Kinetic effects of TiO 2 fine particles and nanoparticles aggregates on the nanomechanical properties of human neutrophils assessed by force spectroscopy

Everton Luis
Santos Da Rosa
Kinetic effects of TiO 2 fine particles and nanoparticles aggregates on the nanomechanical properties of human neutrophils assessed by force spectroscopy
R E S E A R C H A R T I C L E Open Access
Background: Increasing applications of titanium dioxide (TiO 2 ) fine particles (FPs) and nanoparticles (NPs) require coupled knowledge improvement concerning their biokinetic effects. Neutrophils are quickly recruited to titanium implantation areas. Neutrophils mechanical properties display a crucial role on cell physiology and immune responsive functions. Then, micro and nanomechanical characterization assessed by force spectroscopy (FS) technique has been largely applied in this field. Results: Scanning electron microscopy (SEM) images highlighted neutrophils morphological changes along TiO 2 FPs and NPs aggregates exposure time (1, 5, and 30 min) compared to controls. FS approaches showed an increasing on attraction forces to TiO 2 FPs and NPs treated neutrophils. This group depicted stronger stiffness features than controls just at 1 min of exposure. Treated neutrophils showed a tendency to increase adhesive properties after 1 and 5 min of exposure. These cells maintained comparatively higher elasticity behavior for a longer time possibly due to intense phagocytosis and cell stiffness opposing to the tip indentation. Neutrophils activation caused by FPs and NPs uptake could be related to increasing dissipated energy results.Conclusions: Mechanical modifications resulted from TiO 2 FPs and NPs aggregates interaction with neutrophils showed increasing stiffness and also cell morphology alteration. Cells treatment by this metal FPs and NPs caused an increase in attractive forces. This event was mainly observed on the initial exposure times probably regarding to the interaction of neutrophils membrane and phagocytosis. Similar results were found to adhesion forces and dissipated energy outcomes. Treated cells presented comparatively higher elasticity behavior for a longer time. SEM images clearly suggested cell morphology alteration along time course probably related to activation, cytoskeleton rearrangement and phagocytosis. This scenario with increase in stiffness strongly suggests a direct relationship over neutrophil rolling, arrest, and transmigration. Scrutinizing these interactions represents an essential step to clarify the mechanisms involved on treatments containing micro and nanomaterials and their fates on the organisms.

Background

Titanium dioxide (TiO 2 ) has been widely used in automotive and aerospace industry, personal care, food, and even in pharmaceutical and biomedical products [1,2]. Increasingly, this metal and its alloys are used in the biomedical field for medicine and dentistry [3,4]. Pure titanium and some of its alloys are non-toxic and generally have been described as being biocompatible with human tissues [5]. Despite its biocompatibility, toxicological concerns are depicted in the literature related to ions, microparticles (MPs), fine particles and nanoparticles [6][7][8][9][10]. Despite being inert [11], titanium may promote inflammatory reactions by recruiting inflammatory cells [12], such as neutrophils.

Neutrophils represent the first line of innate defense, comprising about 50-70% of all human leukocytes [13]. They are quickly summoned to areas containing titanium micro or nanoparticles [14]. It is well-known that MPs and NPs can be internalized by phagocytic and even non-phagocytic cells [15][16][17], and can interact with cell membranes, subcellular organelles, proteins, and nucleic acids [18,19]. Neutrophils internalize TiO 2 MPs and NPs, leading to a change in shape and motility; these events are modulated by cytoskeleton proteins, and enhance the production of superoxide anion, cytokines, and chemokines due to cell activation [20][21][22][23]. There are different pathways for the uptake of MPs and NPs: micro-sized particles are internalized via phagocytosis by monocytes, macrophages, and neutrophils [17], while NPs (ranging from 1 to 100 nm) seem to use other pathways in spite of the fact that, in some situations, NPs can aggregate or agglomerate, resulting in MPs behaviour, being an important factor in understanding cytotoxicity. However, the effect of the aggregate size of nanoparticles on cells is unclear [24]. Agglomeration (particles are linked by weak forces, eg van der Waals forces, capillary) and aggregation (particles held together by chemical bonds-strong forces) of TiO 2 MPs and NPs are known mechanisms [25] and may occur on the surface of the cells membrane affecting communication with the external environment [26]. Furthermore it is known that the length and chemical nature of the aggregates of TiO 2 are related to the toxicity [27]. In addition to phagocytosis, uptake pathways include clathrin-mediated endocytosis, caveolin-mediated endocytosis, macropinocytosis, and pathways independent of clathrin/caveolin. The passive pathway should also be considered for NPs uptake [28].

Neutrophil recruitment into areas with MPs or NPs proceeds in a cascade process. Rolling of neutrophils inside vessels is mediated by selectins (PSGL-1) and their counter-receptors (L and P selectins) and integrinmediated (LFA-1, Mac-1) arrest [29]. These adhesion mechanisms activate different signaling pathways in the cell, leading to neutrophil extravasation, promoting cytoskeletal rearrangement, and inducing superoxide production and degranulation [30,31]. The behavior of neutrophils and other cells following contact with macro or micro-sized particles are different from following contact with NPs, due to specific surfaces, energy, and physicochemical properties [25,32,33]. When circulating neutrophils encounter a macro-sized TiO 2 surface, the adhesion process starts, mediated by selectins, FcγIII receptor (CD16) and finally integrins (CD11b); cells are not activated to undergo a respiratory burst by TiO 2 surfaces [34]. On the other hand, neutrophils phagocytose TiO 2 NPs and MPs aggregates smaller than the cell [15,23]. Furthermore, TiO 2 MPs and NPs may activate and induce alterations in cell morphology [20,35] and inhibit apoptosis (at 50-100 μg/mL). They also induce the production of several different cytokines/chemokines, mainly IL-8 and Gro-α [20]. Therefore, when this interaction occurs, MPs and NPs play a crucial role in cell physiology and mechanical properties. Moreover, the mechanical properties of cells are important when aiming to understand mechanisms such as adhesion, endothelial transmigration, and diapedesis [36].

Concerning the mechanical properties of cells, a variety of methods and techniques have been used to probe cells such as micropipette aspiration [37], magnetic twisting cytometry (MTC) [38], and atomic force microscopy (AFM). Conceptually, AFM is a type of scanning probe microscopy (SPM) and is perceived as a tool central to the goals of the burgeoning field known as nanotechnology [39]. It represents an effective tool to probe the viscoelastic properties of cells, to study the membrane and subcellular structures, and plays a key role in determining cell function in response to inflammatory stimuli [40,41]. AFM is mainly used for surface topography analysis by using attractive (short-range chemical, van der Waals and electrostatic forces) and repulsive interaction forces among a few atoms attached to the tip of a cantilever and the sample surface (nanoindentation). This technique has increasingly been applied for the characterization of single molecules under tensile or torsional load, cell-to-cell interactions, interactions of surfaces with molecules, and investigations into the mechanical properties of cells [42]. Further details concerning AFM are presented elsewhere [36,43,44]. The nanoindentation technique using force spectroscopy enables researchers to assess living or fixed neutrophils, in a passive or activated state, pointing out the micro-rheology of the entire cell surface (e.g. the cell body, lamellipodia, uropodia, and projections of the cytoskeleton), allowing the analysis of viscoelastic properties [45,46].

Force spectroscopy is the force-versus-distance measurement taken when using AFM in vertical mode only [47]. Cantilever deflection is recorded as it indents the cell, by reflecting a laser off the cantilever into a split photodiode.

Hertzian mechanics gives a linear cell-based elastic model on AFM deflection data to determine cell elasticity, among other mechanical parameters [42,48]. Focusing on neutrophil rheology, Lee and coworkers stated that the neutrophil body is significantly stiffer than the regions closer to the leading edge, while leading edge and tail regions were mechanically indistinguishable [45]. On the other hand, Rosenbluth and coworkers, when comparing neutrophils, myeloid (HL60) and lymphoid (Jurkat) cells, reported that there is a significant cell type effect on stiffness. Neutrophils were significantly softer than HL60 cells and significantly stiffer than Jurkat cells [48].

Increasing applications of FPs and NPs in the dental and medical field (dental implants, orthopedic prostheses, and antineoplastic therapies) have led to concerns regarding the biokinetic effects of micro and nanomaterials and their fate in vivo. Therefore, the present study aimed to analyze some mechanical properties related to the effects of TiO 2 FPs and NPs aggregates exposure on human neutrophils by a force spectroscopy approach over time (1, 5, and 30 min). Parameters like snap-in force, maximum load force, detachment force, Young's modulus, and dissipated energy were depicted by approaching and retracting a non-functionalized tip over adherent cells on local nano-domains in Z-direction. Cell features like stiffness and elasticity were investigated.

Methods

Exposure times

The methodology of human neutrophils interaction with titanium dioxide FPs and NPs was adjusted and various exposure times were tested. Once the literature has few information concerning neutrophils interaction with TiO 2 micro, submicron and nanoparticles by AFM, only the first hour were assessed by FS. However during experiments for this study, cellular analysis intervals tested above thirty minutes, resulted in locking the cantilever tip, by preventing to achieve the force curves measurements. Hence the exposition times used were 1, 5 and 30 minutes.

TiO 2 FPs and NPs characterization

Samples of 99.9% pure titanium oxide FPs and NPs (Sigma-Aldrich, TiO 2 reference number 14027, titanium (IV) oxide puriss) were immersed in 1 mL of Milli-Q water using a 5 mL sterilized disposable syringe. Particle hydrodynamic diameter was analyzed by dynamic light scattering (DLS) in triplicate using a Nanotrac U2131I (Microtec Inc., USA, at Instrutécnica, SP). Moreover, TiO 2 FPs and NPs were sized by Transmission Electron Microscopy (TEM) analysis of the dry powder (JEM-2011, JEOL Ltd.) and also by AFM. Particles were sterilized in an autoclave at 121°C for 20 min [49]. Suspensions containing 200 μg/mL TiO 2 FPs and NPs were submitted to ultrasound bath for 30 min before use, aiming to avoid particles aggregation.

Human neutrophil preparation

Neutrophils were isolated from human whole blood using a density gradient technique as described in a previous study [50]. Briefly, 12 mL of peripheral blood were drawn from three healthy individuals with sterile heparinized syringes and not pooled. Collected blood was added to a Percoll density gradient (60%/70%) and centrifuged. After centrifugation, polymorphonuclear cells were harvested from the interface of the two gradients using a Pasteur pipette and re-suspended in 500 μL of Ca-/Mg-free blood buffer in Hank's Balanced Salt Solution (HBSS; Sigma-Aldrich, St. Louis, Missouri Sigma-Aldrich Corp.). The suspension containing 7 × 10 5 cells was divided into six aliquots: three were incubated (1, 5, and 30 min) with 20 μL of the TiO 2 FPs and NPs suspension at 37°C and three were kept as controls (1, 5, and 30 min). After incubation, cells were fixed with 100 μL of Karnovsky solution (12% paraformaldehyde + 8% glutaraldehyde + 0.2 M cacodylate buffer). Once fixed, cells were washed with PBS for 5 min and centrifuged for 2 minutes, three times (4500 g at 4°C). Each cells aliquot (1, 5, and 30 min) was then placed on separated pre-sterilized circular coverslip. For force spectroscopy measurements, only well-adhered cells were selected.

Ethics and consent statement

All the experiments were performed with the approval of the Research Ethics Committee of Medical Faculty, University of Brasilia (registration number: CEP-CFM-45/2010), in compliance with the Helsinki Declaration. Likewise, adult participants in the study (all human subjects) provided informed consent for blood donation.

Scanning electron microscopy (SEM) analyses

Fixed neutrophils were buffered in 0.1 M sodium cacodylate buffer and 0.1 M osmium tetroxide for 1 h, dehydrated and sputter-coated with gold. A SEM microscope (Jeol 840A operating at an accelerating voltage of 20 kV was used to depict possible morphological changes in neutrophils.

Force spectroscopy analyses

Commercial AFM equipment (SPM-9600, Shimadzu, Japan) operated in air contact mode was used to analyze surface interaction forces between the tip atoms and cell membranes. Circular coverslips (10 mm) containing adhered neutrophils of each sample were fixed on a metallic support using a double-sided adhesive tape. A pyramidal silicon nitride tip (curvature radius < 20 nm) integrated with a cantilever with length of 200 μm (spring constant 0.15 N/m and approximately 24 kHz resonance frequency) was used in this technique. The acquisition rate was 1 Hz with 20 V of amplitude and operating point 3 V. At each incubation time point (1, 5, and 30 min) 10 cells from each group (control and TiO 2 ) were analyzed and five force curves per cell were measured, resulting in 300 events, taking into account the control and experimental groups.

Data analysis

SPIP v. 5.1.5 was used to force curve analysis set at 23°C and 0.15 N/m spring constant, cone indentation (Sneddon), with five fitting points and the approach curve as the baseline correction curve. FS data were further analyzed using Origin 8.6 software by ANOVA and the Tukey test (P < 0.05).

Results and discussion

Particle size is a primary parameter when considering the effect of MPs, FPs and NPs in cellular interactions. According to Watari et al. [35], increasing the specific surface area usually implies a micro or nanosizing effect, which enhances chemical reactivity and consequently leads to serious toxicity for soluble and suspended materials. Watari et al. [51] considered particle size biointeractive below cell size, namely approximately 10 μm or smaller. These authors also point out critical sizes related bioreactivity; the micro or nanomaterial critical size means that, below 200 nm, a particle becomes invasive to the organism's structures, leading to a lack of recognition of this material by the immune system [35,51]. Furthermore, TiO 2 particles and nanoparticles aggregation is related to understanding cytotoxicity. It is known that the surface area, length and chemical nature of the TiO 2 MPs and NPs aggregates are related to the cell toxicity [24,27]. It has been shown that nanoparticles are able to promote a more pronounced toxicity effect than microparticles [52]. Once the MPs and NPs have great potential for agglomeration and aggregation it is unlikely to occur cellular exposure to isolated form of this particles [53]. Okuda-Shimazaki et al. [24] demonstrated large (596 nm) TiO 2 aggregates showed a larger effect on cell viability and gene expression when compared with the small aggregates (166 nm). This suggests that particle aggregate size is related to cellular effects. Bermudez et al. [54] exposed rats to inhalation of TiO 2 NPs (21 nm) for the evaluation of pulmonary toxicity. Even with the aerosols formed was possible to detect the presence of particles aggregates with 1.37 μm, which were able to promote cytotoxicity. Figure 1 shows the TiO 2 FPs and NPs aggregates hydrodynamic diameter distributions (A), where approximately 67% of the particles were about 180 nm in hydrodynamic diameter (see Additional file 1: Table S1-Peak Summary). The TEM image showed TiO 2 aggregates with size from NPs up to FPs and MPs (B). Also AFM demonstrated an average diameter below 170 nm with a peak at 115 nm (C). AFM image of TiO 2 aggregates (D). Figure 2 shows SEM images comparing non-exposed neutrophils (A, B, and C) and TiO 2 particles aggregates exposed neutrophils (D, E, and F) after 1 min (A and D), 5 min (B and E), and 30 min (C and F), respectively. TiO 2 FPs and NPs aggregates are seen close to neutrophils ( Figure 2E, white arrow).

It is possible to observe neutrophil morphological changes with increasing exposure time due to the influence of TiO 2 aggregates. Neutrophils left the quiescent state (Figure 2A,B, and C) and transitioned to an activated form, extending their pseudopodia in order to phagocytose TiO 2 aggregates ( Figure 2D,E, and F). Several other studies [15,23,55] have demonstrated similar morphological alterations resulting from TiO 2 FPs and NPs treatment, characterized by increasing cytotoxic biomarker expression (lactate dehydrogenase, superoxide anion, and proinflammatory cytokines), and inversely related to particle size. However, there is a lack of knowledge concerning the nanomechanical modifications resulting from TiO 2 FPs and NPs aggregates exposure. The next step in this study was to evaluate the nanomechanical effects caused by the presence of TiO 2 aggregates on the neutrophil surface at the same time points, using the FS technique.

The FS results were derived from force-distance curves consisting of an AFM tip approximation and detachment from the sample surface. This event was monitored by an optical laser signal, which reflects from the upper reflective surface of the cantilever. Attractive, repulsive, and/or adhesive forces can manifest while this process occurs. The first event related to tip approximation involves attractive forces (such as van der Waals forces and capillarity interactions) and it is known as snap-in, whereupon the tip "jumps" to establish close contact with the sample surface. Figure 3 shows statistically significant differences in snap-in measurements between control and TiO 2 aggregates treated neutrophils after 1 and 5 min of exposure. The treatments caused an increase in snap-in force measurements when compared to the control. These results could be related to morphological changes caused by neutrophil stimulation, as observed in Figure 2. The control cells showed a time-dependent increase in snap-in force values from 1 to 30 min, while TiO 2 aggregates exposed cells showed increased force values from 1 to 5 min. Moreover, statistically significant differences (P < 0.05) were also found between aggregates exposed cells and their respective controls for the 1 and 5 min incubation times, but not at 30 min ( Figure 3). These data may demonstrate that attractive forces like van der Waals and electrostatic forces on the cell membrane are more intense with longer incubation times. The interaction between FPs and NPs aggregates and the neutrophil membrane and uptake by phagocytosis increase the snap-in forces. Such forces seem to plateau at some point near 30 min, where the interaction with particles aggregates no longer enhances the values. Additional experiments with longer exposure times could demonstrate the behaviour of snap in forces along time.

Variations in sample surface mechanical properties such as viscoelasticity and adhesion forces, as well as fixation solutions, also influence cantilever oscillations [56,57]. On the other hand, one should consider that FPs and NP interference with neutrophil membranes is related to surrounded or attached TiO 2 particles. This event could promote interactions with the tip, considering the ability of FPs and NPs to aggregate into numerous large clusters, as seen in Figure 2D and E [58]. For example, a study Guduru [56] demonstrated that bare polylactic-co-glycolic acid (b-PLGA) NPs interacted with ovarian cancer cells (SKOV-3) and after 24 h of incubation, and most of the particles remained on the cell surface. NPs in close contact with the membrane surface may promote a lack of communication with the external environment [26].

The next event occurring in a force curve experiment is associated with the maximum force applied that relates to sample surface micro and nanostiffness. Maximum loading force measurements comparing control and TiO 2 aggregates treated neutrophils showed statistically significant differences, but only at 1 min of exposure ( Figure 4). Interestingly, mechanical assays showed that activated neutrophils were over two-fold stiffer than quiescent cells [59]. Moreover, under load, the membrane deformed in a non-linear elastic manner and demonstrated binary behavior of stiff and slack conditions [60]. Control cells at 30 min had a statistically significant difference to the respective control at 5 min (P < 0.05), suggesting a possible increase in membrane softness in quiescent neutrophils as time passes. Furthermore, when compared to control cells, TiO 2 treated neutrophils at 1 min depicted stronger forces, which could represent increased cell stiffness due to phagocytosis or TiO 2 aggregates adhesion to the cell membrane. It must be considered that, during phagocytosis, activated neutrophils might undergo increasing membrane cortical tension together with progressive cell surface and cytoskeletal structure hardening [61].

Detachment force measurement represents a relationship with adhesion forces (i.e. hydrogen bonds, ionic attractions, van der Waals forces, hydrophobic interactions, and/or chemical adhesion) [62]. This FS parameter occurs last on a retraction curve, corresponding to the point where the tip and surface lose contact, and it is related to adhesive interactions between them [63]. Figure 5 shows statistically significant differences (P < 0.05) between control and TiO 2 treated neutrophils after 1 and 5 min of exposure, evidencing a tendency to increase this mechanical feature after stimulation. Once stimulated, cells acquire a polarized morphology, with F-actin polymerization in which integrins connect the cytoskeleton with the extracellular matrix [56]. Control cells at 1, 5, and 30 min demonstrated a statistically significant difference from the silica coverslip (P < 0.05). Stronger adhesion forces were seen with the glass than with quiescent neutrophil membrane surfaces, which showed that the adhesion forces were not due to the cell support. Similarly, TiO 2 FPs and NPs aggregates treated neutrophils at 1 and 5 min had lower detachment forces than the silica coverslip. Figure 6 shows a comparison of Young's modulus measurements between control and TiO 2 aggregates treated neutrophils. Generically, this parameter relates to a sample's elasticity/mechanical properties. The Young's modulus of treated samples was smaller than that observed for control cells, demonstrating a comparatively higher elastic behavior over a longer period of time. Control and TiO 2 aggregates treated neutrophils at 1 min demonstrated significant  Maximum load force calculated from force spectroscopy curves measured by AFM operating in contact mode. "Blank" refers to bare circular silica coverslip (10 mm) surface analyses, and "TiO 2 1, 5, and 30 min" refer to force curve analyses obtained from fixed neutrophils adhered to circular silica coverslips (10 mm) and exposed to TiO 2 fine particles and nanoparticles aggregates for 1, 5, and 30 min. "Control 1, 5, and 30 min" refer to fixed neutrophils adhered to circular silica coverslips (10 mm) without any TiO 2 fine particles and nanoparticles treatment, collected at the same time as treated samples (1, 5, and 30 min). The (*) symbol indicates a statistically significant difference between "Blank" and samples (P < 0.05), and (a) indicates a statistically significant difference to the respective control and t5 to "Control 5 min" (p < 0.05).

Figure 3

Snap-in force calculated from force spectroscopy curves measured by AFM operating in contact mode. "Blank" refers to bare circular silica coverslip (10 mm) surface analyses, and "TiO 2 1, 5, and 30 min" refer to force curve analyses obtained from neutrophils adhered to circular silica coverslips (10 mm) and exposed to TiO 2 fine particles and nanoparticles aggregates for 1, 5, and 30 min. "Control 1, 5, and 30 min" refer to neutrophils adhered to circular silica coverslips (10 mm) without any TiO 2 fine particles and nanoparticle treatment, collected at the same time as treated samples (1, 5, and 30 min). The (*) symbol indicates a statistically significant difference between "Blank" and samples (P < 0.05), and (a) indicates a statistically significant difference to the respective control, "t1" to "Control 1 min", "at 1 " to "TiO 2 1 min" and "t 1,5 to "Control 1 min" and "Control 5 min" (P < 0.05).

Figure 5

Detach force calculated from force spectroscopy curves measured by AFM operating in contact mode. "Blank" refers to bare circular silica coverslip (10 mm) surface analyses, and "TiO 2 1, 5, and 30 min" refer to force curve analyses obtained from fixed neutrophils adhered to circular silica coverslips (10 mm) and exposed to TiO 2 fine particles and nanoparticles aggregates for 1, 5, and 30 min. "Control 1, 5, and 30 min" refer to fixed neutrophils adhered to circular silica coverslips (10 mm) without any TiO 2 fine particles and nanoparticles treatment, collected at the same time as treated samples (1, 5, and 30 min). The (*) symbol indicates a statistically significant difference between "Blank" and samples (P < 0.05), and (a) indicates a statistically significant difference to the respective control (P < 0.05). differences (P < 0.05). At the first time point, quiescent cells were much stiffer than treated cells. At 5 min, no statistically significant differences were seen. This may be related to cell activation and assembly of the cytoskeleton in the TiO 2 group, thus increasing cell hardness. Furthermore, a remarkable transition occurred at 30 min. Treated neutrophils showed a statistically significant difference (P < 0.05), where it could be seen that intense phagocytosis and cell stiffening opposed the indentation of the cantilever tip. Similar results were found by Roca-Cusachs and coworkers who described adhered/activated neutrophils as being stiffer than quiescent cells [59].

Cell image analyses at 5 and 30 min ( Figure 2E and F) showed remarkable changes in morphology, representing possible stimulation, F-actin rearrangement and migration to mediate TiO 2 FPs and NPs aggregates phagocytosis. Figure 6 shows that quiescent cells and control cells had a strict time-dependent decay pattern. Control cells at 5 and 30 min showed statistically significant differences to 1 min (P < 0.05). These features points to a quiescent cell elasticity modulus that decreased throughout the time course [36]. By transposing into a biological environment, cell deformation implies high stiffness, favoring neutrophil arrest at the capillary wall and subsequent adhesion. On the other hand, cell softening and slow deformation would facilitate the neutrophil transmigration progress [64].

Finally, Figure 7 shows cells the dissipated energy behavior over time. Dissipated energy results from tip and sample interactions as a function of either the gap distance or applied bias [65]. Vorden et al. [66] described non-conservative forces as being strongly dependent on these interactions. Energy dissipation can be related to sample viscoelastic properties [67]. A major contributor to energy dissipation is related to AFM tip/cell membrane contact with the lipid bilayer and cytoskeleton. Neutrophils do not have a homogeneous surface due to their complex inner composition, including a nucleus (thicker cell region by~5 μm), cytoplasm, and cytoskeleton [57]; thus, different force indentation measurements are expected [44]. The cytoskeleton is known to consist of actin filaments, microtubules, and intermediate filaments with many accessory proteins [68]. In response to TiO 2 stimulation, neutrophils remodel the cytoskeleton and increase cell rigidity, probably contributing to the non-linear and time-dependent mechanical properties. Dissipated energy measurements of TiO 2 FPs and NPs aggregates treated neutrophils showed a strikingly increased tendency over time. A statistically significant difference (P < 0.05) between control and TiO 2 aggregates treated neutrophils after 1 and 5 min of exposure was found, which may correspond to the activation of neutrophils and account for the energy dissipation enhancement as MPs and NPs uptake by Figure 6 Young's modulus calculated from force spectroscopy analysis measured by AFM operating in contact mode. "Blank" refers to bare circular silica coverslip (10 mm) surface analyses, and "TiO 2 1, 5, and 30 min" refer to force curve analyses obtained from fixed neutrophils adhered to circular silica coverslips (10 mm) and exposed to TiO 2 nanoparticles for 1, 5, and 30 min. "Control 1, 5, and 30 min" refer to fixed neutrophils adhered to circular silica coverslip (10 mm) without any TiO 2 fine particles and nanoparticles treatment, collected at the same time as treated samples (1, 5, and 30 min). The (*) symbol indicates a statistically significant difference between "Blank" and samples (P < 0.05), and (a) indicates a statistically significant difference to the respective control and t1 to "Control 1 min" (P < 0.05).

Figure 7

Dissipated energy calculated from force spectroscopy analysis measured by AFM operating in contact mode. "Blank" refers to bare circular silica coverslip (10 mm) surface analyses, and "TiO 2 1, 5, and 30 min" refer to force curve analyses obtained from fixed neutrophils adhered to circular silica coverslips (10 mm) and exposed to TiO 2 nanoparticles for 1, 5 and 30 min. "Control 1, 5, and 30 min" refer to fixed neutrophils adhered to circular silica coverslips (10 mm) without any TiO 2 fine particles and nanoparticles treatment, collected at the same time as treated samples (1, 5, and 30 min). The (*) symbol indicates a statistically significant difference between "Blank" and samples (P < 0.05), and (a) indicates a statistically significant difference to the respective control and t 1,5 to "Control 1 min" and "Control 5 min" (P < 0.05). cells occurred and rigidity increased. Focusing on the controls, the statistical analysis showed significant differences (P < 0.05) between 30 min and both 1 and 5 min control cells. It is possible that quiescent neutrophils could experience some degree of cytoskeletal organization and therefore increasing rigidity along the time course, which would increase the dissipation of energy when the tip is in close contact with the sample.

Conclusions

The present study evaluated the effects of TiO 2 FPs and NPs aggregates on neutrophil nanomechanical properties along a time course. Scanning electron microscopy was used to investigate cell morphological changes. Aiming to correlate the morphological and nanomechanical outcomes, force spectroscopy analyses were performed. To assess the attractive and adhesive interaction patterns between AFM tip and cell surface atoms, parameters like snap-in, detachment, and maximum load force were evaluated, as well as the elasticity modulus (Young's modulus) and dissipated energy. Cells treated with TiO 2 FPs and NPs aggregates showed an increase in attractive force measurements at initial exposure times compared with control cells, which may be related to the AFM tip interaction with activated neutrophils as well as with FPs and NPs aggregates on the cell membrane. Similar results were found for adhesion forces and dissipated energy measurements. Treated neutrophils showed stronger stiffness features than controls at 1 min of exposure and presented comparatively higher elastic behavior for a longer period of time. These results point to increased cellular nanostiffness in response to TiO 2 FPs and NP aggregates treatment. This suggests an intrinsic connection concerning neutrophil morphological alterations with important biological events, such as adhesion and transmigration. A more complete understanding of these interactions will be an indispensable step to elucidate the mechanisms involved in treatments containing nanomaterials, how they interfere with modulation pathways, and the effect on organisms.

Additional file

Additional file 1: Table S1. Peak summary related to the hydrodynamic diameter distributions of TiO 2 fine particles and nanoparticles.

Abbreviations

Competing interests

The author declares that there are no competing interests.

Acknowledgments

The author would like to express deepest gratitude to the Embrapa Genetic Resources and Biotechnology Laboratory of Mass Spectrometry (NTBio), Nanotechnology Room for AFM data acquisition. Thanks also to Prof. Luciano Paulino Silva PhD, and Eduardo Fernandes Barbosa, a PhD student, who kindly shared their expertise on AFM and the nanotechnology field and allowed free access to the available infrastructure, an also shared their experience and knowledge in the preparation of the proposal, execution of experiments, and critical discussion of the manuscript. Many thanks to Prof. Wagner Fontes from the Laboratory of Biochemistry and Protein Chemistry, University of Brasília, for planning the biological experiments and providing critical discussion of the manuscript. Finally, I must express my heartfelt thanks to Prof. José Raimundo Corrêa from the Electron Microscopy Laboratory (Brasilia University) for scanning electron microscopy data acquisition and discussions.

Figure 1
1Characterization of TiO 2 FPs and NPs by DLS, TEM and AFM. Hydrodynamic diameter distributions of TiO 2 particles measured by dynamic light scattering (DLS) analysis (A). TiO 2 fine particles and nanoparticles characterized by TEM (B). Fine particles and nanoparticles (aggregates) average diameter by AFM (C). AFM image of TiO 2 aggregates (D).

Figure 2
2SEM images of controls and TiO2 FPs and NPs aggregates exposed neutrophils. Images of untreated neutrophils or control (A, B, and C) and TiO 2 fine particles and nanoparticles clusters-exposed neutrophils (D, E, and F) obtained by scanning electron microscopy (SEM) analyses. A and D refer to cells collected after 1 min of exposure, B and E after 5 min, and C and F after 30 min.

Figure 4
4Figure 4 Maximum load force calculated from force spectroscopy curves measured by AFM operating in contact mode. "Blank" refers to bare circular silica coverslip (10 mm) surface analyses, and "TiO 2 1, 5, and 30 min" refer to force curve analyses obtained from fixed neutrophils adhered to circular silica coverslips (10 mm) and exposed to TiO 2 fine particles and nanoparticles aggregates for 1, 5, and 30 min. "Control 1, 5, and 30 min" refer to fixed neutrophils adhered to circular silica coverslips (10 mm) without any TiO 2 fine particles and nanoparticles treatment, collected at the same time as treated samples (1, 5, and 30 min). The (*) symbol indicates a statistically significant difference between "Blank" and samples (P < 0.05), and (a) indicates a statistically significant difference to the respective control and t5 to "Control 5 min" (p < 0.05).

TiO 2 :
2Titanium dioxide; MPs: Microparticles; FPs: Fine particles; NPs: Nanoparticles; SPM: Scanning probe microscopy; AFM: Atomic force microscopy; SEM: Scanning electron microscopy; PSGL-1: P-selectin glycoprotein ligand-1; LFA-1: Lymphocyte function-associated antigen 1; Mac-1: Macrophage-1 antigen; FcγIII receptor: Low affinity immunoglobulin gamma Fc region receptor III; CD11b: Cluster of differentiation 11b; CD16: Cluster of differentiation 16; ICAM-1: Intercellular adhesion molecule-1; min.: minute; N/m: Newton per meter; nm: nanometer; μL: microliter; DLS: Dynamic light scattering; HBSS: Hank's balanced salt solution; PBS: Phosphate buffered saline solution; MTC: Magnetic twisting cytometry; kHz: Kilohertz; Hz: Hertz; V: Volt.
http://www.biomedcentral.com/2046-1682/6/11

The influence of fat substitutes based on protein and titanium dioxide on the sensory properties of low fat milk. L Phillips, D Barbano, J Dairy Sci. 80112726Phillips L, Barbano D: The influence of fat substitutes based on protein and titanium dioxide on the sensory properties of low fat milk. J Dairy Sci 1997, 80(11):2726.

Titanium: A Technical Guide, in Titanium: A Technical Guide. M Donachie, ASM InternationalMetals Park OhioDonachie M: Titanium: A Technical Guide, in Titanium: A Technical Guide. Metals Park Ohio: ASM International; 1988.

Enhanced functions of vascular cells on nanostructured Ti for improved stent applications. S Choudhary, K Haberstroh, T Webster, Tissue Eng. 137Choudhary S, Haberstroh K, Webster T: Enhanced functions of vascular cells on nanostructured Ti for improved stent applications. Tissue Eng 2007, 13(7):1421-1430.

Osseointegration and its experimental studies. P-I Branemark, J Prosthetic Dentistry. 50Branemark P-I: Osseointegration and its experimental studies. J Prosthetic Dentistry 1983, 50:399-410.

Surface analysis of titanium maxillofacial plates and screws retrieved from patients. R Langford, J Frame, Int J Oral Maxillofac Surg. 315Langford R, Frame J: Surface analysis of titanium maxillofacial plates and screws retrieved from patients. Int J Oral Maxillofac Surg 2002, 31(5):511-518.

Black staining around titanium alloy prostheses an orthopaedic enigma. J Scales, J Bone Joint Surg Br. 73Scales J: Black staining around titanium alloy prostheses an orthopaedic enigma. J Bone Joint Surg Br 1991, 73:534-536.

Metal wear and tissue response in failed titanium alloy total hip replacements. J Witt, W Swann, J Bone Joint Surg Br. 73Witt J, Swann W: Metal wear and tissue response in failed titanium alloy total hip replacements. J Bone Joint Surg Br 1991, 73:559-563.

A retrieval study on morphological and chemical changes of titanium osteosynthesis plates and adjacent tissues. N Lygidakis, J Cranio-Maxillofacial Surgery. 353Lygidakis N, et al: A retrieval study on morphological and chemical changes of titanium osteosynthesis plates and adjacent tissues. J Cranio-Maxillofacial Surgery 2007, 35(3):168-176.

Wide spread dissemination of metal debris from implants. C Case, J Bone Joint Surg Br. 76Case C, et al: Wide spread dissemination of metal debris from implants. J Bone Joint Surg Br 1994, 76:701-712.

Titanium levels in the organs and blood of rats with a titanium implant, in the absence of wear, as determined by double-focusing ICP-MS. A Sarmiento-González, J R Encinar, J M Marchante-Gayón, A Sanz-Medel, Anal Bioanal Chem. 3931Sarmiento-González A, Encinar JR, Marchante-Gayón JM, Sanz-Medel A: Titanium levels in the organs and blood of rats with a titanium implant, in the absence of wear, as determined by double-focusing ICP-MS. Anal Bioanal Chem 2009, 393(1):335-343.

Titanium". Nature' s Building Blocks: An A-Z Guide to the Elements. J Emsley, Oxford University PressOxford England, UKEmsley J: "Titanium". Nature' s Building Blocks: An A-Z Guide to the Elements. Oxford England, UK: Oxford University Press; 2001.

Method for immunolocalization of extracellular proteins in association with the implant-soft tissue interface. A Rosengren, Biomaterials. 15Rosengren A, et al: Method for immunolocalization of extracellular proteins in association with the implant-soft tissue interface. Biomaterials 1994, 15:17-24.

Análise proteômica comparativa entre neutrófilos quiescentes e estimulados com fator de agregação plaquetária (PAF). E Aquino, Biology Departament. 149University of BrasiliaMaster Degree DissertationAquino E: Análise proteômica comparativa entre neutrófilos quiescentes e estimulados com fator de agregação plaquetária (PAF). Master Degree Dissertation. University of Brasilia, Biology Departament; 2008:149.

Interactions between human whole blood and modified TiO 2 -surfaces: influence of surface topography and oxide thickness on leukocyte adhesion and activation. C Eriksson, J Lausmaa, H Nygren, Biomaterials. 2214Eriksson C, Lausmaa J, Nygren H: Interactions between human whole blood and modified TiO 2 -surfaces: influence of surface topography and oxide thickness on leukocyte adhesion and activation. Biomaterials 2001, 22(14):1987-1996.

Effects of micro/nano particle size on cell function and morphology. K Tamura, Key Eng Mater. Tamura K, et al: Effects of micro/nano particle size on cell function and morphology. Key Eng Mater 2004, 254-256:919-922.

Induction of cell death by TiO 2 nanoparticles: studies on a human monoblastoid cell line. C Vamanu, Toxicol In Vitro. 22Vamanu C, et al: Induction of cell death by TiO 2 nanoparticles: studies on a human monoblastoid cell line. Toxicol In Vitro 2008, 22:1689-1696.

Internalisation of engineered nanoparticles into mammalian cells in vitro: influence of cell type and particle properties. W Busch, J Nanoparticle Res. 131Busch W, et al: Internalisation of engineered nanoparticles into mammalian cells in vitro: influence of cell type and particle properties. J Nanoparticle Res 2011, 13(1):293-310.

Ultrafine particulate pollutants induce oxidative stress and mitochondrial damage. N Li, Environ Health Perspect. 111Li N, et al: Ultrafine particulate pollutants induce oxidative stress and mitochondrial damage. Environ Health Perspect 2003, 111:455-460.

Ultrafine particles cross cellular membranes by nonphagocytic mechanisms in lungs and in cultured cells. M Geiser, Environ Health Perspect. 11311Geiser M, et al: Ultrafine particles cross cellular membranes by nonphagocytic mechanisms in lungs and in cultured cells. Environ Health Perspect 2005, 113(11):1555-1560.

Activation of human neutrophils by titanium dioxide (TiO2) nanoparticles. D M Gonçalves, S Chiasson, D Girard, Toxicol In Vitro. 243Gonçalves DM, Chiasson S, Girard D: Activation of human neutrophils by titanium dioxide (TiO2) nanoparticles. Toxicol In Vitro 2010, 24(3):1002-1008.

Titanium dioxide (TiO2) nanoparticles induce neutrophil influx and local production of several pro-inflammatory mediators in vivo. D M Gonçalves, D Girard, Int Immunopharmacol. 118Gonçalves DM, Girard D: Titanium dioxide (TiO2) nanoparticles induce neutrophil influx and local production of several pro-inflammatory mediators in vivo. Int Immunopharmacol 2011, 11(8):1109-1115.

Function of the cytoskeleton in human neutrophils and methods for evaluation. M Torres, T Coates, J Immunol Methods. 2321-2Torres M, Coates T: Function of the cytoskeleton in human neutrophils and methods for evaluation. J Immunol Methods 1999, 232(1-2):89-109.

Effects of Ti ions and particles on neutrophil function and morphology. R Kumazawa, Biomaterials. 23Kumazawa R, et al: Effects of Ti ions and particles on neutrophil function and morphology. Biomaterials 2002, 23:3757-3764.

Effects of titanium dioxide nanoparticle aggregate size on gene expression. J Okuda-Shimazaki, Int J Mol Sci. 116Okuda-Shimazaki J, et al: Effects of titanium dioxide nanoparticle aggregate size on gene expression. Int J Mol Sci 2010, 11(6):2383-2392.

Estudo do Comportamento de nanopartículas de Dióxido de Titânio em diferentes suspensões. J Pereira, 94LisboaUniversidade de LisboaPereira J: Estudo do Comportamento de nanopartículas de Dióxido de Titânio em diferentes suspensões. Lisboa: Universidade de Lisboa; 2010:94.

Cytotoxicity of colloidal CdSe and CdSe/ZnS nanoparticles. C Kirchner, Nano Lett. 52Kirchner C, et al: Cytotoxicity of colloidal CdSe and CdSe/ZnS nanoparticles. Nano Lett 2005, 5(2):331-338.

Inflammatory response of mice to manufactured titanium dioxide nanoparticles: comparison of size effects through different exposure routes. V Grassian, Nanotoxicology. 13Grassian V, et al: Inflammatory response of mice to manufactured titanium dioxide nanoparticles: comparison of size effects through different exposure routes. Nanotoxicology 2007, 1(3):211-226.

H Arora, Nanomaterials for the Life Sciences in Nanocomposites. C.S.S.R. Kumar. WeinheimWILEY-VCH Verlag GmbH & Co. KGaAArora H, et al: In Nanomaterials for the Life Sciences in Nanocomposites. Edited by C.S.S.R. Kumar. Weinheim: WILEY-VCH Verlag GmbH & Co. KGaA; 2010.

Mechanisms and consequences of neutrophil interaction with the endothelium. A Zarbock, K Ley, Am J Pathol. 1721Zarbock A, Ley K: Mechanisms and consequences of neutrophil interaction with the endothelium. Am J Pathol 2008, 172(1):1-7.

The signaling pathways induced by neutrophil-endothelial cell adhesion. Q Wang, C Doerschuk, Antioxid Redox Signal. 41Wang Q, Doerschuk C: The signaling pathways induced by neutrophil-endothelial cell adhesion. Antioxid Redox Signal 2002, 4(1):39-47.

Neutrophils: molecules, functions and pathophysiological aspects. V Witko-Sarsat, P Rieu, B Descamps-Latscha, P Lesavre, L Halbwachs-Mecarelli, Lab Invest. 80Witko-Sarsat V, Rieu P, Descamps-Latscha B, Lesavre P, Halbwachs-Mecarelli L: Neutrophils: molecules, functions and pathophysiological aspects. Lab Invest 2000, 80:617-653.

Titanium dioxide (P25) produces reactive oxygen species in immortalized brain microglia (BV2): implications for nanoparticle neurotoxicity. T Long, Environ Sci Technol. 4014Long T, et al: Titanium dioxide (P25) produces reactive oxygen species in immortalized brain microglia (BV2): implications for nanoparticle neurotoxicity. Environ Sci Technol 2006, 40(14):4346-4352.

Topographical control of human neutrophil motility on micropatterned materials with various surface chemistry. J Tan, W Saltzman, Biomaterials. 23Tan J, Saltzman W: Topographical control of human neutrophil motility on micropatterned materials with various surface chemistry. Biomaterials 2002, 23:3215-3225.

Adhesion receptors of PMN granulocyteson titanium in contact with whole blood. C Eriksson, H Nygren, J Lab Clin Med. 137Eriksson C, Nygren H: Adhesion receptors of PMN granulocyteson titanium in contact with whole blood. J Lab Clin Med 2001, 137:56-63.

Material nanosizing effect on living organisms: non-specific, biointeractive, physical size effects. F Watari, J R Soc Interface. 6Watari F, et al: Material nanosizing effect on living organisms: non-specific, biointeractive, physical size effects. J R Soc Interface 2009, 6:S371-S388.

Atomic force microscopy probing of cell elasticity. T Kuznetsova, Micron. 388Kuznetsova T, et al: Atomic force microscopy probing of cell elasticity. Micron 2007, 38(8):824-833.

Mechanics and deformation of the nucleus in micropipette aspiration experiment. A Vaziri, M Kaazempur, J Biomech. 40Vaziri A, Kaazempur M: Mechanics and deformation of the nucleus in micropipette aspiration experiment. J Biomech 2007, 40:2053-2062.

Time scale and other invariants of integrative mechanical behavior in living cells. B Fabry, Phys Rev E. 6841914Fabry B, et al: Time scale and other invariants of integrative mechanical behavior in living cells. Phys Rev E 2003, 68:041914.

Applied scanning probe methods I. B Bhushan, S Kawata, S Hosaka, Bhushan BSpringer-VerlagFuchs H, Harald. Berlin-Heidelberg; Berlin HeidelbergBhushan B, Kawata S, Hosaka S: Applied scanning probe methods I. In NanoScience and Technology, P.A.B.B.D.B.K.v.H.S. R.Wiesendanger. Edited by Bhushan B, Fuchs H, Harald. Berlin-Heidelberg: Springer-Verlag Berlin Heidelberg; 2007.

K Hayashi, Tensile Properties and Local Stiffness of Cells in Mechanics of Biological Tissue, R.W.O. Edited by Holzapfel GA. Heidelberg; BerlinSpringerHayashi K: In Tensile Properties and Local Stiffness of Cells in Mechanics of Biological Tissue, R.W.O. Edited by Holzapfel GA. Heidelberg: Springer Berlin; 2006:137-152.

Integrin-specific mechanoresponses to compression and extension probed by cylindrical flat-ended AFM tips in lung cells. I Acerbi, PLoS One. 7232261Acerbi I, et al: Integrin-specific mechanoresponses to compression and extension probed by cylindrical flat-ended AFM tips in lung cells. PLoS One 2012, 7(2):e32261.

Atomic force microscopy and spectroscopy. Y Seo, W Jhe, Rep Prog Phys. 71116101Seo Y, Jhe W: Atomic force microscopy and spectroscopy. Rep Prog Phys 2008, 71(1):016101.

Force measurements with the atomic force microscope: Technique, interpretation and applications. H Butt, B Cappella, M Kappl, Surf Sci Rep. 59Butt H, Cappella B, Kappl M: Force measurements with the atomic force microscope: Technique, interpretation and applications. Surf Sci Rep 2005, 59:1-152.

Determining the elastic modulus of biological samples using atomic force microscopy. T Neumann, JPK.Instruments. AG. Neumann T: Determining the elastic modulus of biological samples using atomic force microscopy. In Edited by JPK.Instruments. AG, Berlin; 2009.

Local rheology of human neutrophils investigated using atomic force microscopy. Y Lee, D Patel, S Park, Int J Biol Sci. 71Lee Y, Patel D, Park S: Local rheology of human neutrophils investigated using atomic force microscopy. Int J Biol Sci 2011, 7(1):102-111.

R Agrawal, K Lee, R Agrawal, K Lee, B: Afm Ponnavolu, Spectroscopy, Micro/Nano Science and Engineering. Evanston-IL,USADepartment of Mechanical Engineering: Northwestern UniversityAgrawal R, Lee K, Agrawal R, Lee K, Ponnavolu B: AFM Spectroscopy, in Micro/Nano Science and Engineering. Evanston-IL,USA: Department of Mechanical Engineering: Northwestern University; 2004.

Atomic force microscopy for high-resolution imaging in cell biology. J Hoh, P Hansma, Trends Cell Biol. 2208213Hoh J, Hansma P: Atomic force microscopy for high-resolution imaging in cell biology. Trends Cell Biol 1992, 2(208):213.

Force microscopy of nonadherent cells: a comparison of leukemia cell deformability. M Rosenbluth, W Lam, D Fletcher, Biophys J. 908Rosenbluth M, Lam W, Fletcher D: Force microscopy of nonadherent cells: a comparison of leukemia cell deformability. Biophys J 2006, 90(8):2994-3003.

Standard Practice for Surface Preparation and Marking of metallic surgical Implants. Astm-F86-04, Publisher-American Society for Testing and MaterialsWest Conshohocken, PAASTM-F86-04: Standard Practice for Surface Preparation and Marking of metallic surgical Implants. West Conshohocken, PA: Publisher-American Society for Testing and Materials; 2009.

Comparison of the neutrophil proteome in trauma patients and normal controls. L Teles, Protein Pept Lett. 196Teles L, et al: Comparison of the neutrophil proteome in trauma patients and normal controls. Protein Pept Lett 2012, 19(6):663-672.

Internal diffusion of micro/nanoparticles inside body. F Watari, Key Eng Mater. Watari F, et al: Internal diffusion of micro/nanoparticles inside body. Key Eng Mater 2007, 361-363:95-98.

Acute toxicity and biodistribution of different sized titanium dioxide particles in mice after oral administration. J Wang, Toxicol Lett. 168Wang J, et al: Acute toxicity and biodistribution of different sized titanium dioxide particles in mice after oral administration. Toxicol Lett 2007, 168:176-185.

Identification of the mechanisms that drive the toxicity of TiO 2 particulates: the contribution of physicochemical characteristics. H Johnston, Part Fibre Toxicol. 633Johnston H, et al: Identification of the mechanisms that drive the toxicity of TiO 2 particulates: the contribution of physicochemical characteristics. Part Fibre Toxicol 2009, 6(33):1-27.

Pulmonary responses of mice, rats, and hamsters to subchronic inhalation of ultrafine titanium dioxide particles. E Bermudez, Toxicol Sci. 772Bermudez E, et al: Pulmonary responses of mice, rats, and hamsters to subchronic inhalation of ultrafine titanium dioxide particles. Toxicol Sci 2004, 77(2):347-357.

Effects of particle size on cell function and morphology in titanium and nickel. K Tamura, Mater Trans. 4312Tamura K, et al: Effects of particle size on cell function and morphology in titanium and nickel. Mater Trans 2002, 43(12):3052-3057.

R Guduru, situ AFM Imaging of Nanoparticle-Cellular Membrane Interaction for a Drug Delivery Study. Miami-FloridaFIU Electronic Thesis and DissertationsPaper 422Guduru R: In situ AFM Imaging of Nanoparticle-Cellular Membrane Interaction for a Drug Delivery Study. Miami-Florida: FIU Electronic Thesis and Dissertations; 2011. Paper 422.

B Smith, Cellular Biomechanics Investigated by Atomic Force Microscopy. Centre for the Physics of Materials, Department of Physics. Montréal: McGill UniversitySmith B: Cellular Biomechanics Investigated by Atomic Force Microscopy, in Centre for the Physics of Materials, Department of Physics. Montréal: McGill University; 2004.

Single nanoparticle spectroscopy: an analysis of sample preparation and microscopy techniques. W Hall, Nanoscape. 21Hall W, et al: Single nanoparticle spectroscopy: an analysis of sample preparation and microscopy techniques. Nanoscape 2005, 2(1):35-41.

Rheology of passive and adhesion-activated neutrophils probed by atomic force microscopy. P Roca-Cusachs, Biophys J. 919Roca-Cusachs P, et al: Rheology of passive and adhesion-activated neutrophils probed by atomic force microscopy. Biophys J 2006, 91(9):3508-3518.

Development of a model to explain the effect of variable membrane compliance on single molecule adhesive bond force. B Maddigan, Massachusetts USA; Massachusetts Institute of technologyMaddigan B: Development of a model to explain the effect of variable membrane compliance on single molecule adhesive bond force. Massachusetts USA: Massachusetts Institute of technology; 2004.

Mechanics of neutrophil phagocytosis: behavior of the cortical tension. M Herant, V Heinrich, M Dembo, J Cell Sci. 1118Pt 9Herant M, Heinrich V, Dembo M: Mechanics of neutrophil phagocytosis: behavior of the cortical tension. J Cell Sci 2005, 1(118(Pt 9)):1789-1797.

Adhesion: molecules and mechanics. K Kendall, Science. 2635154Kendall K: "Adhesion: molecules and mechanics". Science 1994, 263(5154):1720-1725.

Nanoscale characterization of synthetic polymeric porous membranes: Scrutinizing their stiffness, roughness, and chemical composition. E Barbosa, L P Silva, J Membr Sci. Barbosa E, Silva LP: Nanoscale characterization of synthetic polymeric porous membranes: Scrutinizing their stiffness, roughness, and chemical composition. J Membr Sci 2012, 407-408:128-135.

Neutrophil microrheology probed by atomic force microscopy. P Roca-Cusachs, FASEB J. 201296Roca-Cusachs P, et al: Neutrophil microrheology probed by atomic force microscopy. FASEB J 2006, 20(A):1296.

Stomp R: Dissipative and Electrostatic Force Spectroscopy of InAs Quantum Dots by Non-contact Atomic Force Microscopy. 131Centre for the Physics of Materials Department of Physics McGill University Montrèal. Quebec-Canada: McGill UniversityStomp R: Dissipative and Electrostatic Force Spectroscopy of InAs Quantum Dots by Non-contact Atomic Force Microscopy, in Centre for the Physics of Materials Department of Physics McGill University Montrèal. Quebec-Canada: McGill University; 2005:131.

Energy dissipation in dynamic force spectroscopy of PTCDA on Ag-Si(111) √3 × √3 e-J. D Vorden, M Lange, R Moller, Surf Sci Nanotech. 9Vorden D, Lange M, Moller R: Energy dissipation in dynamic force spectroscopy of PTCDA on Ag-Si(111) √3 × √3 e-J. Surf Sci Nanotech 2011, 9:21-25.

Imaging and Stiffness Probing of Neutrophil Using Atomic Force Microscope. Tna Fok, J Tang, Providence, RIHong Kong: Department of Physics, The Chinese University of Hong Kong Physics Department, Brown UniversityFok TNA, Tang J: Imaging and Stiffness Probing of Neutrophil Using Atomic Force Microscope. Hong Kong: Department of Physics, The Chinese University of Hong Kong Physics Department, Brown University, Providence, RI; 2008.

The forces behind cell movement. R Ananthakrishnan, A Ehrlicher, 10.1186/2046-1682-6-11Int J Biol Sci. 35Ananthakrishnan R, Ehrlicher A: The forces behind cell movement. Int J Biol Sci 2007, 3(5):303-317. doi:10.1186/2046-1682-6-11

Cite this article as: da Rosa: Kinetic effects of TiO 2 fine particles and nanoparticles aggregates on the nanomechanical properties of human neutrophils assessed by force spectroscopy. BMC Biophysics. 611Cite this article as: da Rosa: Kinetic effects of TiO 2 fine particles and nanoparticles aggregates on the nanomechanical properties of human neutrophils assessed by force spectroscopy. BMC Biophysics 2013 6:11.


Comparing the Performance of Bread and Breakfast Cereals, Dairy, and Meat in Nutritionally Balanced and Sustainable Diets
June 2018

Marta Wilton Vasconcelos
Hettie Carina Schönfeldt
Ricardo Jorge
Marcelo Tyszler email@example.com
Gerard F H Kramer
Blonk Consultants
GoudaNetherlands

Elsa V Martinez
Blonk Consultants
GoudaNetherlands

Namy D Espinoza-Orias
Nestlé Research Center
LausanneSwitzerland

Karen A Cooper
Nestlé Research Center
LausanneSwitzerland

Marcelo Tyszler
Blonk Consultants
GoudaNetherlands

Hans Blonk
Blonk Consultants
GoudaNetherlands

Universidade Católica Portuguesa
Portugal

South Africa Tânia Gonçalves Albuquerque, Instituto Nacional de Saúde Doutor
University of Pretoria
Portugal

ESB-UCP
Portugal

Comparing the Performance of Bread and Breakfast Cereals, Dairy, and Meat in Nutritionally Balanced and Sustainable Diets

Frontiers in Nutrition | www.frontiersin.org
151June 201810.3389/fnut.2018.00051Received: 25 August 2017 Accepted: 17 May 2018ORIGINAL RESEARCH Edited by: Reviewed by: *Correspondence: Specialty section: This article was submitted to Nutrition and Environmental Sustainability, a section of the journal Frontiers in Nutrition Citation: Kramer GFH, Martinez EV, Espinoza-Orias ND, Cooper KA, Tyszler M and Blonk H (2018) Comparing the Performance of Bread and Breakfast Cereals, Dairy, and Meat in Nutritionally Balanced and Sustainable Diets. Front. Nutr. 5:51.sustainable dietsquadratic optimizationnutrient balanceenvironmental impactbreadbreakfast cerealsdairymeat
Objective: To quantify the performance of food products in a sustainable diet based on the balance of their contribution to nutrient intake and environmental impact, within the context of the Dutch diet.Design: While fixing the quantity of a specific food group at different levels, optimized diets that met nutrient requirements and stayed as close as possible to the current Dutch diet were calculated, in order to understand its potential environmental impact and its nutritional quality. Bread & breakfast cereals, dairy, and meat were compared between 0 and 250% of current intake. Their performance is expressed in the relationship between the quantity of these food products and (1) the environmental impact of diets and (2) the nutrient balance of the diets.Setting: The Netherlands.Subjects: Women aged 31-50.Results: The amount of bread & breakfast cereals in the optimized diets were inversely correlated with their environmental impact. The nutrient balance of the optimized diets was maintained despite varying cereal content, with the expected improvement over the current diet. Increasing amounts of dairy in the optimized diet were associated with an increase in environmental impact and meat with a steep increase. The nutrient balance of optimized diets with varying dairy and meat contents was also maintained at high levels, even at 0% content.Conclusions: Bread and breakfast cereals are sources of nutrients with a better environmental performance compared to dairy or meat within the context of the Dutch diet. It is possible to optimize diets for environmental impact whilst maintaining a high nutrient balance.

INTRODUCTION

It is evident that the food system has an important role in global greenhouse gas emissions (1,2) and the depletion of natural resources (3). This has led to an increased scientific interest in the sustainability of diets. It is widely recognized that many diets need to change to reduce the environmental impact (4,5). How this can be done, while respecting nutritional quality and local dietary habits, is the focus of an increasing number of studies (6)(7)(8). These studies showed that the performance of specific food products in sustainable diets is determined by the balance between their nutritional quality and environmental impact.

In particular, for food manufacturing companies, it is important to know how their current food products contribute to a sustainable diet and how the performance of new food products can be optimized. Several metrics have been suggested to capture this integral performance (9,10) ideally combining a metric for nutritional quality or nutrient density with environmental metrics from Life Cycle Assessment (LCA).

Methodology in this area is evolving rapidly. We contribute to this developing research field by presenting a method that evaluates the balance between nutritional quality and environmental impact within the context of a total diet. Relative to the current diet, we show the effects of increasing and decreasing the intake of a specific food product or food group on the environmental impact of the total diet. This is done by optimizing the current diet iso-calorically so that nutritional requirements are met while holding the amounts of the food (group) of interest fixed at different levels. Consequently, other food products in the diet will either be replaced or have their amounts changed. If an optimized diet with increased amounts from a food (group) of interest has a lower environmental impact and a better nutrient profile, the food (group) can be regarded as a source of nutrients with a better environmental performance than the food products replaced or changed. We optimize the diets by minimizing the changes to the current diet (11,12) until all nutrient requirements are met. We use a current Dutch diet as starting point and examine the performance of three food groups: bread and breakfast cereals, dairy, and meat. These three groups are key food groups in the average Dutch diet and have recently been subject to question about their role in a sustainable and/or healthy diet in both scientific and gray literature. Dairy and meat are typically questioned about their high environmental impact, even though they are sources of high biological value proteins, while bread & breakfast cereals have positive recommendation from public health authorities (13), in contrast with negative recommendation from popular media (14,15).

The above exercise revealed that bread and breakfast cereals has the best environmental performance of the three groups, whilst maintaining a good nutrient balance. The exercise was not exhaustive and did not compare the performance other groups, which could be an extension of the present study.

METHODS

Starting from a current diet, the amounts of a food group of interest were fixed in varying levels in steps of 25% between 0 and 250% of the quantity in the current diet. In each of the 11 steps, after fixing the amount of the food group of interest, the diet was iso-calorically optimized. Notice that within the food group of interest changes are also allowed, as long as the total mass in grams stays at the set level. In the case of dairy, the current diet ratio of liquid dairy products to cheese was kept constant at 8.76 g:1 g in order to avoid artificial environmental improvements by substituting cheese for milk. The exercise resulted in 11 iso-caloric optimized diets satisfying nutritional requirements and with associated nutritional, environmental, and optimization metrics.

We detail below the elements of the optimization and metrics computed.

Current Diet

An average weekly diet (current diet) for Dutch women aged 31-50 was derived from the Dutch National Food Consumption Survey (DNFCS, 2007-2010) (16,17). The procedure is described elsewhere (18). The analysis included a database of a total of 208 food products containing both nutritional composition (19) and environmental impacts. These are representative for food products consumed nowadays in the Netherlands (20)(21)(22), excluding brands. The list contained a limited number of fortified products, such as soy drink, meat replacer, and (iodine fortified) bread. Cereals included were mainstream food products and not fortified (19).

Environmental Impacts

Life Cycle Assessment (LCA) methodology (23) was applied to calculate the environmental impacts (20,24) [Greenhouse Gas Emissions (GHGe), Fossil Energy Use (FEU), and Land Occupation (LO)] associated with each of the 208 food products and the optimized diets. The source database is of high quality and been reviewed externally by Centre for Design and Society, RMIT University, Melbourne, Australia, and by the Netherlands National Institute for Public Health and the Environment (23). The scope of the LCAs in this study included agricultural production (in the Netherlands and abroad), transport, processing, distribution, retail (lighting, cooling), consumer phase (e.g., cooling and cooking), and waste treatment. The Carbon Footprint for a selection of products is shown in Table 1.

Nutrient Balance Concept (NBC)

The NBC (10, 25) is a nutrient profiling concept that evaluates the nutritional values of multiple food products in meals and total diets. The NBC advances on nutrient density by adding the metric of nutrient balance (NB) to qualifying (QI) and disqualifying (DI) indices (25). The QI is defined as the ratio of 28 essential nutrients contained in 2,000 kcal of a given food product relative to the country reference intakes for those nutrients. The DI is defined as the ratio of 7 public health sensitive nutrients contained in 2,000 kcal of a given food product, relative to the Maximal Reference Values (MRV) for those nutrients. If the QI value is >1, the food product is considered nutrient dense; if the QI value is smaller than 1, the food product is considered energy dense. If the DI value is >1, the food product is deemed compromised because it contains disqualifying nutrients in values higher than the MRV relative to the energy content of the food product. Finally, the NB score is calculated as the average proportion of daily values for qualifying nutrients (QI) present in 2,000 kcal of a given food, truncated at 1 for each qualifying nutrient. A NB score of 100% is achieved if every qualifying nutrient satisfies 100% or more of its daily requirement. Table 2 summarizes the calculations of QI, DI, and NB.

To compute NBC metrics comparable with previous NBC studies, the 208 food products in these diets were matched with an equivalent food from the USDA Food Composition Database (Release 28).

Diet Optimization

The Optimeal R software (Blonk Consultants, Gouda, The Netherlands) was used to compute the diets most similar to the current diet and simultaneously satisfying the set of specified nutritional requirements. This is known as quadratic programming. As described previously (18,26), the Optimeal R software contains all required data on food composition (19), nutrient requirements (27,28), and environmental impacts of food products consumed in the Netherlands.

In the model, the nutritional requirements are: (a) the Dutch (27) Recommended Dietary Allowance (RDA) or Adequate Intake (AI) (when an RDA cannot be determined), defined as lower limits; and (b) the Tolerable Upper Intake Level (UL), defined as upper limit (see Supplementary Table S1). The sugar limit used in the NBC metrics was taken from the Institute of Medicine (29) as no formal recommendation on total sugars is provided in the Netherlands, only on total carbohydrates. To avoid confounding effects, all diets were iso-caloric with 1,995 kcal per day.

The similarity of two diets can be measured by the Euclidian distance (ED) between them, i.e., by the square root of the sum of the square differences (in grams) of the amounts of each food product in the two diets. The Euclidian distance is the generalization of the shortest path between two points. It is assumed that a similar diet is easier to adopt by consumers than one that is more deviating from their current diet. At a population level, shifts in consumption of food products are relatively gradual.

RESULTS

The results of the assessment of the three food groups are shown graphically in Figure 1. The slopes in the graphs with environmental impacts are indicative of the environmental performance of a food group as a source of nutrients. Figure 2 shows the NB, QI, and DI of the optimized diets, being indicative of the nutrient balance.

Overall, increased amounts of meat products were associated with higher environmental impacts. Relative to the current diet, GHGe, FEU, and LO increased 49, 21, and 53%, respectively at the 250% level. In contrast, when the content of meat in an optimized diet was reduced, the environmental impacts decreased rapidly and attained the lowest values observed in this study. The NB score ranged from 94.5 to 97.1 with the meat content variation. Notably, the best NB score was achieved with the 0% meat level. At this level, the optimized diets also had the lowest DI and highest QI, suggesting a potentially positive balance can be achieved through dietary optimization after removal of meat products from the diets. The optimized diet with 250% level of current dietary dairy resulted in an increase of 16% in GHGe and 13% in FEU relative to the current diet, but in a 9% reduction of LO. The latter coincides with a shift in the type of land use from cropland to pasture, which, in general, is associated with a lower environmental impact. The NB score also varied from 94.8 to 96.6. The optimized diet with 0% dairy also had the highest QI overall, and the lowest DI from the dairy-optimized diets.

The results show that bread and breakfast cereals had the lowest impact when its amounts were increased. The optimized diet with 250% level of the current level of bread and breakfast cereals reduced 14% in GHGe, 12% in FEU, and 15% in LO, compared to the current diet. In contrast, decreasing bread and breakfast cereals resulted in the highest environmental impact compared to the other two food groups studied. Overall, the amounts of bread and breakfast cereals were inversely correlated with the levels of GHGe and FEU. Both increased and decreased amounts of bread and breakfast cereals were associated with lower levels of LO. The NB score varied from 94.9 to 96.0, QI varied from 1.5 to 1.7 and DI from 1 to 1.1, indicating a small positive relationship between the amounts of bread and breakfast cereals and the nutrient balance of the diet. Since bread and breakfast cereal was the food group with better performance, we investigated it in more detail. Table 3 shows the changes in the most important food groups in the 11 optimized diets with varying amounts of bread and breakfast cereals. Among the nutrients in bread and breakfast cereals that have to be replaced at 0% (see Supplementary Table S1) are total energy, iodine, fiber, and vitamin B1. This explains increased amounts of fish (iodine), legumes (energy, fiber, B1), pasta/rice (energy, B1), and vegetables (fiber, B1).

It is also relevant to understand the changes within the food group. Table 4 presents the ratio of wholegrain to other varieties of bread and breakfast cereals. In the current diet the ratio was 0.69. Absolute amounts of both wholegrain and other varieties increase as the total amount of bread and breakfast cereals increase. The ratio of wholegrain to other varieties, however, decreases.

DISCUSSION

The relative order in performance of meat, dairy and bread and breakfast cereals obtained can be explained by the balance between nutritional performance and environmental performance. In particular, wholegrain cereal products combine a high nutrient balance (25) score with a low environmental impact, whereas meat combines a medium nutrient balance score with a high environmental impact (30). It is important to realize, however, that bread is an iodine fortified product. Dairy has an intermediate position in both metrics (nutritional and environmental performance), which is in agreement with previous studies (6,31). This observation can also be explained by the fact that cheese and liquid dairy were introduced in a fixed ratio.

The NB scores vary minimally with changes in cereals content, due to the fact that all the optimized diets were high scoring, i.e., above 90%, and so in order to facilitate an increase, the changes in cereals and resultant overall optimized diets would need to address the small undersupply of just a few nutrients, which in this case were fiber, choline, vitamin E, and vitamin D. It is possible that if the wholegrain content of the optimized diets had been increased at the same rate as total cereals, some of these shortfall nutrients might have been met (fiber, vitamin E, iron).

Interestingly, iron was undersupplied in the optimized diets with <150% of current intake of bread and breakfast cereals. However, in all the optimized diets, fat and sugar were oversupplied. The fact that the NB score of the optimized diets was always very high (circa 94-95%), is not surprising, as they are all optimized to meet Dutch nutrient recommendations.

NB scores were also calculated for exemplar foods from each of the three food groups of interest (Figure 3). Results show that whole wheat bread and low-fat milk had a higher NB score, and Gouda cheese and beef a lower NB score. Differences in contents of dietary fiber, saturated fat and salt explain most of the differences, for instance between white bread and whole wheat bread. The different types of meat, beef in particular, performed worse than both types of bread. As for dairy, low-fat milk performed much better than Gouda cheese.

Even though all optimized diets are plausible in the sense that they satisfy all nutritional requirements and are as close as possible to the current diet, attainability is a question. In earlier studies (18,26) with the Optimeal R tool, Linear Programming was applied, i.e., the distance between two diets was measured by a linear transformation, the sum of the absolute differences of the normalized amounts. The disadvantage of that technique is that it tends to prefer large shifts in a limited number of food products in the diet, for instance by adding large quantities of legumes (18). This seems unrealistic at population level, as shifts in national diets tend to be gradual. By minimizing the Euclidian Distance, large shifts in a single food are more heavily penalized, resulting in more realistic solutions.

Moreover, the proposed method provides insight in the attainability of changes, by investigating distance between diets. This analysis shows that meat seems to be replaced more easily than bread and breakfast cereals and dairy. As mentioned already, bread is an iodine fortified product. Studying the effects of (iodine) fortification was out of scope and is a limitation of this study. Replacement of dairy is more difficult to attain since these products are nutrient dense and it would require more additional food products to be included in the optimized diet to provide the missing nutrients such as calcium and vitamins B2 and B12. In most cases these alternative products are not as nutrient dense as dairy. This reinforces the advice that a varied diet is the best way to achieve adequate nutrient intake.  This study is limited by the environmental data. Only GHGe, EU, and LO were analyzed, ignoring other environmental impacts. For example, the reduction of LO at lower levels of bread and breakfast cereals can be explained by the replacement of bread and breakfast cereals by fish, which is one of the few other food sources of iodine. Wild-caught fish has no LO, but is associated with other specific environmental impacts such as marine resource depletion (32), which was not included here. In general, the effects and interactions of general shifts on bioavailability is a limitation of this study.

Moreover, the life cycle inventories used affect the results obtained. Nutrient quality, if understood as the losses of nutrient quality along the life cycle of a food product or due to storage and preparation methods, is addressed as follows: as closely as possible, the data picked from food tables for each food item represented the food item as it was described in the Dutch diet. For example, if an item was mentioned as "raw vegetable, " then its nutritional composition was taken as such. On the other hand, if a food item was described as "cooked vegetable, " then the food item was chosen as cooked. The purpose of this assessment was not to  0  35  70  106  141  176  211  245  282  317  352 b   Cheese  37  17  18  20  21  22  21  21  22  22  22  21  Eggs  11  16  14  13  13  12  12  13  13  14  14  14  Fish  17  92  73  52  36  28  28  27  27  27  27  29  Fruits  120  175  167  160  152  145  139  133  126  118  109  102  Legumes  4  68  61  54  46  39  33  28  22  16  9  7  Meat  88  69  74  79  85  86  86  85  84  82  76  59  Dairy  328  335  328  321  317  312  311  310  309  307  302  296  Pasta/Rice  52  60  58  56  55  53  52  51  49  46  43  39  Vegetables  134  381  352  329  299  274  254  237  222  206  191   capture the losses of nutrient quality. Assessing the nutritional composition of food items will always be dependent on data availability in food tables, unless the study actually measures this. Similarly, as environmental impacts of food products are dependent on country of origin and production system, they are highly variable, which could be a barrier for the extension of this method to other countries. Variability in the results also depends on the methodology used to develop the life cycle inventories. Valuable work is taking place globally among concerned stakeholders (academia, researchers in public and private organizations, agricultural producers, food manufacturers and regulators, to name a few) to harmonize as much as possible those methodologies and to incorporate pragmatically significant developments in environmental science, environmental impact modeling, and big data acquisition and management. All these factors contribute to rapid changes in how the environmental impacts of food products are assessed, understood, and communicated.

Finally, the choices made by the optimization algorithm are only an approximation of actual behavior and the attainability of replacement options.

CONCLUSIONS

We present an innovative method to compare the performance of food groups in a diet: fixing the amounts of a selected food FIGURE 3 | Exemplar products to demonstrate relative positions and scores from the Nutrient Balance Concept analyses. The NB scores per product are shown in the blue circles. These results were calculated by the authors for the purposes of this article. group at different levels and optimizing the remaining diet so that nutritional requirements are met and the changes to the initial diet are minimized. For a set of optimizations we compute environmental (CF, EU, LO) and nutritional metrics (NB). The method is flexible in the number of included nutrient requirements and environmental impacts.

In this paper we compared bread and breakfast cereals, meat, and dairy. Performance of other food groups could be done as an extension of the present study.

The analysis suggested that bread and breakfast cereals are sources of nutrients with a better environmental performance compared to dairy or meat within the context of the Dutch diet. More specifically, amounts of bread and breakfast cereals have an inverse correlation with the environmental impact of the optimized diet. This indicates that they are a source of nutrients with a better environmental performance relative to other foods in the diet. When overall bread and breakfast cereal intake is low in the optimized diet, wholegrain cereal products are the preferred choice due to their nutritional performance.

AUTHOR CONTRIBUTIONS

GK was the lead researcher in the study, computing, and analyzing the results. EM provided scientific and technical support in the calculations and optimization. NE-O and KC led the application of the Nutrient Balance Concept and its interpretation. MT developed the original optimization algorithm and led the writing and organizing of the final manuscript for scientific publication. HB conceptualized the study and led the Environmental Impact analyzes. All authors were involved in the different drafts and revisions of the article.

FUNDING

This study was funded by members of the HealthGrain Forum: Nestlé, Cereal Partners Worldwide, Fazer, and GoodMills Innovation.

FIGURE 1 |
1Environmental Indicators with varying amounts of dairy, meat and bread and breakfast cereals, relative to the current intake of each (100%). (A) Carbon footprint. (B) Fossil energy use. (C) Land occupation. These results were calculated by the authors for the purposes of this article. Frontiers in Nutrition | www.frontiersin.org

FIGURE 2 |
2QI, DI, and NB (shown in the blue circles) for the optimized diets. (A) Bread and breakfast cereals. (B) Meat. (C) Dairy. These results were calculated by the authors for the purposes of this article.

196 a
196141 g consists of 0.6 tablespoons of cereals, 0.4 slices of crispbread, and 3.5 slices of bread. b 352 g consists of 8.4 tablespoons of cereals, 4.7 slices of crispbread, and 6.5 slices of bread.These results were calculated by the authors for the purposes of this article.

TABLE 1 |
1Carbon Footprint of a selection of products available in the diet.These results were calculated by the authors for the purposes of this article.Product
GHGe

(kg CO 2 -eq/kg)

Beef
46.7

Cheese, Gouda
9.2

Pork
7.7

Chicken
5.1

Salmon
3.9

Egg
3.3

Herring
2.0

Tomato
1.7

Cashew nuts
1.6

Milk, semi-skimmed
1.2

Crispbread
1.0

Bread, white
1.0

Bread, rye
0.9

Bread, wholemeal
0.9

Carrots
0.7

Potatoes
0.7

Cereal, wholegrain
0.7

Apple
0.5

TABLE 2 |
2Calculation of QI, DI, and NB(25).Equations
Description
Nutrients considered

QI =
E d
E p
·

Nq
j = 1

a q,j

r q,j

Nq

E d = daily energy need (kcal)
E p = energy in the qtty. of food analyzed (kcal)
a q,j = qtty. of qualifying nutrient (g)
a d,j = qtty. of disqualifying nutrient (g)
r q,j = DRI of qualifying nutrient (g/day)
r d,j = MRV of disqualifying nutrient (g/day)
N q = Number of qualifying nutrients evaluated
N d = Number of disqualifying nutrients
evaluated
QI = Qualifying index
DI = Disqualifying index
NB = Qualifying nutrient balance score

For QI:
folate; niacin; panthotenic acid; riboflavin;
thiamin; vitamins A, B6, B12, C, D, E, K;
Ca, Cu, F, Fe, Mg, Mn, K, P, Se, Zn;

α-linolenic acid; linolenic acid; choline;
dietary fiber; protein; water.
For DI:
total fat; saturated fat; cholesterol; trans
fatty acids; total sugars; Na; alcohol.

DI =
E d
E p
·

N d
j = 1

a d,j

r d,j

N d

NB = 100 ·

Nq

i = 1 QI i
Nq

TABLE 3 |
3Shifts in food groups for different percentages of bread and breakfast cereals (women 31-50 years) relative to current intake (=100%).Food Group [g/day]
Starting diet
0%
25%
50%
75%
100%
125%
150%
175%
200%
225%
250%

Bread and Breakfast Cereals
141 a

TABLE 4 |
4Ratio of wholegrain to refined with different amounts of Bread and breakfast cereals.These results were calculated by the authors for the purposes of this article.Diet
Wholegrain (g)
Refined (g)
Ratio Wholegrain:refined

Starting diet
57
83
0.69
0%
0
0
0.00
25%
24
12
2.04
50%
39
31
1.26
100%
80
61
1.31
150%
112
99
1.12
200%
138
144
0.96
250%
168
184
0.92

Frontiers in Nutrition | www.frontiersin.org
June 2018 | Volume 5 | Article 51
ACKNOWLEDGMENTSWe thank Jan-Willem van der Kamp (TNO, The Netherlands) for inspiring this study and organizing the funds.SUPPLEMENTARY MATERIALThe remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.This study was partially funded by the Healthgrain Forum, an association, based in Europe, of universities, institutes and industries interested in grain and grain based products. The HGF was not involved in the writing of the article and supported the scientific approach regardless of the results.Copyright © 2018 Kramer, Martinez, Espinoza-Orias, Cooper, Tyszler and Blonk. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Climate change and food systems. S J Vermeulen, B M Campbell, Jsi Ingram, 10.1146/annurev-environ-020411-130608Annu Rev Environ Resour. 37Vermeulen SJ, Campbell BM, Ingram JSI. Climate change and food systems. Annu Rev Environ Resour. (2012) 37:195-222. doi: 10.1146/annurev-environ- 020411-130608

Where are the best opportunities for reducing greenhouse gas emissions in the food system (including the food chain)? Food Policy. T Garnett, 10.1016/j.foodpol.2010.10.01036Garnett T. Where are the best opportunities for reducing greenhouse gas emissions in the food system (including the food chain)? Food Policy (2011) 36:S23-32. doi: 10.1016/j.foodpol.2010.10.010

Environmental impacts of changes to healthier diets in Europe. A Tukker, R A Goldbohm, A De Koning, M Verheijden, R Kleijn, O Wolf, 10.1016/j.ecolecon.2011.05.001Ecol Econ. 70Tukker A, Goldbohm RA, de Koning A, Verheijden M, Kleijn R, Wolf O, et al. Environmental impacts of changes to healthier diets in Europe. Ecol Econ. (2011) 70:1776-88. doi: 10.1016/j.ecolecon.2011. 05.001

Environmental impact of dietary change: a systematic review. E Hallström, A Carlsson-Kanyama, P Börjesson, 10.1016/j.jclepro.2014.12.008J Clean Prod. 91Hallström, E, Carlsson-Kanyama A, Börjesson P. Environmental impact of dietary change: a systematic review. J Clean Prod. (2015) 91:1-11. doi: 10.1016/j.jclepro.2014.12.008

FAO. Sustainable Diets and Biodiversity Sustainable Diets And Biodiversity. Burlingame B, Dernini S, editors. RomeFAOFAO. Sustainable Diets and Biodiversity Sustainable Diets And Biodiversity. Burlingame B, Dernini S, editors. Rome: FAO (2010).

Sustainable diets for the future: can we contribute to reducing greenhouse gas emissions by eating a healthy diet?. J I Macdiarmid, J Kyle, G W Horgan, J Loe, C Fyfe, A Johnstone, 10.3945/ajcn.112.038729Am J Clin Nutr. 96Macdiarmid JI, Kyle J, Horgan, GW, Loe J, Fyfe C, Johnstone A, et al. Sustainable diets for the future: can we contribute to reducing greenhouse gas emissions by eating a healthy diet? Am J Clin Nutr. (2012) 96:632-9. doi: 10.3945/ajcn.112.038729

The global warming potential of two healthy Nordic diets compared with the average Danish diet. H Saxe, T M Larsen, L Mogensen, 10.1007/s10584-012-0495-4Clim Change. 116Saxe H, Larsen TM, Mogensen L. The global warming potential of two healthy Nordic diets compared with the average Danish diet. Clim Change (2013) 116:249-62. doi: 10.1007/s10584-012-0495-4

Exploring dietary guidelines based on ecological and nutritional values: a comparison of six dietary patterns. C Van Dooren, M Marinussen, H Blonk, H Aiking, P Vellinga, 10.1016/j.foodpol.2013.11.002Food Policy. 44van Dooren C, Marinussen, M, Blonk H, Aiking H, Vellinga P. Exploring dietary guidelines based on ecological and nutritional values: a comparison of six dietary patterns. Food Policy (2014) 44:36-46. doi: 10.1016/j.foodpol.2013.11.002

Toward a life cycle-based, dietlevel framework for food environmental impact and nutritional quality assessment: a critical review. M C Heller, G A Keoleian, W C Willett, 10.1021/es4025113Environ Sci Technol. 47Heller MC, Keoleian GA, Willett WC. Toward a life cycle-based, diet- level framework for food environmental impact and nutritional quality assessment: a critical review. Environ Sci Technol. (2013) 47:12632-47. doi: 10.1021/es4025113

Connecting the dots : assessing sustainable nutrition at Nestlé. N Espinoza-Orias, A Roulin, H Watzke, K Cooper, 9th International Conference on Life Cycle Assessment in Food. San Fransisco, CAEspinoza-Orias, N, Roulin, A, Watzke, H, Cooper, K. Connecting the dots : assessing sustainable nutrition at Nestlé. In: 9th International Conference on Life Cycle Assessment in Food. San Fransisco, CA (2014).

Modelling the likely impact of healthy eating guidelines on agricultural production and land use in England and Wales. M H Arnoult, P J Jones, R B Tranter, W B Tiffin, J Tzanopoulos, 10.1016/j.landusepol.2010.02.001Land Use Policy. 27Arnoult MH, Jones PJ, Tranter RB, Tiffin WB, Tzanopoulos J. Modelling the likely impact of healthy eating guidelines on agricultural production and land use in England and Wales. Land Use Policy (2010) 27:1046-55. doi: 10.1016/j.landusepol.2010.02.001

Diet changea solution to reduce water use?. M Jalava, M Kummu, M Porkka, S Siebert, O Varis, 10.1088/1748-9326/9/7/074016Environ Res Lett. 974016Jalava, M., Kummu, M., Porkka, M., Siebert S, Varis O. Diet change- a solution to reduce water use? Environ Res Lett. (2014) 9:074016. doi: 10.1088/1748-9326/9/7/074016

Health Council of the Netherlands. Dutch Dietary Guidelines. 2015/24E. The Hague:Health Council of the Netherlands. 13. Health Council of the Netherlands. Dutch Dietary Guidelines 2015, Publication no. 2015/24E. The Hague:Health Council of the Netherlands (2015).

The Food Hourglass. K Verburgh, Harper Collins PublishersLondonVerburgh K. The Food Hourglass. London: Harper Collins Publishers (2014).

. W Davis, Wheat, Belly, RodaleNew York, NYDavis W. Wheat Belly. New York, NY: Rodale (2011).

Dutch National Food Consumption Survey. Ctm Van Rossum, H P Fransen, J Verkaik-Kloosterman, Ejm Buurma-Rethans, M C Ocke, RIVMBilthovenVan Rossum CTM, Fransen HP, Verkaik-Kloosterman J, Buurma-Rethans EJM, Ocke MC. Dutch National Food Consumption Survey 2007-2010. Bilthoven: RIVM (2011).

dat Version 20111125, Part of the Dutch National Food Consumption Survey. Rivm Consumption_Food_Nut, BilthovenRIVM. Consumption_food_nut.dat Version 20111125, Part of the Dutch National Food Consumption Survey 2007-2010. Bilthoven (2012).

Just eating healthier is not enough: studying the environmental impact of different diet scenarios for the Netherlands by Linear Programming. M Tyszler, Gfh Kramer, H Blonk, 9th International Conferene on Life Cycle Assesment in Food. San Fransisco, CATyszler M, Kramer GFH, Blonk H. Just eating healthier is not enough: studying the environmental impact of different diet scenarios for the Netherlands by Linear Programming. In: 9th International Conferene on Life Cycle Assesment in Food. San Fransisco, CA (2014).

. Rivm Nevo-Online, Versie, 40RIVM. NEVO-Online Versie 2013/4.0 (2013). Available online at http://nevo- online.rivm.nl/

The Agri-Footprint Database Methodology and Data. Blonk Consultants, Gouda: Blonk Consultants. Blonk Consultants. The Agri-Footprint Database Methodology and Data. Gouda: Blonk Consultants (2014).

Reducing our environmental footprint and improving our health: greenhouse gas emission and land use of usual diet and mortality in EPIC-NL: a prospective cohort study. S Biesbroek, B H Bueno-De-Mesquita, Phm Peeters, W M Verschuren, Y T Van Der Schouw, G F Kramer, 10.1186/1476-069X-13-27Environ Health. 1327Biesbroek S, Bueno-de-Mesquita BH, Peeters PHM, Verschuren WM, van der Schouw YT, Kramer GF, et al. Reducing our environmental footprint and improving our health: greenhouse gas emission and land use of usual diet and mortality in EPIC-NL: a prospective cohort study. Environ Health (2014) 13:27. doi: 10.1186/1476-069X-13-27

Greenhouse gas emission of diets in the Netherlands and associations with food, energy and macronutrient intakes. Ehm Temme, I B Toxopeus, Gfh Kramer, M C Brosens, J M Drijvers, M Tyszler, 10.1017/S1368980014002821.Public Health Nutr. 18Temme EHM, Toxopeus IB, Kramer GFH, Brosens MC, Drijvers JM, Tyszler M, et al. Greenhouse gas emission of diets in the Netherlands and associations with food, energy and macronutrient intakes. Public Health Nutr. (2014) 18:2433-45. doi: 10.1017/S13689800140 02821.

Blonk Agri-footprint BV. Agri-Footprint 2.0 -Part 1 -Methodology and Basic Principles. Gouda: Blonk Agri-footprint BV. Blonk Agri-footprint BV. Agri-Footprint 2.0 -Part 1 -Methodology and Basic Principles. Gouda: Blonk Agri-footprint BV (2015).

A Life Cycle Impact Assessment Method Which Comprises Harmonised Category Indicators at the Midpoint and The Endpoint Level. M Goedkoop, R Heijungs, M Huijbregts, De Schryver, A Struijs, J , Van Zelm, R , RIVMThe HagueFirst editionGoedkoop M, Heijungs R, Huijbregts M, De Schryver A, Struijs, J, Van Zelm, R, et al. ReCiPe 2008 A Life Cycle Impact Assessment Method Which Comprises Harmonised Category Indicators at the Midpoint and The Endpoint Level, First edition. The Hague: RIVM (2013).

The nutrient balance concept: a new quality metric for composite meals and diets. E B Fern, H Watzke, D V Barclay, A Roulin, A Drewnowski, 10.1371/journal.pone.0130491PLoS ONE. 10130491Fern EB, Watzke H, Barclay DV, Roulin A, Drewnowski A. The nutrient balance concept: a new quality metric for composite meals and diets. PLoS ONE (2015) 10:e0130491. doi: 10.1371/journal.pone.01 30491

Comparing apples with oranges: on the functional equivalence of food products for comparative LCAs. M Tyszler, G Kramer, H Blonk, Tyszler M, Kramer G, Blonk H. Comparing apples with oranges: on the functional equivalence of food products for comparative LCAs.

. 10.1007/s11367-014-0762-xInt J Life Cycle Assess. 19Int J Life Cycle Assess. (2014) 19:1482-7. doi: 10.1007/s11367-014-0 762-x

Netherlands Nutrition Centre. Aanbevelingen Voor Vitamines , Mineralen en Spoorelementen (Recommendations for Vitamins, Minerals and Trace Elements). The Hague: Netherlands Nutrition Centre. Netherlands Nutrition Centre. Aanbevelingen Voor Vitamines , Mineralen en Spoorelementen (Recommendations for Vitamins, Minerals and Trace Elements). The Hague: Netherlands Nutrition Centre (2014).

Dietary Reference Intakes: Energy, Proteins, Fats, and Digestible Carbohydrates; Publication no. 2001/19R. The Hague: Health Council of the. NetherlandsHealth Council of the NetherlandsHealth Council of the Netherlands. Dietary Reference Intakes: Energy, Proteins, Fats, and Digestible Carbohydrates; Publication no. 2001/19R. The Hague: Health Council of the Netherlands (2001).

10.17226/10490Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein, and Amino Acids. Washington, DCThe National Academies PressInstitute of Medicine. Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein, and Amino Acids. Washington, DC: The National Academies Press. (2005). doi: 10.17226/10490

A novel nutrition-based functional equivalency metric for comparative life cycle assessment of food. M C Heller, G A Keoleian, Procedings 8th International Conference LCA Agri-Food Sect 1-4. edings 8th International Conference LCA Agri-Food Sect 1-4Saint MaloHeller MC, Keoleian GA. A novel nutrition-based functional equivalency metric for comparative life cycle assessment of food. In: Procedings 8th International Conference LCA Agri-Food Sect 1-4 Oct 2012. Saint Malo (2012).

Greenhouse gas emissions of realistic dietary choices in Denmark: the carbon footprint and nutritional value of dairy products. L B Werner, A Flysjö, T Tholstrup, 10.3402/fnr.v58.20687Food Nutr Res. 58Werner LB, Flysjö A, Tholstrup T. Greenhouse gas emissions of realistic dietary choices in Denmark: the carbon footprint and nutritional value of dairy products. Food Nutr Res. (2014) 58:1-16. doi: 10.3402/fnr.v58.20687

Accounting for overfishing in life cycle assessment: new impact categories for biotic resource use. A Emanuelsson, F Ziegler, L Pihl, M Sköld, U Sonesson, 10.1007/s11367-013-0684-zInt. J. Life Cycle Assess. 19Emanuelsson A, Ziegler F, Pihl L, Sköld, M, Sonesson U. Accounting for overfishing in life cycle assessment: new impact categories for biotic resource use. Int. J. Life Cycle Assess. (2014) 19:1156-68. doi: 10.1007/s11367-013-0684-z