Universal Biomaterial-on-Chip: a versatile platform for evaluating cellular responses on diverse biomaterial substrates.

Microfluidics has emerged as a promising approach for assessing cellular behavior in vitro, providing more physiologically relevant cell culture environments with dynamic flow and shear stresses. This study introduces the Universal Biomaterial-on-Chip (UBoC) device, which enables the evaluation of cell response on diverse biomaterial substrates in a 3D-printed microfluidic device. The UBoC platform offers mechanical stimulation of the cells and monitoring of their response on diverse biomaterials, enabling qualitative and quantitative in vitro analysis both on- and off-chip. Cell adhesion and proliferation were assessed to evaluate the biocompatibility of materials with different physical properties, while mechanical stimulation was performed to investigate shear-dependent calcium signaling in pre-osteoblasts. Moreover, the applicability of the UBoC platform in creating more complex in vitro models by culturing multiple cell types was demonstrated, establishing a dynamic multicellular environment to investigate cellular interfaces and their significance in biological processes. Overall, the UBoC presents an adaptable tool for in vitro evaluation of cellular behavior, offering opportunities for studying various biomaterials and cell interactions in microfluidic environments.

A microfluidic-based approach to investigate the inflammatory response of macrophages to pristine and drug-loaded nanostructured hydroxyapatite.

The in vitro biological characterization of biomaterials is largely based on static cell cultures. However, for highly reactive biomaterials such as calcium-deficient hydroxyapatite (CDHA), this static environment has limitations. Drastic alterations in the ionic composition of the cell culture medium can negatively affect cell behavior, which can lead to misleading results or data that is difficult to interpret. This challenge could be addressed by a microfluidics-based approach (i.e. on-chip), which offers the opportunity to provide a continuous flow of cell culture medium and a potentially more physiologically relevant microenvironment. The aim of this work was to explore microfluidic technology for its potential to characterize CDHA, particularly in the context of inflammation. Two different CDHA substrates (chemically identical, but varying in microstructure) were integrated on-chip and subsequently evaluated. We demonstrated that the on-chip environment can avoid drastic ionic alterations and increase protein sorption, which was reflected in cell studies with RAW 264.7 macrophages. The cells grown on-chip showed a high cell viability and enhanced proliferation compared to cells maintained under static conditions. Whereas no clear differences in the secretion of tumor necrosis factor alpha (TNF-α) were found, variations in cell morphology suggested a more anti-inflammatory environment on-chip. In the second part of this study, the CDHA substrates were loaded with the drug Trolox. We showed that it is possible to characterize drug release on-chip and moreover demonstrated that Trolox affects the TNF-α secretion and morphology of RAW 264.7 â€‹cells. Overall, these results highlight the potential of microfluidics to evaluate (bioactive) biomaterials, both in pristine form and when drug-loaded. This is of particular interest for the latter case, as it allows the biological characterization and assessment of drug release to take place under the same dynamic in vitro environment.

Experimental Characterization and Mathematical Modeling of the Adsorption of Proteins and Cells on Biomimetic Hydroxyapatite.

Biomaterial development is a long process consisting of multiple stages of design and evaluation within the context of both in vitro and in vivo testing. To streamline this process, mathematical and computational modeling displays potential as a tool for rapid biomaterial characterization, enabling the prediction of optimal physicochemical parameters. In this work, a Langmuir isotherm-based model was used to describe protein and cell adhesion on a biomimetic hydroxyapatite surface, both independently and in a one-way coupled system. The results indicated that increased protein surface coverage leads to improved cell adhesion and spread, with maximal protein coverage occurring within 48 h. In addition, the Langmuir model displayed a good fit with the experimental data. Overall, computational modeling is an exciting avenue that may lead to savings in terms of time and cost during the biomaterial development process.

A practical guide for evaluating the osteoimmunomodulatory properties of biomaterials.

Biomaterials offer a promising approach to repair bone defects. Whereas traditional studies predominantly focused on optimizing the osteogenic capacity of biomaterials, less focus has been on the immune response elicited by them. However, the immune and skeletal systems extensively interact, a concept which is referred to as 'osteoimmunology'. This realization has fuelled the development of biomaterials with favourable osteoimmunomodulatory (OIM) properties, aiming to modulate the immune response and to support bone regeneration, thereby affecting the success of an implant. Given the plethora of in vitro assays used to evaluate the OIM properties of biomaterials, it may be challenging to select the right methods to produce conclusive results. In this review, we aim to provide a comprehensive and practical guide for researchers interested in studying the OIM properties of biomaterials in vitro. After a concise overview of the concept of osteoimmunology, emphasis is put on the methodologies that are regularly used to evaluate the OIM properties of biomaterials. First, a description of the most commonly used cell types and cell culture media is provided. Second, typical experimental set-ups and their relevant characteristics are discussed. Third, a detailed overview of the generally used methodologies and readouts, including cell type-specific markers and time points of analysis, is given. Finally, we highlight the promise of advanced approaches, namely microarrays, bioreactors and microfluidic-based systems, and the potential that these may offer to the osteoimmunology field. STATEMENT OF SIGNIFICANCE: Osteoimmunology focuses on the connection and communication between the skeletal and immune systems. This interaction has been recognized to play an important role in the clinical success of biomaterials, which has resulted in an increasing amount of research on the osteoimmunomodulatory (OIM) properties of biomaterials. However, the amount of literature makes it challenging to extract the information needed to design experiments from beginning to end, and to compare obtained results to existing work. This article intends to serve as a guide for those aiming to learn more about the commonly used experimental approaches in the field. We cover early-stage choices, such as cell types and experimental set-ups, but also discuss specific assays, including cell markers and time points of analysis.

A microfluidics-based method for culturing osteoblasts on biomimetic hydroxyapatite.

The reliability of conventional cell culture studies to evaluate biomaterials is often questioned, as in vitro outcomes may contradict results obtained through in vivo assays. Microfluidics technology has the potential to reproduce complex physiological conditions by allowing for fine control of microscale features such as cell confinement and flow rate. Having a continuous flow during cell culture is especially advantageous for bioactive biomaterials such as calcium-deficient hydroxyapatite (HA), which may otherwise alter medium composition and jeopardize cell viability, potentially producing false negative results in vitro. In this work, HA was integrated into a microfluidics-based platform (HA-on-chip) and the effect of varied flow rates (2, 8 and 14 µl/min, corresponding to 0.002, 0.008 and 0.014 dyn/cm2, respectively) was evaluated. A HA sample placed in a well plate (HA-static) was included as a control. While substantial calcium depletion and phosphate release occurred in static conditions, the concentration of ions in HA-on-chip samples remained similar to those of fresh medium, particularly at higher flow rates. Pre-osteoblast-like cells (MC3T3-E1) exhibited a significantly higher degree of proliferation on HA-on-chip (8 µl/min flow rate) as compared to HA-static. However, cell differentiation, analysed by alkaline phosphatase (ALP) activity, showed low values in both conditions. This study indicates that cells respond differently when cultured on HA under flow compared to static conditions, which indicates the need for more physiologically relevant methods to increase the predictive value of in vitro studies used to evaluate biomaterials. STATEMENT OF SIGNIFICANCE: There is a lack of correlation between the results obtained when testing some biomaterials under cell culture as opposed to animal models. To address this issue, a cell culture method with slightly enhanced physiological relevance was developed by incorporating a biomaterial, known to regenerate bone, inside of a microfluidic platform that enabled a continuous supply of cell culture medium. Since the utilized biomaterial interacts with surrounding ions, the perfusion of medium allowed for shielding of these changes similarly as would happen in the body. The experimental outcomes observed in the dynamic platform were different than those obtained with standard static cell culture systems, proving the key role of the platform in the assessment of biomaterials.

Effect of the Addition Frequency of 5-Azacytidine in Both Micro- and Macroscale Cultures.

INTRODUCTION: Human mesenchymal stem cells (hMSCs) have a great clinical potential for tissue regeneration purposes due to its multilineage capability. Previous studies have reported that a single addition of 5-azacytidine (5-AzaC) causes the differentiation of hMSCs towards a myocardial lineage. The aim of this work was to evaluate the effect of 5-AzaC addition frequency on hMSCs priming (i.e., indicating an early genetic differentiation) using two culture environments. METHODS: hMSCs were supplemented with 5-AzaC while cultured in well plates and in microfluidic chips. The impact of 5-AzaC concentration (10 and 20 µM) and addition frequency (once, daily or continuously), as well as of culture period (2 or 5 days) on the genetic upregulation of PPARγ (adipocytes), PAX3 (myoblasts), SOX9 (chondrocytes) and RUNX2 (osteoblasts) was evaluated. RESULTS: Daily delivering 5-AzaC caused a higher upregulation of PPARγ, SOX9 and RUNX2 in comparison to a single dose delivery, both under static well plates and dynamic microfluidic cultures. A particularly high gene expression of PPARγ (tenfold-change) could indicate priming of hMSCs towards adipocytes. CONCLUSIONS: Both macro- and microscale cultures provided results with similar trends, where addition frequency of 5-AzaC was a crucial factor to upregulate several genes. Microfluidics technology was proven to be a suitable platform for the continuous delivery of a drug and could be used for screening purposes in tissue engineering research.

Exploring microfluidics as a tool to evaluate the biological properties of a titanium alloy under dynamic conditions.

To bring novel biomaterials to clinical use, reliable in vitro models are imperative. The aim of this work was to develop a microfluidic tool to evaluate the biological properties of biomaterials for bone repair. Two approaches to embed medical grade titanium (Ti6Al4V) on-chip were explored. The first approach consisted of a polydimethylsiloxane microfluidic channel placed onto a titanium disc, held together by an additively manufactured fixture. In the second approach, a titanium disc was assembled onto a microscopic glass slide, using a double-sided tape microfluidic channel. Both approaches demonstrated potential for maintaining MC3T3-E1 preosteoblast-like cell cultures on-chip, as was shown by the vast majority of living cells after 1 day. In addition, the cells cultured on-chip showed a more elongated morphology compared to cells grown under static conditions and a tendency to align to the direction of the flow. For longer-term (i.e. 10 days) studies, the glass-based chip was selected. Assessment of cell viability showed a high number of living cells during the entire experimental period. Cell proliferation and differentiation studies revealed an increase in cell proliferation on-chip, suggesting that proliferation was the dominating process at the detriment of differentiation in this micrometric dynamic environment. These results illustrate the importance of optimizing in vitro cell culture conditions and how these may affect biomaterial testing outcomes. Overall, this work provides a step towards more in vivo-like microfluidic testing platforms, which are expected to provide more reliable in vitro screening of biomaterials.

Development and characterisation of bilayered periosteum-inspired composite membranes based on sodium alginate-hydroxyapatite nanoparticles.

BACKGROUND AND AIM: Membranes for guided bone regeneration should have a mechanical structure and a chemical composition suitable for mimicking biological structures. In this work, we pursue the development of periosteum-inspired bilayered membranes obtained by crosslinking alginate with different amounts of nanohydroxyapatite. EXPERIMENTS: Alginate-nanohydroxyapatite interaction was studied by rheology and infrared spectroscopy measurements. The membranes were characterized regarding their tensile strength, degradation and surface morphology. Finally, cell cultures were performed on each side of the membranes. FINDINGS: The ionic bonding between alginate polysaccharide networks and nanohydroxyapatite was proven, and had a clear effect in the strength and microstructure of the hydrogels. Distinct surface characteristics were achieved on each side of the membranes, resulting in a highly porous fibrous side and a mineral-rich side with higher roughness and lower porosity. Moreover, the effect of amount of nanohydroxyapatite was reflected in a decrease of the membranes' plasticity and an increment of degradation rate. Finally, it was proved that osteoblast-like cells proliferated and differentiated on the mineral-rich side, specially when a higher amount of nanohydroxyapatite was used, whereas fibroblasts-like cells were able to proliferate on the fibrous side. These periosteum-inspired membranes are promising biomaterials for guided tissue regeneration applications.

A novel strategy to enhance interfacial adhesion in fiber-reinforced calcium phosphate cement.

Calcium phosphate cements (CPCs) are extensively used as synthetic bone grafts, but their poor toughness limits their use to non-load-bearing applications. Reinforcement through introduction of fibers and yarns has been evaluated in various studies but always resulted in a decrease in elastic modulus or bending strength when compared to the CPC matrix. The aim of the present work was to improve the interfacial adhesion between fibers and matrix to obtain tougher biocompatible fiber-reinforced calcium phosphate cements (FRCPCs). This was done by adding a polymer solution to the matrix, with chemical affinity to the reinforcing chitosan fibers, namely trimethyl chitosan (TMC). The improved wettability and chemical affinity of the chitosan fibers with the TMC in the liquid phase led to an enhancement of the interfacial adhesion. This resulted in an increase of the work of fracture (several hundred-fold increase), while the elastic modulus and bending strength were maintained similar to the materials without additives. Additionally the TMC-modified CPCs showed suitable biocompatibility with an osteoblastic cell line.

Fabrication of user-friendly and biomimetic 1,1'-carbonyldiimidazole cross-linked gelatin/agar microfluidic devices.

We have developed a straightforward technique for fabricating user-friendly and biomimetic microfluidic devices out of a gelatin/agar gel cross-linked with 1,1'-carbonyldiimidazole. The fabrication procedure requires only inexpensive starting materials such as glass capillaries and wires to mold 3D cylindrical channels into the gel with the possibility of achieving channel diameters of 375µm and 1000µm. We demonstrate that the channel absent of gel injury can retain fluid within its dimensions for at least 7h. We also show that the device material does not autofluoresce nor provide hindrances with fluorescent imaging. A discussion of the chemical linkage identities of cross-linked gelatin/agar is included via ATR-FTIR studies. Crosslinking of the gelatin/agar is further confirmed by the lack of a gel to sol transition at physiological temperature as assessed by DSC measurements. SEM micrographs that demonstrate the 100nm mean pore width of the cross-linked gelatin/agar are provided. This device is considered biomimetic because it represents components present in the natural extracellular matrix such as collagen and proteoglycans in the form of cross-linked gelatin/agar.

Influence of Si substitution on the reactivity of α-tricalcium phosphate.

Silicon substituted calcium phosphates have been widely studied over the last ten years due to their enhanced osteogenic properties. Notwithstanding, the role of silicon on α-TCP reactivity is not clear yet. Therefore, the aim of this work was to evaluate the reactivity and the properties of Si-α-TCP in comparison to α-TCP. Precursor powders have similar properties regarding purity, particle size distribution and specific surface area, which allowed a better comparison of the Si effects on their reactivity and cements properties. Both Si-α-TCP and α-TCP hydrolyzed to a calcium-deficient hydroxyapatite when mixed with water but their conversion rates were different. Si-α-TCP exhibited a slower setting rate than α-TCP, i.e. kSSA for Si-TCP (0.021g·m-2·h-1) was almost four times lower than for α-TCP (0.072g·m-2·h-1). On the other hand, the compressive strength of the CPC resulting from fully reacted Si-α-TCP was significantly higher (12.80±0.38MPa) than that of α-TCP (11.44±0.54MPa), due to the smaller size of the entangled precipitated apatite crystals.

Selenium- and Tellurium-Based Antioxidants for Modulating Inflammation and Effects on Osteoblastic Activity.

Increased oxidative stress plays a significant role in the etiology of bone diseases. Heightened levels of H2O2 disrupt bone homeostasis, leading to greater bone resorption than bone formation. Organochalcogen compounds could act as free radical trapping agents or glutathione peroxidase mimetics, reducing oxidative stress in inflammatory diseases. In this report, we synthesized and screened a library of organoselenium and organotellurium compounds for hydrogen peroxide scavenging activity, using macrophagic cell lines RAW264.7 and THP-1, as well as human mono- and poly-nuclear cells. These cells were stimulated to release H2O2, using phorbol 12-myristate 13-acetate, with and without organochalogens. Released H2O2 was then measured using a chemiluminescent assay over a period of 2 h. The screening identified an organoselenium compound which scavenged H2O2 more effectively than the vitamin E analog, Trolox. We also found that this organoselenium compound protected MC3T3 cells against H2O2-induced toxicity, whereas Trolox did not. The organoselenium compound exhibited no cytotoxicity to the cells and had no deleterious effects on cell proliferation, viability, or alkaline phosphatase activity. The rapidity of H2O2 scavenging and protection suggests that the mechanism of protection is due to the direct scavenging of extracellular H2O2. This compound is a promising modulators of inflammation and could potentially treat diseases involving high levels of oxidative stress.

Cytotoxicity of modified glass ionomer cement on odontoblast cells.

Recently a modified glass ionomer cement (GIC) with enhanced bioactivity due to the incorporation of wollastonite or mineral trioxide aggregate (MTA) has been reported. The aim of this study was to evaluate the cytotoxic effect of the modified GIC on odontoblast-like cells. The cytotoxicity of a conventional GIC, wollastonite modified GIC (W-mGIC), MTA modified GIC (M-mGIC) and MTA cement has been evaluated using cement extracts, a culture media modified by the cement. Ion concentration and pH of each material in the culture media were measured and correlated to the results of the cytotoxicity study. Among the four groups, conventional GIC showed the most cytotoxicity effect, followed by W-mGIC and M-mGIC. MTA showed the least toxic effect. GIC showed the lowest pH (6.36) while MTA showed the highest (8.62). In terms of ion concentration, MTA showed the largest Ca(2+) concentration (467.3 mg/L) while GIC showed the highest concentration of Si(4+) (19.9 mg/L), Al(3+) (7.2 mg/L) and Sr(2+) (100.3 mg/L). Concentration of F(-) was under the detection limit (0.02 mg/L) for all samples. However the concentrations of these ions are considered too low to be toxic. Our study showed that the cytotoxicity of conventional GIC can be moderated by incorporating calcium silicate based ceramics. The modified GIC might be promising as novel dental restorative cements.

Role of pore size and morphology in musculo-skeletal tissue regeneration.

Biomaterials in the form of scaffolds hold great promise in the regeneration of diseased tissues. The scaffolds stimulate cellular adhesion, proliferation and differentiation. While the scaffold composition will dictate their biocompatibility, their porosity plays a key role in allowing proper cell penetration, nutrient diffusion as well as bone ingrowth. Porous scaffolds are processed with the help of a wide variety of techniques. Designing scaffolds with the appropriate porosity is a complex issue since this may jeopardize other physico-chemical properties. From a macroscopic point of view, parameters such as the overall architecture, pore morphology, interconnectivity and pore size distribution, have unique roles in allowing bone ingrowth to take place. From a microscopic perspective, the adsorption and retention of proteins in the microporosities of the material will dictate the subsequent cell adhesion. Therefore, the microstructure of the substrate can determine cell proliferation as well as the expression of specific osteogenic genes. This review aims at discussing the effect of micro- and macroporosity on the physico-chemical and biological properties of scaffolds for musculo-skeletal tissue regeneration.

Impact of Porosity and Electrolyte Composition on the Surface Charge of Hydroxyapatite Biomaterials.

The success or failure of a material when implanted in the body is greatly determined by the surface properties of the material and the host tissue reactions. The very first event that takes place after implantation is the interaction of soluble ions, molecules and proteins from the biological environment with the material surface leading to the formation of an adsorbed protein layer that will later influence cell attachment. In this context, the particular topography and surface charge of a material become critical as they influence the nature of the proteins that will adsorb. However, very limited information is available on the surface charge of porous substrates. Only until very recently was the determination of the zeta potential on porous membranes accurately determined. The goal of this work was to implement the previous findings for the determination of the zeta potential of a series of porous hydroxyapatite (HA) substrates and to assess how porosity affects the measurements. In addition, studies using various electrolytes were also performed to prove how the specific affinity of certain ions for HA can further impact surface charge. The results showed that all materials exhibited very similar external surface charge (approximately -23 mV), consistent with their almost identical topographies. However, the presence of interconnected pores underneath the sample surface resulted in an additional internal zeta potential that varied with the porosity content. Measurements with different electrolytes confirmed the selectivity of divalent ions for HA underlying the importance of testing biomaterials using relevant electrolytes.

Evaluation of Biocompatibility and Release of Reactive Oxygen Species of Aluminum Oxide-Coated Materials.

Surface properties of biomaterials can strongly influence biomaterial-host interactions. For this reason, coating processes open a wide range of possibilities to modulate the fate of a biomaterial in the body. This study evaluates the effect of a coating material intended for drug delivery capsules on biocompatibility and the release of reactive oxygen species (ROS), that is, respiratory burst in macrophages that indicates acute inflammation. In parallel with a new approach to develop drug-delivery capsules by directly coating solid-state drug particles, in this study, glass slides and silicon nanoparticles (NPs) were coated with aluminum oxide (Al2O3) using atomic layer deposition. Different sizes of NPs (20 and 310 nm) were suspended at different concentrations (10, 100, and 1000 µg/mL) and were evaluated. The homogeneous coating of slides was proved using X-ray photoelectron spectroscopy, and the coating on NP was observed using transmission electron microscopy. Human dermal fibroblasts and human osteoblasts were able to proliferate on the coated slides and in the presence of a suspension of coated NPs (20 and 310 nm) at a low concentration (10 µg/mL). The macrophages released ROS only when in contact with NPs at a concentration of 1000 µg/mL, where the 20 nm NPs caused a higher release of ROS than the 310 nm NPs. This study shows that Al2O3 coatings do not affect the cells negatively and that the cell viability was compromised only when in contact with a high concentration (1000 µg/mL) of smaller (20 nm) NPs.

Changes in the drug release pattern of fresh and set simvastatin-loaded brushite cement.

Calcium phosphate cements are synthetic bone graft substitutes able to set at physiological conditions. They can be applied by minimally invasive surgery and can also be used as drug delivery systems. Consequently, the drug release pattern from the cement paste (fresh cement) is of high clinical interest. However, previous studies have commonly evaluated the drug release using pre-set cements only. Therefore, the aim of this work was to determine if the time elapsed from cement preparation until immersion in the solution (3 min for fresh cements, and 1h and 15 h for pre-set cements) had an influence on its physical properties, and correlating these to the drug release profile. Simvastatin was selected as a model drug, while brushite cement was used as drug carrier. This study quantified how the setting of a material reduces the accessibility of the release media to the material, thus preventing drug release. A shift in the drug release pattern was observed, from a burst-release for fresh cements to a sustained release for pre-set cements.

Simvastatin and zinc synergistically enhance osteoblasts activity and decrease the acute response of inflammatory cells.

Several ceramic biomaterials have been suggested as promising alternatives to autologous bone to replace or restore bone after trauma or disease. The osteoinductive potential of most scaffolds is often rather low by themselves and for this reason growth factors or drugs have been supplemented to these synthetic materials. Although some growth factors show good osteoinductive potential their drawback is their high cost and potential severe side effects. In this work the combination of the well-known drug simvastatin (SVA) and the inorganic element Zinc (Zn) is suggested as a potential additive to bone grafts in order to increase their bone regeneration/formation. MC3T3-E1 cells were cultured with Zn (10 and 25 µM) and SVA (0.25 and 0.4 µM) for 10 days to evaluate proliferation and differentiation, and for 22 days to evaluate secretion of calcium deposits. The combination of Zn (10 µM) and SVA (0.25 µM) significantly enhanced cell differentiation and mineralization in a synergetic manner. In addition, the release of reactive oxygen species (ROS) from primary human monocytes in contact with the same concentrations of Zn and SVA was evaluated by chemiluminescence. The combination of the additives decreased the release of ROS, although Zn and SVA separately caused opposite effects. This work shows that a new combination of additives can be used to increase the osteoinductive capacity of porous bioceramics.

In Vitro and In Vivo Response to Low-Modulus PMMA-Based Bone Cement.

The high stiffness of acrylic bone cements has been hypothesized to contribute to the increased number of fractures encountered after vertebroplasty, which has led to the development of low-modulus cements. However, there is no data available on the in vivo biocompatibility of any low-modulus cement. In this study, the in vitro cytotoxicity and in vivo biocompatibility of two types of low-modulus acrylic cements, one modified with castor oil and one with linoleic acid, were evaluated using human osteoblast-like cells and a rodent model, respectively. While the in vitro cytotoxicity appeared somewhat affected by the castor oil and linoleic acid additions, no difference could be found in the in vivo response to these cements in comparison to the base, commercially available cement, in terms of histology and flow cytometry analysis of the presence of immune cells. Furthermore, the in vivo radiopacity of the cements appeared unaltered. While these results are promising, the mechanical behavior of these cements in vivo remains to be investigated.

Effect of a Bromo Substituent on the Glutathione Peroxidase Activity of a Pyridoxine-like Diselenide.

In search for better mimics of the glutathione peroxidase enzymes, pyridoxine-like diselenides 6 and 11, carrying a 6-bromo substituent, were prepared. Reaction of 2,6-dibromo-3-pyridinol 5 with sodium diselenide provided 6 via aromatic nucleophilic substitution of the 2-bromo substituent. LiAlH4 caused reduction of all four ester groups and returned 11 after acidic workup. The X-ray structure of 6 showed that the dipyridyl diselenide moiety was kept in an almost planar, transoid conformation. According to NBO-analysis, this was due to weak intramolecular Se···O (1.1 kcal/mol) and Se···N-interactions (2.5 kcal/mol). That the 6-bromo substituent increased the positive charge on selenium was confirmed by NPA-analysis and seen in calculated and observed (77)Se NMR-shifts. Diselenide 6 showed a more than 3-fold higher reactivity than the corresponding des-bromo compound 3a and ebselen when evaluated in the coupled reductase assay. Experiments followed for longer time (2 h) confirmed that diselenide 6 is a better GPx-catalyst than 11. On the basis of (77)Se-NMR experiments, a catalytic mechanism for diselenide 6 was proposed involving selenol, selenosulfide and seleninic acid intermediates. At low concentration (10 µM) where it showed only minimal toxicity, it could scavenge ROS produced by MNC- and PMNC-cells more efficiently than Trolox.