Microfluidically Aligned Collagen to Maintain the Phenotype of Tenocytes In Vitro.

Tendon is a highly organized tissue that transmits forces between muscle and bone. The architecture of the extracellular matrix of tendon, predominantly from collagen type I, is important for maintaining tenocyte phenotype and function. Therefore, in repair and regeneration of damaged and diseased tendon tissue, it is crucial to restore the aligned arrangement of the collagen type I fibers of the original matrix. To this end, a novel, user-friendly microfluidic piggyback platform is developed allowing the controlled patterned formation and alignment of collagen fibers simply on the bottom of culture dishes. Rat tenocytes cultured on the micropatterns of aligned fibrous collagen exhibit a more elongated morphology. The cells also show an increased expression of tenogenic markers at the gene and protein level compared to tenocytes cultured on tissue culture plastic or non-fibrillar collagen coatings. Moreover, using imprinted polystyrene replicas of aligned collagen fibers, this work shows that the fibrillar structure of collagen per se affects the tenocyte morphology, whereas the biochemical nature of collagen plays a prominent role in the expression of tenogenic markers. Beyond the controlled provision of aligned collagen, the microfluidic platform can aid in developing more physiologically relevant in vitro models of tendon and its regeneration.

A 96-wells fluidic system for high-throughput screenings under laminar high wall shear stress conditions.

The ability of endothelial cells to respond to blood flow is fundamental for the correct formation and maintenance of a functional and hierarchically organized vascular network. Defective flow responses, in particular related to high flow conditions, have been associated with atherosclerosis, stroke, arteriovenous malformations, and neurodegenerative diseases. Yet, the molecular mechanisms involved in high flow response are still poorly understood. Here, we described the development and validation of a 96-wells fluidic system, with interchangeable cell culture and fluidics, to perform high-throughput screenings under laminar high-flow conditions. We demonstrated that endothelial cells in our newly developed 96-wells fluidic system respond to fluid flow-induced shear stress by aligning along the flow direction and increasing the levels of KLF2 and KLF4. We further demonstrate that our 96-wells fluidic system allows for efficient gene knock-down compatible with automated liquid handling for high-throughput screening platforms. Overall, we propose that this modular 96-well fluidic system is an excellent platform to perform genome-wide and/or drug screenings to identify the molecular mechanisms involved in the responses of endothelial cells to high wall shear stress.

Generating fast-twitch myotubes in vitro with an optogenetic-based, quantitative contractility assay.

The composition of fiber types within skeletal muscle impacts the tissue's physiological characteristics and susceptibility to disease and ageing. In vitro systems should therefore account for fiber-type composition when modelling muscle conditions. To induce fiber specification in vitro, we designed a quantitative contractility assay based on optogenetics and particle image velocimetry. We submitted cultured myotubes to long-term intermittent light-stimulation patterns and characterized their structural and functional adaptations. After several days of in vitro exercise, myotubes contract faster and are more resistant to fatigue. The enhanced contractile functionality was accompanied by advanced maturation such as increased width and up-regulation of neuron receptor genes. We observed an up-regulation in the expression of fast myosin heavy-chain isoforms, which induced a shift towards a fast-twitch phenotype. This long-term in vitro exercise strategy can be used to study fiber specification and refine muscle disease modelling.

Competition for endothelial cell polarity drives vascular morphogenesis in the mouse retina.

Blood-vessel formation generates unique vascular patterns in each individual. The principles governing the apparent stochasticity of this process remain to be elucidated. Using mathematical methods, we find that the transition between two fundamental vascular morphogenetic programs-sprouting angiogenesis and vascular remodeling-is established by a shift of collective front-to-rear polarity of endothelial cells in the mouse retina. We demonstrate that the competition between biochemical (VEGFA) and mechanical (blood-flow-induced shear stress) cues controls this collective polarity shift. Shear stress increases tension at focal adhesions overriding VEGFA-driven collective polarization, which relies on tension at adherens junctions. We propose that vascular morphogenetic cues compete to regulate individual cell polarity and migration through tension shifts that translates into tissue-level emergent behaviors, ultimately leading to uniquely organized vascular patterns.

3D Lung-on-Chip Model Based on Biomimetically Microcurved Culture Membranes.

A comparatively straightforward approach to accomplish more physiological realism in organ-on-a-chip (OoC) models is through substrate geometry. There is increasing evidence that the strongly, microscale curved surfaces that epithelial or endothelial cells experience when lining small body lumens, such as the alveoli or blood vessels, impact their behavior. However, the most commonly used cell culture substrates for modeling of these human tissue barriers in OoCs, ion track-etched porous membranes, provide only flat surfaces. Here, we propose a more realistic culture environment for alveolar cells based on biomimetically microcurved track-etched membranes. They recreate the mainly spherical geometry of the cells' native microenvironment. In this feasibility study, the membranes were given the shape of hexagonally arrayed hemispherical microwells by an innovative combination of three-dimensional (3D) microfilm (thermo)forming and ion track technology. Integrated in microfluidic chips, they separated a top from a bottom cell culture chamber. The microcurved membranes were seeded by infusion with primary human alveolar epithelial cells. Despite the pronounced topology, the cells fully lined the alveoli-like microwell structures on the membranes' top side. The confluent curved epithelial cell monolayers could be cultured successfully at the air-liquid interface for 14 days. Similarly, the top and bottom sides of the microcurved membranes were seeded with cells from the Calu-3 lung epithelial cell line and human lung microvascular endothelial cells, respectively. Thereby, the latter lined the interalveolar septum-like interspace between the microwells in a network-type fashion, as in the natural counterpart. The coculture was maintained for 11 days. The presented 3D lung-on-a-chip model might set the stage for other (micro)anatomically inspired membrane-based OoCs in the future.

Droplet microfluidics as a tool for production of bioactive calcium phosphate microparticles with controllable physicochemical properties.

Affordable and therapeutically effective biomaterials are required for successful treatment of orthopaedic critical-size bone defects. Calcium phosphate (CaP) ceramics are widely used for bone repair and regeneration, however, further optimization of their properties and biological performance is still required. To improve the existing CaP bone graft substitutes, novel synthesis and production approaches are needed that provide a fine control over the chemical and physical properties and versatility in the delivery format. In this study, a microfluidic strategy for production of CaP microparticles with different sizes derived from highly monodisperse droplets is proposed for the controlled synthesis of bioactive CaP ceramics. Microfluidic droplets, that served as microreactors for CaP precipitation, allowed the production of different CaP phases, as well as strontium-substituted CaP. By varying the concentration of the precursor solution, microparticles with different porosity were obtained. The droplet microfluidic system allowed direct visualization and quantification of the reaction kinetics. Upon production and purification of the microparticles, the biocompatibility and bioactivity were tested in vitro using human mesenchymal stromal cells (hMSCs). Cell attachment was analysed by imaging of the cytoskeleton and focal adhesions Moreover, cell proliferation, metabolic activity, alkaline phosphatase activity and mRNA expression of a set of osteogenic markers were quantified. We demonstrated that droplet microfluidics is a functional technique for the synthesis of a range of bioactive CaP-based ceramics with controlled properties. STATEMENT OF SIGNIFICANCE: Calcium phosphate (CaP) ceramics are widely applied synthetic biomaterials for repair and regeneration of damaged bone; yet, CaP bone graft substitutes require further improvement to fully replace natural bone grafts in challenging clinical situations. To this end, novel synthesis and production approaches are needed that provide a fine control over the chemical and physical properties. Here, we developed a microfluidic platform for production of CaP microparticles with different size, composition and porosity, derived from monodisperse droplets. We demonstrated that CaP microparticles produced using this platform supported growth and differentiation of human mesenchymal stromal cells. This platform is a useful tool for developing a variety of CaPs in a controlled manner to study their physicochemical properties in relation to their bioactivity.

Guiding mesenchymal stem cell differentiation using mesoporous silica nanoparticle-based films.

The development of smart interfaces that can guide tissue formation is of great importance in the field of regenerative medicine. Nanoparticles represent an interesting class of materials that can be used to enhance regenerative treatments by enabling close control over surface properties and directing cellular responses. Moreover, nanoparticles can be used to provide temporally controlled delivery of (multiple) biochemical compounds. Here, we exploited the cargo loading and surface functionalization properties of mesoporous silica nanoparticles (MSNs) to design films that can guide human mesenchymal stem cell (hMSC) differentiation towards the osteogenic lineage. We developed biocompatible MSN-based films that support stem cell adhesion and proliferation and demonstrated that these MSN films simultaneously allowed efficient local delivery of biomolecules without effecting film integrity. Films loaded with the osteogenesis-stimulating drug dexamethasone (Dex) were able to induce osteogenic differentiation of hMSCs in vitro. Dex delivery from the films led to increased alkaline phosphatase levels and matrix mineralization compared to directly supplementing Dex to the medium. Furthermore, we demonstrated that Dex release kinetics can be modulated using surface modifications with supported lipid bilayers. Together, these data demonstrate that MSN films represent an interesting approach to create biomaterial interfaces with controllable biomolecule release and surface properties to improve the bioactivity of biomaterials. STATEMENT OF SIGNIFICANCE: Engineering surfaces that can control cell and tissue responses is one of the major challenges in biomaterials-based regenerative therapies. Here, we demonstrate the potential of mesoporous silica nanoparticles (MSNs) as drug-delivering surface coatings. First, we show differentiation of mesenchymal stem cells towards the bone lineage when in contact with MSN films loaded with dexamethasone. Furthermore, we demonstrate that modification of MSNs with supported lipid bilayer allows control over drug release dynamics and cell shape. Given the range of loadable cargos and the tunability of release kinetics, MSN coatings can be used to mimic the sequential appearance of bioactive factors during tissue regeneration, which will ultimately lead to biomaterials with improved bioactivity.

Lipid surface modifications increase mesoporous silica nanoparticle labeling properties in mesenchymal stem cells.

BACKGROUND: Nanoparticles have emerged as promising cell-labeling tools, as they can be precisely tailored in terms of chemical and physical properties. Mesoporous silica nanoparticles (MSNs), in particular, are easily tunable with regard to surface and core chemistry, and are able to confine dyes and drug molecules efficiently. PURPOSE: The aim of this study was to investigate the effect of lipid and polyethylene glycol (PEG) surface modifications on MSN stem-cell-tracking abilities. METHODS: Lipid and PEG surface functionalized MSNs were synthesized and the effect of surface functionalization on cell internalization, proliferation, differentiation and cell proteomics was investigated in patient derived mesenchymal stem cells (MSCs). RESULTS: MSNs and lipid surface-modified MSNs were internalized by >80% of the MSC population, with the exception of nanoparticles modified with short PEG chains (molecular weight 750 [MSN-PEG750]). Lipid-modified MSNs had higher labeling efficiency with maximum uptake after 2 hours of exposure and were in addition internalized 17 times higher compared to unmodified MSNs, without negatively affecting differentiation capacity. Using a mass-spectrometry-based label-free quantitative proteomics approach, we show that MSN labeling leads to the up- and downregulation of proteins that were unique for the different surface-modified MSNs. In addition, functional enrichments were found in human MSCs labeled with MSNs, MSN-PEG750, and lipid-modified MSNs. SUMMARY: Here we show that organic modifications with lipids and PEGylation can be used as a promising strategy to improve MSN labeling capabilities. In particular, we show that lipid modifications can optimize such probes in three distinct ways: significantly improved signal strength, a barrier for sustained release of additional probes, and improved stem-cell-labeling efficiency.

Development of a shear stress-free microfluidic gradient generator capable of quantitatively analyzing single-cell morphology.

Microfluidics, the science of engineering fluid streams at the micrometer scale, offers unique tools for creating and controlling gradients of soluble compounds. Gradient generation can be used to recreate complex physiological microenvironments, but is also useful for screening purposes. For example, in a single experiment, adherent cells can be exposed to a range of concentrations of the compound of interest, enabling high-content analysis of cell behaviour and enhancing throughput. In this study, we present the development of a microfluidic screening platform where, by means of diffusion, gradients of soluble compounds can be generated and sustained. This platform enables the culture of adherent cells under shear stress-free conditions, and their exposure to a soluble compound in a concentration gradient-wise manner. The platform consists of five serial cell culture chambers, all coupled to two lateral fluid supply channels that are used for gradient generation through a source-sink mechanism. Furthermore, an additional inlet and outlet are used for cell seeding inside the chambers. Finite element modeling was used for the optimization of the design of the platform and for validation of the dynamics of gradient generation. Then, as a proof-of-concept, human osteosarcoma MG-63 cells were cultured inside the platform and exposed to a gradient of Cytochalasin D, an actin polymerization inhibitor. This set-up allowed us to analyze cell morphological changes over time, including cell area and eccentricity measurements, as a function of Cytochalasin D concentration by using fluorescence image-based cytometry.

Cell-instructive high-resolution micropatterned polylactic acid surfaces.

Micro and nanoscale topographical structuring of biomaterial surfaces has been a valuable tool for influencing cell behavior, including cell attachment, proliferation and differentiation. However, most fabrication techniques for surface patterning of implantable biomaterials suffer from a limited resolution, not allowing controlled generation of sub-cellular three-dimensional features. Here, a direct laser lithography technique based on two-photon absorption was used to construct several patterns varying in size between 500 nm and 15 µm. Through replication via an intermediate mold, the patterns were transferred into polylactic acid (PLA), a widely used biomedical polymer, while retaining the original geometry. An osteoblast-like cell line, MG-63 was used for characterizing the morphological response to the topographical patterns. The results indicated that semi-continuous (dashed) lines, with a height of 1 µm were able to induce cell elongation in the direction of the lines. However, when dashes with a height of 0.5 µm were combined with perpendicularly crossing continuous lines (rails) with a height of 8 µm, the contact guidance effect of the dashes was lost and elongation of the cells was observed in the direction of the larger features. A second pattern, consisting of different arrays of pillars showed that, depending on the pillar height, the cells were either able to spread over the pattern or were confined between the pattern features. These differences in the ability of cells to spread further resulted in the formation of tension forces through stress fibers and displacement of vimentin. The method for high-resolution micropatterning of PLA as presented here can also be applied to other biomedical polymers, making it useful both for fundamental studies and for designing new biomaterials with improved functionality.

High throughput screening of photocatalytic conversion of pharmaceutical contaminants in water.

The susceptibility for photon-induced degradation of over 800 pharmaceutical compounds present in the LOPAC1280 library, was analyzed by UV/Vis spectroscopy in the absence or presence of TiO2 P25 in water. In general, few compounds were effectively degraded in the absence of the TiO2 photocatalyst (3% of all compounds tested), while in the presence of TiO2, the majority of compounds was converted, often to a large degree. Differences in degree of degradation are evaluated on the basis of molecular weight, as well as the chemical nature of the drug compounds (functional groups and pharmacological classes). In general, if the molecular weight increases, the degradation efficacy decreases. Relatively high degrees of conversion can be achieved for (relatively small) molecules with functional groups such as aldehydes, alcohols, ketones and nitriles. A low degree of conversion was observed for compounds composed of conjugated aromatic systems. Trends in degradation efficacy on the basis of pharmacological class, e.g. comparing hormones and opioids, are not obvious.

Controlling Growth and Osteogenic Differentiation of Osteoblasts on Microgrooved Polystyrene Surfaces.

Surface topography is increasingly being recognized as an important factor to control the response of cells and tissues to biomaterials. In the current study, the aim was to obtain deeper understanding of the effect of microgrooves on shape and orientation of osteoblast-like cells and to relate this effect to their proliferation and osteogenic differentiation. To this end, two microgrooved polystyrene (PS) substrates, differing in the width of the grooves (about 2 µm and 4 µm) and distance between individual grooves (about 6 µm and 11 µm, respectively) were fabricated using a combination of photolithography and hot embossing. MG-63 human osteosarcoma cells were cultured on these microgrooved surfaces, with unpatterned hot-embossed PS substrate as a control. Scanning electron- and fluorescence microscopy analyses showed that on patterned surfaces, the cells aligned along the microgrooves. The cells cultured on 4 µm-grooves / 11 µm-ridges surface showed a more pronounced alignment and a somewhat smaller cell area and cell perimeter as compared to cells cultured on surface with 2 µm-grooves / 6 µm-ridges or unpatterned PS. PrestoBlue analysis and quantification of DNA amounts suggested that microgrooves used in this experiment did not have a strong effect on cell metabolic activity or proliferation. However, cell differentiation towards the osteogenic lineage was significantly enhanced when MG-63 cells were cultured on the 2/6 substrate, as compared to the 4/11 substrate or unpatterned PS. This effect on osteogenic differentiation may be related to differences in cell spreading between the substrates.

Independent effects of the chemical and microstructural surface properties of polymer/ceramic composites on proliferation and osteogenic differentiation of human MSCs.

UNLABELLED: Within the general aim of finding affordable and sustainable regenerative solutions for damaged and diseased tissues and organs, significant efforts have been invested in developing synthetic alternatives to natural bone grafts, such as autografts. Calcium phosphate (CaP) ceramics are among widely used synthetic bone graft substitutes, but their mechanical properties and bone regenerative capacity are still outperformed by their natural counterparts. In order to improve the existing synthetic bone graft substitutes, it is imperative to understand the effects of their individual properties on a biological response, and to find a way to combine the desired properties into new, improved functional biomaterials. To this end, we studied the independent effects of the chemical composition and surface microstructure of a poly(lactic acid)/hydroxyapatite (PLA/HA) composite material on the proliferation and osteogenic differentiation of clinically relevant bone marrow-derived human mesenchymal stromal cells (hMSCs). While the molecular weight of the polymer and presence/absence of the ceramic phase were used as the chemical variables, a soft embossing technique was used to pattern the surfaces of all materials with either pits or pillars with identical microscale dimensions. The results indicated that, while cell morphology was affected by both the presence and availability of HA and by the surface microstructure, the effect of the latter parameter on cell proliferation was negligible. The osteogenic differentiation of hMSCs, and in particular the expression of bone morphogenetic protein 2 (BMP-2) and osteopontin (OP) were significantly enhanced when cells were cultured on the composite based on low-molecular-weight PLA, as compared to the high-molecular-weight PLA-based composite and the two pure polymers. The OP expression on the low-molecular-weight PLA-based composite was further enhanced when the surface was patterned with pits. Taken together, within this experimental set up, the individual effect of the chemistry, and in particular of the presence of CaP, was more pronounced than the individual effect of the surface microstructure, although their combined effects were, in some cases, synergistic. The approach presented here opens new routes to study the interactions of biomaterials with the biological environment in greater depths, which can serve as a starting point for developing biomaterials with improved bioactivity. STATEMENT OF SIGNIFICANCE: The aim of the this study was to obtain insight into independent effects of the chemical composition and surface microstructure of a poly(lactic acid)/hydroxyapatite (PLA/HA) composite material on the morphology, proliferation and osteogenic differentiation of clinically relevant bone marrow-derived human mesenchymal stromal cells (hMSCs). While the need for synthetic alternatives for natural bone in bone regenerative strategies is rapidly increasing, the clinical performance of synthetic biomaterials needs to be further improved. To do this successfully, we believe that a better understanding of the relationship between a property of a material and a biological response is imperative. This study is a step forward in this direction, and we are therefore convinced that it will be of interest to the readers of Acta Biomaterialia.

High-throughput screening approaches and combinatorial development of biomaterials using microfluidics.

From the first microfluidic devices used for analysis of single metabolic by-products to highly complex multicompartmental co-culture organ-on-chip platforms, efforts of many multidisciplinary teams around the world have been invested in overcoming the limitations of conventional research methods in the biomedical field. Close spatial and temporal control over fluids and physical parameters, integration of sensors for direct read-out as well as the possibility to increase throughput of screening through parallelization, multiplexing and automation are some of the advantages of microfluidic over conventional, 2D tissue culture in vitro systems. Moreover, small volumes and relatively small cell numbers used in experimental set-ups involving microfluidics, can potentially decrease research cost. On the other hand, these small volumes and numbers of cells also mean that many of the conventional molecular biology or biochemistry assays cannot be directly applied to experiments that are performed in microfluidic platforms. Development of different types of assays and evidence that such assays are indeed a suitable alternative to conventional ones is a step that needs to be taken in order to have microfluidics-based platforms fully adopted in biomedical research. In this review, rather than providing a comprehensive overview of the literature on microfluidics, we aim to discuss developments in the field of microfluidics that can aid advancement of biomedical research, with emphasis on the field of biomaterials. Three important topics will be discussed, being: screening, in particular high-throughput and combinatorial screening; mimicking of natural microenvironment ranging from 3D hydrogel-based cellular niches to organ-on-chip devices; and production of biomaterials with closely controlled properties. While important technical aspects of various platforms will be discussed, the focus is mainly on their applications, including the state-of-the-art, future perspectives and challenges. STATEMENT OF SIGNIFICANCE: Microfluidics, being a technology characterized by the engineered manipulation of fluids at the submillimeter scale, offers some interesting tools that can advance biomedical research and development. Screening platforms based on microfluidic technologies that allow high-throughput and combinatorial screening may lead to breakthrough discoveries not only in basic research but also relevant to clinical application. This is further strengthened by the fact that reliability of such screens may improve, since microfluidic systems allow close mimicking of physiological conditions. Finally, microfluidic systems are also very promising as micro factories of a new generation of natural or synthetic biomaterials and constructs, with finely controlled properties.

Development of Highly Functional Biomaterials by Decoupling and Recombining Material Properties.

Development of functional biomaterials by a design-driven approach is described, whereby individual properties are first decoupled to investigate their sole effects on a biological process. Following this investigation, they are recombined in such a way that the overall performance and applicability of the biomaterial is improved. This is in contrast to classical, processing-driven biomaterials development where the properties of a material are mainly determined by the possibilities of the technique used to produce it.

Microfluidic platform with four orthogonal and overlapping gradients for soluble compound screening in regenerative medicine research.

We present here a screening method based on a microfluidic platform, which can generate four orthogonal and overlapping concentration gradients of soluble compounds over a monolayer of cells, in combination with automated and in situ image analysis, for use in regenerative medicine research. The device includes a square chamber in which cells are grown, and four independent supply channels along the sides of the chamber, which are connected through an array of small diffusion channels. Compounds flown through the supply channels diffuse through diffusion channels into the chamber to create a gradient over the cell culture area. Further, the chamber is connected to two channels intended for introduction of cells and in situ staining. In this study, the dimensions of the different channels were optimized through finite element modeling to yield stable gradients, and two designs were used with gradients spanning 2.9-2.4 µM and 3.4-2.0 µM. Next, overlapping gradients were generated using four rhodamine-derived fluorescent dyes, and imaged using confocal microscopy. Finally, the platform was applied to assess the concentration-dependent response of an osteoblastic cell line exposed to a hypoxia-mimicking molecule phenanthroline, using an in situ fluorescent staining assay in combination with image analysis, applicable to closed microfluidic devices. The on-chip assay yielded results comparable to those observed in conventional culture, where a range of concentrations was tested in independent microwells. In the future, we intend to use this method to complement or replace current research approaches in screening soluble compounds for regenerative medicine, which are often based on one-sample-for-one-experiment principle.

High throughput analysis of photocatalytic water purification.

We present a novel high throughput photocatalyst efficiency assessment method based on 96-well microplates and UV-vis spectroscopy. We demonstrate the reproducibility of the method using methyl orange (MO) decomposition and compare kinetic data obtained with those provided in the literature for larger conventional photoreactors. To demonstrate the capabilities of the method, we rapidly screened the effects of salts, potentially present in wastewater, on kinetic rates of MO decomposition and briefly discuss the obtained data on the basis of existing literature.

Microtiter plate-sized standalone chip holder for microenvironmental physiological control in gas-impermeable microfluidic devices.

We present a microtiter plate-sized standalone chip holder for precise control of physiological conditions inside closed microfluidic cell culture systems, made from gas-impermeable materials. Specifically, we demonstrate the suitability of the holder to support cell growth in a glass chip, to allow time-lapse imaging of live cells and the creation of a hypoxic environment, all relevant for applications in regenerative medicine research.

Bio-microfluidic platform for gold nanoprobe based DNA detection--application to Mycobacterium tuberculosis.

We have projected and fabricated a microfluidic platform for DNA sensing that makes use of an optical colorimetric detection method based on gold nanoparticles. The platform was fabricated using replica moulding technology in PDMS patterned by high-aspect-ratio SU-8 moulds. Biochips of various geometries were tested and evaluated in order to find out the most efficient architecture, and the rational for design, microfabrication and detection performance is presented. The best biochip configuration has been successfully applied to the DNA detection of Mycobacterium tuberculosis using only 3 µl on DNA solution (i.e. 90 ng of target DNA), therefore a 20-fold reduction of reagents volume is obtained when compared with the actual state of the art.

Bioactive compounds from endemic plants of Southwest Portugal: inhibition of acetylcholinesterase and radical scavenging activities.

CONTEXT: Natural products are reported to have substantial neuroprotective activity due to their radical scavenging capacity, and also acetylcholinesterase (AChE) inhibitory capacity, both activities important in neurodegeneration. OBJECTIVE: The undesirable side effects of compounds in pharmacological use make it important to identify natural neuroprotective molecules. This work assesses the potential of five endemic Portuguese plants as sources of neuroprotective compounds. MATERIALS AND METHODS: Antioxidant capacity for peroxyl radical was determined by Oxygen Radical Absorbance Capacity method and for hydroxyl by Electron Paramagnetic Resonance, as well as AChE inhibitory capacity of the plant hydroethanolic extracts. The molecules responsible for these valuable properties were also tentatively identified by HPLC. RESULTS AND DISCUSSION: Armeria rouyana and Thymus capitellatus presented some of the highest phenolic contents (76.60 ± 7.19 and 12.82 ± 0.24 mg GAE g−1 dw, respectively) and antioxidant capacities (592 ± 116 and 449 ± 57 µmol TE g−1 dw, respectively). The flavonoids were identified as the phytomolecules related to the antioxidant capacity of these plant extracts; in the case of A. rouyana, l-ascorbic acid also made an important contribution (3.27 ± 0.26 mg g−1 dw). Plant extracts clearly demonstrated effective AChE inhibitory activity (480 ± 98 and 490 ± 46 µg mL−1, respectively), that could be associated to polyphenols. CONCLUSIONS: The extracts of A. rouyana and T. capitellatus and their active components, especially polyphenols, demonstrate interesting neuroprotective potential. They, therefore, deserve further study as their phytomolecules are promising sources of either natural neuroprotective products and/or novel lead compounds.