Strategies of leaf water uptake based on anatomical traits.

The ability of leaves to absorb fog water can positively contribute to the water and carbon balance of plants in montane ecosystems, especially in periods of soil water deficit. However, the ecophysiological traits and mechanisms responsible for variations in the speed and total water absorption capacity of leaves are still poorly known. This study investigated leaf anatomical attributes of seven species occurring in seasonal tropical high-altitude ecosystems (rocky outcrop and forest), which could explain differences in leaf water uptake (LWU) capacities. We tested the hypothesis that different sets of anatomical leaf attributes will be more marked in plant individuals living under these contrasting environmental conditions. Anatomical variations will affect the initial rate of water absorption and the total storage capacity, resulting in different strategies for using the water supplied by fog events. Water absorption by leaves was inferred indirectly, based on leaf anatomical structure and visual observation of the main access routes (using an apoplastic marker), the diffusion of water through the cuticle, and non-glandular or glandular trichomes in all species. The results suggest that three LWU strategies coexist in the species studied. The different anatomical patterns influenced the speed and maximum LWU capacity. The three LWU strategies can provide different adaptive advantages to adjust to temporal and spatial variations of water availability in these tropical high-altitude environments.

Toxicity of the bionematicide 1,4-naphthoquinone on non-target soil organisms.

The main goal of the present study was to evaluate the ecotoxicological effects of 1,4-naphthoquinone (1,4-NTQ), a natural-origin compound presenting nematicidal activity, that can be obtained from walnut husk, in plants and soil invertebrates, including non-target soil nematode communities. This research was part of an ongoing project that aims to develop environmentally-friendly nematicides obtained from agricultural residues. The battery of ISO tests included emergence and growth of corn (Zea mays) and rape (Brassica napus); avoidance with the earthworm Eisenia andrei and the collembolan Folsomia candida; and reproduction with the previous species plus the enchytraeid Enchytraeus crypticus. A novel soil nematode community assay was also performed. ISO tests and nematode assays were conducted using a natural uncontaminated soil that was spiked with a range of 1,4-NTQ concentrations. Toxicity of 1,4-NTQ was found for all test-species and the most sensitive were F. candida and E. andrei. After 7 days of exposure to 1,4-NTQ, nematode abundance decreased along the concentration gradient, and a partial recovery was observed after 14 days (1,4-NTQ <48 mg kg-1 soil). The number of nematode families consistently decreased in both periods. Overall, results indicate that a 1,4-NTQ concentration of <20 mg kg-1 could be environmentally safe but preliminary data suggest that it might be ineffective for the target-nematodes, root-knot nematodes, Meloidogyne spp., and root-lesion nematodes, Pratylenchus spp. In addition, if higher dosages of 1,4-NTQ bionematicide are necessary, the potential recovery of non-target organisms under real field scenarios also needs to be assessed.

Multifactor analysis on the effect of collagen concentration, cross-linking and fiber/pore orientation on chemical, microstructural, mechanical and biological properties of collagen type I scaffolds.

This work evaluates the effect of processing variables on some physicochemical and mechanical properties of multi- and unidirectional laminar collagen type I scaffolds. The processing variables considered in this study included microstructure orientation (uni- and multidirectional fiber/pore controlled by freeze-drying methodology), cross-linking (chemical - using genipin and glutaraldehyde, and physical - using a dehydrothermal method), and collagen concentration (2, 5 and 8mg/ml). The biocompatibility of the scaffolds obtained in each of the evaluated manufacturing processes was also assessed. Despite previous research on collagen-based platforms, the effects that these processing variables have on the properties of collagen scaffolds are still not completely understood. Unidirectional scaffolds presented higher resistance to failure under stress than multidirectional ones. The cross-linking degree was found to decrease when the concentration of collagen increased whilst using chemical cross-linkers, and to increase with the concentration of collagen for the dehydrothermal cross-linked scaffolds. Pore orientation indexes of both unidirectional and multidirectional scaffolds were not influenced by collagen concentration. Cross-linked scaffolds were more hydrophobic than non-cross-linked ones, and presented water vapor permeability adequate for use in low-to-moderate exuding wounds. Pore size ranges were compatible with cell in-growth, independently of the employed cross-linking and freezing methodologies. Moreover, scaffolds cross-linked with glutaraldehyde presented higher in-growth of primary oral mucosa fibroblasts than those cross-linked with genipin or with the dehydrothermal treatment. This multi-factor analysis is expected to contribute to the design of collagen type I platforms, which are usable on several potential soft tissue-engineering applications.

Dexamethasone-loaded poly(ε-caprolactone)/silica nanoparticles composites prepared by supercritical CO2 foaming/mixing and deposition.

A supercritical carbon dioxide (scCO2)-assisted foaming/mixing method (SFM) was implemented for preparing dexamethasone (DXMT)-loaded poly(ε-caprolactone)/silica nanoparticles (PCL/SNPs) composite materials suitable for bone regeneration. The composites were prepared from PCL and mesoporous SNPs (MCM-41/SBA-15) by means of scCO2-assisted SFM at several operational pressures, processing times and depressurization conditions. DXMT was loaded into SNPs (applying a scCO2 solvent impregnation/deposition method - SSID) and into PCL/SNPs composites (using the SFM method). The effects of the employed operational and compositional variables on the physicochemical and morphological features as well as in the in vitro release profiles of DXMT were analyzed in detail. This work demonstrates that the above-referred scCO2-based methods can be very useful for the preparation of DXMT-loaded PCL/SNPs composites with tunable physicochemical, thermomechanical, morphological and drug release properties and suitable for hard-tissue regeneration applications.

Development of natural-based wound dressings impregnated with bioactive compounds and using supercritical carbon dioxide.

Film- and foam-like structures of N-carboxybutylchitosan (CBC) and of agarose (AGA) were prepared and characterized in order to evaluate their potential application as topical membrane-type wound dressing materials, mostly regarding their sustained release capacities and fluid handling properties. Polymeric biomaterials were loaded with two natural-origin bioactive compounds (quercetin and thymol, which present anti-inflammatory and anaesthetic properties, respectively), separately or as a mixture of these two substances, and using a supercritical solvent impregnation (SSI) method. Impregnation experiments were carried out with supercritical carbon dioxide (scCO2) at 10 and 20 MPa, and at 303 and 323 K. Ethanol (10%, v/v) was employed as a co-solvent whenever quercetin was used. Release kinetic studies were performed for all prepared systems and the obtained results showed that higher amounts of quercetin and/or thymol were loaded when higher pressures and temperatures were employed. Results showed that the separated and the simultaneous SSI loading of these two bioactive substances into CBC and AGA is a feasible and advantageous process and that the relative loaded amounts of these substances can be "tuned" simply by changing the operational pressure-temperature conditions. Quercetin presented more sustained release profiles which can be justified by its higher molecular volume and by its lower water solubility as well as by the specific favourable interactions that can be established between quercetin and CBC. Obtained results showed that the employed SSI process also promoted the size reduction of loaded quercetin particles which can significantly improve the solubility of this compound in aqueous solutions. In addition, prepared systems presented adequate water sorption and water vapor sorption capacities as well as water vapor transmission rates that were in the typical and desired ranges for commercial wound dressings.

Preparation and chemical and biological characterization of a pectin/chitosan polyelectrolyte complex scaffold for possible bone tissue engineering applications.

In this work, porous scaffolds obtained from the freeze-drying of pectin/chitosan polyelectrolyte complexes were prepared and characterized by FTIR, SEM and weight loss studies. Additionally, the cytotoxicity of the prepared scaffolds was evaluated in vitro, using human osteoblast cells. The results obtained showed that cells adhered to scaffolds and proliferated. The study also confirmed that the degradation by-products of pectin/chitosan scaffold are noncytotoxic.

Astra blue and basic fuchsin double staining of plant materials.

Methods for double staining plant materials using astra blue and basic fuchsin are described here. These methods can be applied to free hand and microtome sections embedded in paraffin, paraplast or historesin. Also, they can be used to study isolated epidermal peels and pollen preparations. Temporary, semipermanent and permanent preparations were studied. Astra blue stained polysaccharides of the cell wall such as cellulose and pectins. Basic fuchsin showed an affinity for lignified, suberized or cutinized walls. The easy preparation of the reagents, excellent color contrast of the histological preparations, and brief staining times of some methods makes them useful for both routine research and didactic purposes. Also, excellent color or black and white photomicrography can be obtained after the double staining described here.