBCG and BCG (p < 0.001). The intra-group analysis showed a statistically significant increase in new bone formation (p < 0.05) at 60 days. The influence of the duration of the experimentation in regards to the bone neoformation rate in each group was reported in 2 = 0.72%; p < 0.001), followed by the MBCG, with a daily rate of 0.24% , and BCG with a daily rate of 0.18% . The highest daily increase of bone formation was described in The comparative analysis of the percentage of newly bone formation was expressed in The present experimental study reports on the successful application of a BCP biomaterial in critical size defects to investigate the osteoconductive potential of HA/β-TCP association. Biphasic HA/β-TCP is a bone substitute produced by a single process to prevent clustering and to establish a homogeneous molecule. Its specific 60%/40% HA/β-TCP ratio gives it two phases of activity. The association of β-TCP/HA provided a good level of osteoconduction favoring and facilitating the bone formation process. In all groups, a trend towards an increasing amount of newly formed bone was observed within the study periods. It is worthy of note that the defects filled with the BCP biomaterial reached the maximum of bone formation, closing the defect created in all animals within 60 days. The quantitative analysis is corroborated by the histological findings, showing a complete maturation of the defect, with the critical area entirely occupied by newly formed bone, in the BCPG group. Although the degree of reabsorption of biomaterial granules was not measured in this study, but it is possible to compare the degree of resorption of granules between the periods  of each group through the histological images.An important aspect is related to the HA/β-TCP ratio, which might influence the resorption activity of osteoclasts by modifying the solubility of ceramic. The resorption and precipitation processes are regarded as major factors implicated in their bioactive properties. Resorption activity was observed on pure β-TCP and BCP with 25% HA and 75% β-TCP. Conversely, osteoclasts did not resorb BCP with a 75%/25% HA/β-TCP ratio or pure HA .Interestingly, scaffolds presenting a HA/β-TCP weight ratio of 60%/40% have shown beneficial effects for cells growth, as a confirmation of their biocompatibility, and thus might be considered as good candidates for bone tissue engineering applications . SurprisWhen transferred to the maxillary sinus compartment, BCP in the form of 60%/40% HA/β-TCP used as a bone substitute, exhibited biocompatibility and osteoconductive properties, promoting new bone formation after sinus elevation surgeries. It is noteworthy that the amount of new bone formation was significantly higher in the autogenous group compared to the BCP group . Our resMicroscopic structure of HA/β-TCP offers granules with a porosity of 90%, and interconnected pores ranging from 100 to 500 μm in diameter to support cellular penetration. Actually, the graft material used in the present study presented 67% porosity, composed of 84% microporosity (<10 μm), 8% mesoporosity (10–100 μm), and 8% macroporosity (>100–150 μm). Interestingly, in evaluating the impact of total porosity of BCP on cell microenvironment, a similar porosity of 65% displayed a great odontoblastic differentiation strongly suggesting it can support human dental pulp cells differentiation for dentin tissue regeneration . A part A particulated graft serves as a scaffold that promotes a tridimensional matrix, which will stabilize and maintain the organization of the blood clot. It also allows and supports cell migration and angiogenesis, resulting in new bone formation during repair. On the other hand, compressive forces by the surrounding soft tissues might dislodge the biomaterial from the defect, hampering the healing process. For this reason, the placement of an occlusive membrane has been introduced to prevent connective tissue cells from colonizing the defect and at the same time provide enough space to allow for bone regeneration of the entire defect volume preventing micromovements and compression . The preIn conclusion, the results suggested that BCP composed of 60%/40% HA/β-TCP promoted a new bone formation by osteoconduction and might be considered a valid alternative in bone regeneration procedures. Further in vivo studies are needed to evaluate the optimal HA/β-TCP ratio and the ideal porosity of BCP-based bone substitutes.


Despite their importance, there is less understanding of how microbial community assemblages will respond to environmental changes. In this study, a manipulative mesocosm experiment was employed, using next-generation sequencing to assess changes in microbial communities under future environmental change scenarios. Illumina sequencing generated over 11 million 16S rRNA gene sequences (using a primer set biased toward bacteria) and revealed Bacteroidetes and Proteobacteria dominated the total bacterial community of sediment samples. In this study, the sequencing coverage and depth revealed clear changes in species abundance within some phyla. Bacterial community composition was correlated with simulated environmental conditions, and species level community composition was significantly influenced by the mean temperature of the environmental regime (p = 0.002), but not by variation in CO2 or diurnal temperature variation. Species level changes with increasing mean temperature corresponded with changes in NH4 concentration, suggesting there is no functional redundancy in microbial communities for nitrogen cycling. Marine coastal biogeochemical cycling under future environmental conditions is likely to be driven by changes in nutrient availability as a direct result of microbial activity.Marine sediments are important sites for global biogeochemical cycling, mediated by macrofauna and microalgae. However, it is the microorganisms that drive these key processes. There is strong evidence that coastal benthic habitats will be affected by changing environmental variables (rising temperature, elevated CO Predictions of future temperature elevation are often referred to as a mean global rise, and the diurnal variability of temperature in this experimental design represents the change in both mean temperature, but also the extremes experienced particularly in coastal and tidal ecosystems. The 16S rRNA gene was sequenced from environmental DNA extracted from the incubated intertidal cohesive sediment samples at the end of the experiment. This provides insight into microbial responses toward environmental change, and we discuss the implications on marine biogeochemical cycling. This study harnesses advanced sequencing technology to provide essential understanding of the global consequences of climate change on microbial community composition. We hypothesize that shifts in microbial community assemblages will be a response to changing environmental conditions, and this may be synergistic or additive.To-date, few studies have examined the effects of combined environmental stressors on microbial benthic communities , as previously described  on the surface of sediment in each mesocosm, and topped up with seawater to an overlying depth of 20 cm. This water was replaced after 24 h to reduce any biogeochemical fluxes associated with sediment homogeneity and mesocosm assembly  was collected from the Ythan Estuary tidal mud flats in, Aberdeenshire, Scotland, UK  in spring/early summer 2008 and sieved (500 μm) in a seawater bath  to remove macrofauna. The sediment was left to settle for 48 h before the supernatant was removed. Additional microphytobenthos (MPB)-rich sediment was collected at the same time, and placed under constant light in a shallow tray for 48 h. The sediment was then homogenized and placed in perspex mesocosms to a depth of 10 cm  and 1,000 ppmv ]. The experiments were run on a 12 h light-12 h dark (L/D) cycle using high intensity discharge sodium lamps  to allow MPB photosynthesis. Eighteen unique environmental regimes were used, consisting of three mean temperatures , two atmospheric CO2 concentrations, and three fluctuating temperature regimes  around the mean (one complete fluctuation cycle every 24 h). Experimental design included three replicates (n = 3) per environmental regime , with each chamber running at one of two COme Table .2 concentrations were maintained as previously described . Sediment samples were individually wrapped in foil and immediately stored in a −80°C freezer until DNA extraction.MPB biomass was measured in each mesocosm prior to sediment and water sampling, using non-invasive Pulse Amplitude Modulated (PAM) fluorometry to estimate chlorophyll content, following the methodology described in Consalvey et al.  and Hick4, NOx, (nitrate and nitrite) and PO4 concentration were measured using a flow through injector analyser (FIA Star 5010 series) with an artificial seawater carrier solution  were taken from the overlying water in each mesocosm at the end of the experiment. NHn = 3) were named according to their environmental treatment e.g., H6-1 represents High CO2; 6°C mean temperature; and 1°C temperature fluctuation  at 99% sequence identity. A minimum cluster size was set to remove clusters containing fewer than four sequences. OTU-picking was done using QIIME to cluster sequences, remove chimeras and define OTU abundance. This final dataset was then clustered at 97% sequence similarity to identify taxonomy from the Greengenes database, version 12.8  with a small number of archaeal taxa  and no eukaryotic taxa due to the bias of the primers used to target the V4 region of the 16S rRNA gene  passed the quality control filters and all 52 samples were pooled into a single metadata file, which was processed for metagenomic analysis using QIIME , with the nutrient concentrations  as additional explanatory variables. A separate CCA was carried out with the same explanatory variables on larger groupings of OTUs (to either Class or Phylum) to compare trends at lower species resolution.Canonical Correspondence Analysis was carried out in R  using QIIME and three measures of diversity were estimated: Chao1; the observed number of species; and the phylogenetic diversity (PD). These estimates were plotted as rarefaction curves using QIIME scripts Figure . The rarTo compare the similarity of samples based on the bacterial sequences, the distance between each sample was calculated using a UniFrac metric. The distance was defined as the sum of the unshared branch lengths between two samples divided by the total branch length (shared and unshared) of two samples .OTU clustering was visualized using an Unweighted Pair Group Method with Arithmetic Mean (UPGMA) tree Figure  with Jacn Figure . From bon Figure . The nutF = 18.7059, p = 0.005), MPB community  and PO4 concentration  was found to significantly influence the species level bacterial community composition  and nitrate-nitrite (NOx) concentrations were also insignificant  in the CCA. The raw data for each nutrient concentration is presented in boxplots according to treatment, and did not change with mean temperature, CO2 or fluctuating regime , followed by PO4 concentration  and MPB biomass . CO2, fluctuating regime and NH4 and NOx concentration were again found to have no significant effect  examined the effect of environmental variables on the bacterial community composition. Using the species (OTU) level bacterial composition , mean temperature (4), the species level community composition under each of the three experimental temperatures were compared (Table Flavobacteriae), with over 50% of total relative abundance attributed to this phylum across all mean temperature treatments, peaking at 59% at 12°C  were both absent at 18°C mean temperature, and Lutibacter spp. were only found under the 6°C mean temperature treatments . In contrast, Alphaproteobacteria remained relatively constant (~6%) across all treatments, and Deltaproteobacteria increased in abundance with increasing mean temperature, so although the overall phylum abundance decreased, this observation masked changes in the lower taxonomic levels.Proteobacteria was the second most abundant phylum after Bacteroidetes, making up 30% of relative abundance at 6°C, but dropping to 25% at 12°C and 23% at 18°C. The s was present in all samples, with an average constant abundance of ~5% which increased slightly as mean temperature increased  and Phycisphaerales (~1%) dominating the lower temperature regimes within this phylum. However, as mean temperature increases, the relative abundance of Pirellulaceae increases, rising from 0.2% at 6°C to 2% at 18°C. In general, Cyanobacteria abundance was low in all samples; with Cyanobacteria sp. found at all mean temperatures. As observed for other phyla, this general trend was underpinned by specific species dynamics. At a mean temperature of 12°C . This was also reflected where samples with Planktothrix at the mean temperature of 12°C were clustered together  may be altered with environmental change, particularly with warming temperature , supporting previous work , and together with Plactomycetes, they play a vital role in benthic carbon cycling as the initial organic matter degraders , and a corresponding decrease in Eudoraea adriatica and Lutibacter species . Species within the Robiginitalea genus are known to have a thermal preference above 10–15°C  and (increasing) phosphate (PO4) with increasing mean temperature support this concept. Here we demonstrate an increase in sulfate reducing bacteria (Deltaproteobacteria) as mean temperature increases, and the presence of thermophilic bacteria (Deinococcus-Thermus) at the highest mean temperature treatment (18°C). Sulfate reducing bacteria (SRB) are often found in cohesive sediments  within the Betaproteobacteria, their abundance was less than 1% across all treatments. The corresponding decrease in NH4 concentration in the overlying water suggests there may be changes in the nitrogen cycling, possibly influenced by the absence of Betaproteobacteria, and NOx levels remain low across all treatments  and corresponding decrease of Desulfuromonadaceae with increasing mean temperature, suggest that changes to the sulfur cycle may not be noticed at the ecosystem service level, despite a change in species assemblage. Targeted future work should address how changes in some species  may be reflected in a broad range of biogeochemical processes, such as integrating measurements relating to sulfur, nitrogen and carbon cycles. Sediment profiles of oxygen and H2S would provide insight into potential shifting from oxic to anoxic (sulfate reduction) conditions, and this linked to corresponding microbial communities would provide direct biogeochemical information on coastal sediment dynamics. This study has focused on intertidal cohesive sediments, but the microbial response will vary with sediment type, driven by changes in oxygen penetration depth  appears to drive community assemblage change. Whilst there are limitations in realistically interpreting results from artificial mesocosm systems, and caution must be taken in interpreting responses, these experiments are valuable in providing insights on how complex ecosystems may respond to warming or elevated CO2 (Benton et al., In conclusion, changes in microbial assemblage were only found between the mean temperature treatment, and not in response to changes in diurnal temperature variability or elevated CONH and DP conceived and designed the experiments. NH ran the experiments and collected the samples. NH and KD performed DNA extraction using a protocol developed. KD, XL, RG, JK, AL, and LL ran the bioinformatics, including sampling, quality control, data processing and sequence assignment. XL performed additional analysis on genomic results. NH, KD, and DP wrote the manuscript, with input from all co-authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Polylactic acid (PLA) is one of the most produced polymeric materials, due to its exceptional chemical and mechanical properties. Some of them, such as biodegradability and biocompatibility, make them attractive for biomedical applications. Conversely, the major drawback of PLA in the biomedical field is their vulnerability to bacterial contamination. This study focuses on the immobilization of saccharides onto the PLA surface by a multistep approach, with the aim of providing antibacterial features and evaluting the synergistic effect of these saccharides. In this approach, after poly (acrylic acid) (PAA) brushes attached non-covalently to the PLA surface via plasma post-irradiation grafting technique, immobilization of glucosamine (GlcN) and chondroitin sulfate (ChS) to the PAA brushes was carried out. To understand the changes in surface properties, such as chemical composition, surface topography and hydrophilicity, the untreated and treated PLA films were analyzed using various characterization techniques . In vitro cytotoxicity assays were investigated by the methyl tetrazolium test. The antibacterial activity of the PLA samples was tested against Escherichia coli and Staphylococcus aureus bacteria strains. Plasma-treated films immobilized with ChS and GlcN, separately and in combination, demonstrated bactericidal effect against the both bacteria strains and also the results revealed that the combination has no synergistic effect on antibacterial action. Among all sustainable polymers, polylactic acid (PLA) is one of the most promising and most produced bioplastics due to its superior chemical and mechanical properties, such as biodegradability, good biocompatibility, and low immunogenicity ,2,3. TheThe future of PLA utilization in medical applications not only depends on overcoming intrinsic drawbacks, but also on appropriate modification of the surface to achieve new targeted properties or enhance the present features. In this regard, the most important challenges are the activation of the chemically inert PLA surface to reduce hydrophobicity and introduce a new molecules to possess significant physical and mechanical properties, in order to function properly when in contact with blood . TherefoIn the medical field, especially for implants, microbial colonization and biofilm formation are critical clinical problems, which may lead to long-term treatments, replacement of infected implants, amputations, and even patient death ,18,19. IAmongst these antibacterial moieties, natural-origin saccharides, such as chondroitin sulfate (ChS) and glucosamine (GlcN) are attractive options, since the carbohydrate moieties can imprint both hydrophilic and biocompatibility properties to the material surface .ChS is the most abundant mucopolysaccharide in all of the vertebrates . This naGlcN is a naturally-produced amino sugar, which is essential for glycoproteins, glycolipids, and glycosaminoglycans (GAG). This positively charged saccharide has anti-inflammatory, antioxidant, anti-ageing, anti-cancer, and antibacterial activities ,35,36,37GlcN is also used in biomedical applications in combination with ChS to achieve a synergistic effect ,41. For Thus, the present study aims to develop a PLA-based antibacterial platform using two common saccharides, which also have cell proliferation and adhesion features, and also to compare the antibacterial and cytotoxic activity of GlcN and CS, individually and in combination. While clinical trials and most in vitro studies have focused on the synergistic effects of glucosamine and CS in treatment of osteoarthritis, the present study focuses on the prevention of bacterial growth on polymer biomaterials by using saccharides. For this purpose, low-pressure radio frequency plasma (RF) method was utilized to activate the PLA surfaces and the molecules of GlcN and ChS were immobilized on the surface of the PLA films to improve in antibacterial activity. The bacterial adhesion of surface modified PLA films was systematically investigated by antibacterial activity against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus).Poly(lactic acid) (PLA) 4032 D in pellets form was purchased from Nature Works . Acrylic acid (99%), sodium metabisulfite (99%), D-glucosamine hydrochloride (99%), chondroitin sulfate A sodium salt , and sodium hydroxide (98%) were analytical grade reagents and were supplied by Sigma Aldrich .PLA pellets were first dried in a desiccator at 60 °C overnight to eliminate humidity. Then, to obtain PLA sheets, the compression molding technique was used. PLA pellets were hot-pressed at 180 °C for 20min and then the molding plates were placed in another press for cooling. Sheets of PLA about 430 µm thick and 125 × 125 mm square shaped were obtained and then cut into samples of 25 × 25 mm for further surface treatments. The specimens were rinsed with a detergent solution and subsequently dried at room temperature prior to plasma exposure. The PLA films were treated using radio frequency (13.56 MHz), low-pressure plasma equipment . Briefly, the sample was first inserted into an evacuated to 60 Pa vacuum chamber and 20 sccm of air flowed into the chamber. Each side of the sample was treated by air plasma generated at power 50 W for 60 s.Acrylic acid (AAc) solution was prepared by dissolving 10% of AAc in distilled water with sodium metabisulfite. PLA films were immersed immediately into this homopolymer solution after the irradiation step. Following the grafting reaction, the homopolymer was removed, then the samples were placed into the aqueous solution of 1 w% sodium hydroxide for neutralization.For immobilization of antibacterial agents, AAc grafted samples were immersed into either 1 w% GlcN or 1 w% ChS solutions for 24 h. Thereafter, each sample was washed with deionised water. Subsequently, GlcN immobilized samples were lastly placed into 1 w% ChS solution. The films were then removed, washed in distilled water and left for drying overnight for further characterization. All the process is graphically presented in The elemental analysis of untreated and treated PLA films was conducted with X-ray photoelectron spectroscopy (XPS) using the Thermo Scientific K-Alpha XPS system . A monochromic Al Kα X-ray source (1486.6 eV) line was applied as the photoemission excitation with a 400µm spot size at a power of 72 W.The change in hydrophobicity induced by surface modifications was analysed through static contact angle measurements by the Surface Energy Evaluation System . As a testing liquid, deionized water was used and digital images of a 2 μL water droplet on the surface were captured by the charged-coupled device (CCD) camera system. Three different spots on the droplet’s image were determined and the contact angle value was obtained for each reading. Each representative contact angle was calculated by averaging at least 10 separate readings for each sample.The surface morphology of the specimens was observed using a NANOSEM 450  scanning electron microscope at an accelerating voltage of 5.0 kV after sputter coating with gold/palladium.TM; NIH/3T3) with the disinfected (30 min of exposure to a UV-radiation source) PLA samples were compared by MTT assay. The ATCC-formulated Dulbecco’s Modified Eagle’s Medium  containing 10% of calf serum and 100 U mL−1 penicillin/streptomycin  was used as the culture medium. The cells were seeded onto square samples (10 × 10 mm) in concentration 1 × 105 cells/mL in volume 200 µL for 1h incubation. The amount of 800 µL of culture medium was added after 1 h incubation and the cells were incubated at 37 ± 1 °C for 72 h. In this study, the cells cultured on polystyrene were used as a reference. The proliferation of mouse embryonic fibroblast cells (ATCC CRL-1658−1). Following the removal of all the medium, dimethyl sulfoxide  was added for dissolution of the formed formazan crystals on the sample surfaces. The absorbance was measured at a wavelength of 570 nm (test) and 690 nm (reference) by using an Infinite M200 Pro NanoQuant microplate reader . Cell viability was shown as the percentage of viable cells after exposure to the tested solutions relative to the reference (100% viability).Cytotoxicity testing was conducted according to the international standard EN ISO 10993-5 with modification. After 72 h incubation, all culture medium was removed and incubated for 4 h with 900µL fresh medium which included 100 µL tetrazolium dye MTT solution  were placed, in groups of tree, into Petri dishes and 0.4 mL of the test inoculum was pipetted onto each sample, which was then covered with a polypropylene (PP) film (20 × 20 mm). The Petri dishes were incubated for 24 h at 35 °C. After the incubation, both the samples and PP covers were washed with 10 mL of neutralizing broth. The recovered bacterial suspensions were diluted to 10-fold concentrations. Each diluted and undiluted bacterial solution was placed into Petri dishes with 15 mL of count plate agar. The number of bacteria colonies were counted after 24 h of incubation.Briefly, PLA films were sterilized by immersion in 70% ethanol for 24 h. Test bacteria were transferred to nutrient broth (NB) and for using as test inoculations, the number of bacteria was adjusted to certain concentrations, which is between 2.5 × 10Through static contact angle measurements, surface modification, which induced a change in hydrophilicity, was analyzed . As can Typical SEM micrographs of the pristine and modified PLA films are illustrated in For the analysis of surface chemical compositions of the PLA surfaces, XPS measurements were carried out before and after the treatments. The elemental content of carbon, oxygen, nitrogen, and sulfur in each sample is summarized in Cell viability was quantified by an MTT assay as an indicator of mitochondrial succinate dehydrogenase enzyme activity. The viability of the reference corresponds to 100% survival of cells in the absence of the tested substances in the cultivation medium. According to the EN ISO 10993-5, values above 80% compared to reference are assigned to ‘no cytotoxicity’, values from 60% to 80% ‘mild cytotoxicity’, and values below 40% ‘severe cytotoxicity’ .p < 0.05. It can be seen that all of the relative cytotoxicity values (%)-except for untreated PLA-are higher than 80%, independent of polymer modification. One possible reason for this phenomenon is the increased hydrophilicity of untreated PLA with various modifications . The GlcN grafted samples display 85% cell viability, while the ChS immobilized samples have more than 120% viability of the cells. For the combination of these two saccharides, the cell viability increases to 148%, which shows better biocompatibility than either saccharide alone.Cytotoxicity data are presented in Other studies reported the evaluation of toxicity of chondroitin sulfate and glucosamine. Most of the studies claim little or no cytotoxicity of either GlcN or ChS for concentrations below 5.0 mg/mL ,48. One Staphylococcus aureus and Escherichia coli strains, and analyzed by comparing the number of viable cells in the agar plates after 24h of incubation time. As shown in 2. The difference in counts between GlcN combined with ChS immobilized samples and separately attached saccharides are small and this combination results in a destruction of more than 99.99% of the inoculation, which generally is accepted as the definition of bactericidal agents [The antibacterial activity of the PLA films was evaluated against l agents ,52. The antibacterial surface modification of PLA films was achieved through the immobilization of GlcN and ChS on film surfaces via plasma treatment technique, followed by AAc grafting. The contact angle and XPS results verify successful immobilization of the saccharides. In the survey scan XPS spectra increases in characteristic elements (N and S) of ChS and GlcN were observed. SEM images showed that the saccharide aggregates partially covered the PLA film surfaces. The antibacterial testing results demonstrated that PLA films coated with ChS exhibited the highest antimicrobial activity against E. coli. Besides, only-GlcN immobilized PLA films showed the best bactericidal effect against S.aureus. When combined with ChS the degree of both bacteria growth inhibition was still up to 99.99%.This study definitely proved that the developed GlcN/ChS coated PLA films are excellent bactericide agents against representative gram-positive and gram-negative bacteria. Furthermore, the combination of these two saccharides should be highlighted in the current study, due to increased cell viability, which could make it easier to bring the developed medical devices to the market.