Superhydrophobic platforms for the combinatorial analysis of biomaterials-cells interactions using arrays of 3D scaffolds with distinct mechanical and morphological properties

High-throughput studies of cells mechanotransduction are usually performed using 2D biomaterials. However, cells-extracellular matrix interactions in the body occur in 3D environment. By using cells entrapped in hydrogels, it is not easy to isolate the mechanical effect from the chemical cues of biomaterials. We used a cytocompatible and non-expensive platform based in the patterning of wettable spots in superhydrophobic surfaces to deposit porous biomaterials in these regions. Freeze-dried alginate/chitosan scaffolds were used to create an array of mechanical properties and porosities. Adaptation of dynamic mechanic analysis equipment allowed performing on-chip single scaffold analysis. Micro-computed tomography allowed acquir- ing data for whole chips simultaneously. Results were validated using individual scaffolds and single-formulation chips. A sub-array with combined modulus/porosity properties was selected. Fibronectin (Fn) in different concentration was adsorbed in the scaffolds, and fibroblasts and osteoblast-like cells were seeded. The independent study of variables influence in cell response was performed by image-based methods. In the absence of Fn fibroblasts did not respond to mechani- cal properties in the chip range. Osteoblast-like cells showed higher cell adhesion in stiffer substrates. The adsorption of Fn was studied qualita- tively by image methods. Results related with Fn amount and scaffolds’ mechanical properties were analyzed for each cell type.</p

Novel strategies for preventing dysbiosis in the oral cavity

Oral diseases affect over three billion people worldwide, making it one of the most common infections. Recent studies show that one approach to reducing the risk of chronic infections, such as caries, gingivitis, periodontitis, and halitosis, is to control the ecology of the oral microbiome instead of completely removing both the harmful and beneficial microorganisms. This is based on the knowledge that oral diseases are not caused by a single pathogen but rather by a shift in the homeostasis of the entire microbiota, a process known as dysbiosis. Consequently, it is of the utmost importance to implement strategies that are able to prevent and control oral dysbiosis to avoid serious complications, including heart, lung, and other systemic diseases. Conventional treatments include the use of antibiotics, which further disrupt the equilibrium in the oral microbiota, together with the mechanical removal of the decayed cavity area following its formation. Therefore, it is imperative to implement alternative strategies with the potential to overcome the disadvantages of the current therapy, namely, the use of broad-spectrum antibiotics. In this sense, probiotics and postbiotics have received particular attention since they can modulate the oral microbiota and decrease the dysbiosis rate in the oral cavity. However, their mechanisms of action need to be addressed to clarify and drive their possible applications as preventive strategies. In this sense, this review provides an overview of the potential of probiotics and postbiotics, focusing on their antimicrobial and antibiofilm activities as well as their ability to modulate the inflammatory response. Finally, it also showcases the main advantages and disadvantages of orodispersible films—a promising delivery mechanism for both probiotics and postbiotics to target oral dysbiosis.info:eu-repo/semantics/publishedVersio

Phase equilibrium data and modeling of ethylic biodiesel, with application to a non-edible vegetable oil

Contributing to extending the knowledge for the design and operation of biodiesel production processes, isobaric PTxy vapor-liquid equilibria data of ethanol + ethyl hexanoate, 1-pentanol + ethyl hexanoate and 1-pentanol + ethyl octanoate at two different pressures are reported for the first time. Consistency tests were applied to attest the quality of the collected data, for these especially complex measurements. Besides that, vapor pressures of the pure ethyl esters have also been measured. For modeling purposes, the Lyngby and Dortmund UNIFAC variants were used to predict the VLE phase diagrams. Generally, the predictions are of very good quality, being the UNIFAC-Do (Dortmund) better, as the deviations in temperature and vapor compositions are always lower to 1.0 K and 0.020, respectively. Checking for the viability for extrapolations in pressure, CPA EoS was also applied to the modeling of the experimental data with very good results. Finally, aiming at examining the model capabilities to describe multicomponent systems, VLE measurements involving two alcohols and the fatty acid ethyl ester mixture obtained from non-edible vegetable oil have been carried out showing the good performance of the predictive models.This work was developed in the scope of the Projects POCI-01- 0145-FEDER-006984 – Associate Laboratory LSRE-LCM and POCI- 01-0145-FEDER-007679 – CICECO-Aveiro Institute of Materials (FCT Ref. UID/CTM /50011/2013), funded by FEDER (European Union) through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) – and by national funds through FCT (Portugal) – Fundação para a Ciência e a Tecnologia. Also, the authors express their acknowledgment to Frédéric Roze from Université de Lorraine – ENSIC (France) for his technical support during the work.info:eu-repo/semantics/publishedVersio

Rheological and mechanical properties of acellular and cellladen methacrylated gellan gum hydrogels

Tissue engineered hydrogels hold great potential as nucleus pulposus substitutes (NP), as they promote intervertebral disc (IVD) regeneration and re-establish its original function. But, the key to their success in future clinical applications greatly depends on its ability to replicate the native 3D micro-environment and circumvent their limitation in terms of mechanical performance. In the present study, we investigated the rheological/mechanical properties of both ionic- (iGG-MA) and photo-crosslinked methacrylated gellan gum (phGG-MA) hydrogels. Steady shear analysis, injectability and confined compression stress-relaxation tests were carried out. The injectability of the reactive solutions employed for the preparation of iGG-MA and phGG-MA hydrogels was firstly studied, then the zero-strain compressive modulus and permeability of the acellular hydrogels were evaluated.In addition, human intervertebral disc (hIVD) cells encapsulated in both iGG-MA and phGG-MA hydrogels were cultured in vitro, and its mechanical properties also investigated under dynamic mechanical analysis at 37ºC and pH 7.4. After 21 d of culturing, hIVD cells were alive (Calcein AM) and the E’ of ionic-crosslinked hydrogels and photo-crosslinked was higher than that observed for acellular hydrogels. Our study suggests that methacrylated gellan gum hydrogels present promising mechanical and biological performance as hIVD cells were producing extracellular matrix

In Vivo High-Content Evaluation of Three-Dimensional Scaffolds Biocompatibility

While developing tissue engineering strategies, inflammatory response caused by biomaterials is an unavoidable aspect to be taken into consideration, as it may be an early limiting step of tissue regeneration approaches. We demonstrate the application of flat and flexible films exhibiting patterned high-contrast wettability regions as implantable platforms for the high-content in vivo study of inflammatory response caused by biomaterials. Screening biomaterials by using high-throughput platforms is a powerful method to detect hit spots with promising properties and to exclude uninteresting conditions for targeted applications. High-content analysis of biomaterials has been mostly restricted to in vitro tests where crucial information is lost, as in vivo environment is highly complex. Conventional biomaterials implantation requires the use of high numbers of animals, leading to ethical questions and costly experimentation. Inflammatory response of biomaterials has also been highly neglected in high-throughput studies. We designed an array of 36 combinations of biomaterials based on an initial library of four polysaccharides. Biomaterials were dispensed onto biomimetic superhydrophobic platforms with wettable regions and processed as freeze-dried three-dimensional scaffolds with a high control of the array configuration. These chips were afterward implanted subcutaneously in Wistar rats. Lymphocyte recruitment and activated macrophages were studied on-chip, by performing immunocytochemistry in the miniaturized biomaterials after 24 h and 7 days of implantation. Histological cuts of the surrounding tissue of the implants were also analyzed. Localized and independent inflammatory responses were detected. The integration of these data with control data proved that these chips are robust platforms for the rapid screening of early-stage in vivo biomaterials’ response

In vitro and in vivo biological performance of modified gellan gum-based hydrogels for nucleus pulposus tissue engineering

Ionic- (iGG-MA) and photo-crosslinked (phGG-MA) methacrylated gellan gum hydrogels have been proposed as biomaterials for supporting nucleus pulposus (NP) regeneration and/or repair. In this study, the mechanical stability and biocompatibility of these hydrogels have been evaluated in vitro. Human intervertebral disc cells obtained from herniated patients were cultured within both hydrogels, for 1–21 days. Dynamic mechanical analysis and biological characterization (Live/ Dead assay, ATP and DNA quantification, PCR and immunocytochemistry) were performed after specific times of culturing. The in vitro study showed that both cell loading and culturing time do not affect the mechanical properties of hydrogels. In addition, the iGG-MA and phGG-MA hydrogels showed to be effective on supporting cells encapsulation and viability up to 21 days of culturing. In vivo biocompatibility screening was also performed, by subcutaneous implantation of both hydrogels in Lewis rats for the period of 10 and 18 days. Haematoxylin & eosin staining revealed that the hydrogels do not elicit necrosis, calcification or acute inflammatory reaction. The present study demonstrates that the iGG-MA and phGG-MA hydrogels support cells encapsulation and viability, and are well-tolerated, stable and non-cytotoxic in vitro and in vivo, thus possessing promising features for finding application as viable NP substitutes

Development of an injectable system based on elastin-like recombinamer particles for tissue engineering applications

An elastin-like recombinamer (ELR) containing the RGD cell adhesion domain was used to fabricate microparticles by an innovative and affordable process based on the use of superhydrophobic surfaces. Two microparticles types with different crosslinking extents were prepared. The biological response was tested using an osteoblast-like cell line (SaOs-2) performing proliferation and alkaline phosphatase (ALP) quantification tests, as well as assessing cytotoxicity, morphology and cell distribution on the particles. The main goal of the work was the assessment of the in vitro formation of cell-induced microparticle aggregates that could provide indications for the possible formation of an in situ-forming scaffold upon implantation. ELR microparticles have been successfully obtained by deposition of a polymeric solution on bioinspired polystyrene superhydrophobic surfaces and two different crosslinking extents were achieved by controlling the time of exposure to the crosslinker. The crosslinking extent affected swelling behavior and the dynamic mechanical properties of the particles. SaOs-2 morphology, ALP expression, spatial distribution and ability to bind the microparticles together were dependent on the physicochemical properties of the microparticles: the more crosslinked condition was the most favorable for cell proliferation and to form a cell-induced aggregation scaffold, making these particles suitable to be applied in bone tissue engineering

Mechanical performance and biocompatibility study of methacrylated Gellan gum hydrogels with potential for nucleus pulposus regeneration

Methacrylated gellan gum hydrogels, obtained either by ionic- (iGGMA) and photo-crosslinking (phGG-MA), have been investigated as potential biomaterials for supporting nucleus pulposus (NP) regeneration and/or repair [1,2]. In previous work, some advantages were attributed to GG-MA hydrogels, such as: (i) the possibility to control endothelial cells infiltration and blood vessel ingrowth’s, (ii) tunable and improved mechanical properties, and (iii) in situ gelation, within seconds to few minutes. In this study, the mechanical and biological performance of these hydrogels was firstly evaluated in vitro. Human intervertebral disc (hIVD) cells obtained from herniated patients were cultured within both hydrogels, for 1 up to 21 days. Dynamic mechanical analysis and biological characterization (calcein-AM staining, ATP and DNA quantification and PCR) were performed after specific times of culturing. A biocompatibility study was also performed in vivo, by subcutaneous implantation of acellular iGG-MA and phGG-MA hydrogels in Lewis rats for the period of 10 and 18 days. Tissue response to the hydrogels implantation was determined by histological analysis (haematoxylin-eosin staining). The in vitro study showed that both cell loading and culturing time do not have an effect on the mechanical properties of the hydrogels. Regarding their biological performance, the iGG-MA and phGG-MA hydrogels showed to be effective on supporting hIVD cells encapsulation and viability up to 21 days of culturing. Human IVD cells were homogeneously distributed within the hydrogels and maintained its round-shape morphology during culturing time. The in vivo biocompatibility study showed that iGG-MA and phGG-MA hydrogels do not elicit any deleterious effect, as denoted by the absence of necrosis and calcification, or acute inflammatory reaction. A thin fibrous capsule was observed around the implanted hydrogels. The results presented in this study indicate that the iGG-MA and phGG-MA hydrogels are stable in vitro and in vivo, support hIVD cells encapsulation and viability, and were found to be well-tolerated and non-cytotoxic in vivo, thus being potential candidates for NP regeneration

XRD and FTIR analysis of Ti–Si–C–ON coatings for biomedical applications

Ti–Si–C–ON films were deposited by DC reactive magnetron sputtering using different partial pressure ratio of oxygen (pO2) and nitrogen (pN2). Compositional analysis revealed the existence of three different growth zones for the films; (I) N/Ti = 2.1 (high atomic ratio) and low oxygen content; (II) 0.76 < N/Ti < 2.1 (intermediate atomic ratio) and (III) N/Ti ≤ 0.12 (low ratio) and high oxygen content. For high N/Ti atomic ratio (N/Ti = 2.1) the XRD pattern exhibits reflections that correspond to a mixture of two different phases: a metallic-like Ti and a fcc NaCl type structure. Its electrical resistivity presents a metallic character and, consequently, has high infrared reflectivity. For the intermediate N/Ti ratio (0.76 < N/Ti < 2.1), the films crystallize in a B1-NaCl crystal structure typical for TiC0.2N0.8. Their FTIR spectra present C–N modes, besides the TiN ones, that indicate a progressive substitution of nitrogen by carbon atoms with increasing oxygen content (and lowering N/Ti ratio). For the highest oxygen content (and lower N/Ti ratio) the presence of the Ti–O–Ti stretching mode shows the formation of highly resistive Ti–O compounds consistent with the semiconductor character of this film. Biofilm formation as well as material cytotoxicity seemed to be related with the presence of the Ti

Development of biofunctional textiles by the application of resveratrol to cotton, bamboo and silk

The goal of this work was to create a new generation of greener fabrics made of natural materials. For that, resveratrol (Res), obtained from Polygonum cuspidatum extract and known to have antibacterial, antifungal, and anti-inflammatory activity, was applied by an exhaustion method to cotton, bamboo, and silk knit fabrics. The fabrics adsorption behavior was tested and the amount of Res adsorbed was determined by its decrease on the immersion solutions with time and measured by spectrophotometry at 350 nm. The maximum adsorption capacity was observed for silk and it was independent of pH conditions used (50.5 % at pH=7 and 58.3 % at pH=5 of the initial Res concentration). At acidic pH conditions, cotton adsorbed 51.2 % of Res and Bamboo adsorbed only 28.1 % in 15 min. However, neither cotton nor bamboo adsorbed Res at pH=7. The release behavior was also analyzed and the highest Res release was observed for cotton in alkaline sweat and urine mimic solutions. The lowest release was achieved by cotton in water (1.0 ng/ml). Moreover, no relation was found between the amounts of Res adsorbed or released and cell viability. In conclusion, this work shows that it is possible to obtain cotton, bamboo, and silk functionalized with resveratrol. The incorporating process here described is simple and silk-Res can be presented as a good combination