S Lenz

M Bonini

S K Nett

M C Lechmann

S G J Emmerling

R S Kappes

M Memesa

A Timmann

S V Roth

J S Gutmann
email: gutmann@mpip-mainz.mpg.de

Global scattering functions: a tool for grazing

come   L'archive ouverte pluridisciplinaire

scattering resulting from GISAXS. In this context the applicability and limit of its application to diffuse scattering in the GISAXS geometry is discussed. Furthermore diffuse q || scattering from different ideal particle types is simulated and compared with fitted results. To verify our approach, fit results from experimental GISAXS curves obtained for real samples are compared with results from Scanning Probe Microscopy and Scanning Electron Microscopy studies. The samples investigated range from evaporated Au films to hybrid TiO 2 /Polymer films and demonstrate the usefulness in the structural analysis of complex films. PACS. 61.05.cc Theories of x-ray diffraction and scattering 68.55.-a Thin film structure and morphology Short Title: Scattering functions for GISAXS analysis of lateral structures

Introduction

Grazing Incidence Small Angle X-Ray Scattering (GISAXS) can probe structural film properties perpendicular and parallel to the sample plane. Information on film structures perpendicular to the sample plane is usually obtained by the analysis of the specular reflected beam in q ┴ direction. In addition lateral film structures can be studied by either analyzing scattering parallel to the sample's surface at the specular beam position or by analyzing additional off specular scattering.

Our aim is to probe lateral length scales ranging from a few nanometres up to hundreds of nanometres 1,2 of colloidal systems with a defined mean particle dimension and typical polydispersities with standard deviations σ = 0 -0.6, which is often sufficient to understand material film properties 3 . Polydispserse particles can be located as islands at the film/air interface or buried within a thin film matrix. Especially for buried particle systems high penetration depths into the film matrix are recommended. Thus, incident angles α i , which are sufficiently higher than the critical angle α c of the film matrix, should be chosen. We demonstrate under which conditions our approach yields results for mean particle dimensions with deviations in the range of the particles polydispersity at GISAXS taken at α i > α c .

At these experimental conditions, it is possible to benefit from a separation of the reflected intensity into two peaks in q ┴ direction. One is the specular reflected beam at an angle of the exit beam α f equal to α i . The second is the material dependent Yoneda peak 4,5 at α f ≈ α c 6,7 .

Diffuse scattering at this peak position can be explained within the Distorted Wave Born Approximation (DWBA) 8 . For practical reasons a separation of the two peak positions is useful, because scattering at the specular beam position, can be separated from diffuse scattering occurring at the Yoneda peak. Hence, deconvolution of the primary reflected beam profile, scattering at the specular peak position and diffuse scattering at the Yoneda peak can be avoided.

Transverse detector scans along the reciprocal q || (q x , q y ) scattering plane at the Yoneda peak position can be performed to obtain lateral film information. Occurring peaks on the transverse q || scans are often analyzed with a generalized Bragg interference function , which is widely used for Born approximated scattering. However, for a rigorous data analysis of GISAXS one should use the DWBA. Theories for the most important film systems containing rough surfaces 8,10,11 , buried particles 9 and supported islands 12 were previously derived. To compute and simulate GISAXS data, Lazzari developed the simulation and fitting software IsGISAXS a, 13,14 . In this software the mentioned theories and approximations are included.

Nevertheless, simulation of scattering data will only be meaningful if additional information on the film system is available, including an exact model of the particle shape and lattice. In cases of film systems composed of typical polydisperse colloidal particles, which a www.insp.jussieu.fr/axe2/Oxydes/IsGISAXS/isgisaxs.htm are not arranged on a grid, the use of well defined form and structure factors will become questionable.

Comparable weakly correlated colloidal powder systems and polymer solutions were analysed in the past with unified fit approaches in transmission geometry [START_REF] Guinier | Small-Angle Scattering of X-rays[END_REF][START_REF] Beaucage | [END_REF][17][START_REF] Porod | Small-Angle X-ray Scattering[END_REF] . This model describes scattering from a specimen in terms of multiple structural levels. It is based on a combination of exponential laws, power laws and Bragg based interference functions and describes fractal scattering objects by their fractal dimensions, radius of gyrations and mean centre to centre distances. Compared to full DWBA simulation approaches, a unified approach would make GISAXS data analysis of the latter film systems easier. However, before the application of such BA based models, it is of high importance to discuss the applicability and limits towards DWBA described q || scattering in detail.

Our considerations combine theoretical discussions with simulation and experimental studies. Comparisons of simulations computed for BA based transmission SAXS and DWBA based GISAXS demonstrate the principal applicability of the unified formalism to GISAXS.

Based on these comparisons, an optimum experimental range for the incident angle is proposed for particle island systems at the film/air interface and buried particle systems.

While in this range refraction effects cannot be neglected, it is nevertheless possible to obtain fit results deviating less than 10% from their theoretical values for monodisperse particle samples. For polydisperse film systems mean deviations are not exceeding σ or usual experimental error intervals.

Experimental GISAXS studies were performed on polydisperse Au particle islands prepared by chemical vapour deposition, as a sample system for polydisperse particles located at a free interface. The practical usefulness of the unified fit approach is further demonstrated for buried particles. The study of a model TiO 2 /polymethylmethacrylate (PMMA) hybrid material film demonstrates the applicability of the unified fit approach to buried particle systems. The usefulness of unified fits to unravel new physical phenomena is demonstrated in an investigation of percolating networks in hybrid barrier layers used in solar cell applications. Here the analysis of GISAXS measurements using the Unified Fit approach is able to prove the existence of fractal 3D networks proposed from conductive SPM (Scanning Probe Microscopy) experiments [START_REF] Memesa | [END_REF] . It therefore establishes a structural model which explains the physics of charge carrier transport in these systems.

Experimental Methods

GISAXS with the help of µ-sized x-ray beams was performed on three exemplary film systems. First the adaptability of the unified fit approach towards q || scattering on rough Au films, with low lateral correlations shall be studied. To this end we prepared one Au film of a thickness of 20 nm deposited on a base cleaned Si-substrate by thermal evaporation methods (BALTEC MED 020, BALTEC, Balzers, Lichtenstein), which was carried out under a chamber base pressure of 1.7 • 10 -5 mbar with an evaporation rate of < 1 Å/s and room temperature substrates. GISAXS on a film of polydisperse TiO 2 particles buried in a polymer matrix was performed, in order to demonstrate the usefulness of the unified model for buried particle systems. TiO 2 particles (0.01 mL, Solaronix T) were dispersed in a PMMA (0.1 g) solution in THF (5 ml), which resulted in a film with ~1%wt TiO 2 . The film was spin coated on Si substrates for 60 s using a Süss Micro Tec Delta 80 spin coater under ambient conditions (rotation speed of 1000 rpm; acceleration speed of 1000 rpm/s). The thickness of the studied film was 180 nm ± 10 nm, as measured with a Tencor P-10 surface profiler. In addition, one percolating TiO 2 hybrid material film was prepared by sol-gel-chemistry using a templating (PEO)MA-PDMS-MA(PEO) tri-block-co-polymer. The film was plasma treated and ceramized at 600°C as described previously [START_REF] Memesa | [END_REF] . The thickness of the studied film was 20 ± 1 nm, as measured by X-Ray reflectivity with a Seifert θ-θ XRD 3003 X-Ray reflectometer.

GISAXS experiments were carried out at the beamline BW4/HASYLAB using the microfocus option [20][21][22] , with a sample-to-detector distance of 1.9 m, a wavelength λ = 0.138 nm, and a beam size of 32 x 17 µm 2 (horizontal x vertical). A compromise between a sufficient separation of specular reflected peaks and Yoneda peaks, a minimized theoretical deviation between DWBA and BA theory was made (See section "Results and Discussion") and available experimental conditions was made. Consequently an incident angle of α i equal to 0.7° was chosen for GISAXS studies on the Au film. GISAXS on the TiO 2 /PMMA film GISAXS scattering patterns were measured at α i = 0.55°, while TiO 2 /(PEO)MA-PDMS-MA(PEO) films were measured at α i = 0.7°. Transverse q || scans were performed at the In order to compare GISAXS data and the obtained fit parameters with comparative methods, SPM and SEM studies were performed on Au, pure TiO 2 particle films and TiO 2 /(PEO)MA-PDMS-MA(PEO), respectively.

SPM images of the Au films were taken in tapping mode at constant amplitude (environmental SPM, Veeco Instruments, Santa Barbara, CA). For these studies silicon cantilevers (Olympus, OMCL-AC240TS, Japan) having a nominal spring constant of 2 N/m and a resonance frequency of around 70 kHz were used. For operation of the SPM we used a NanoScope IIIa controller (Veeco Instruments, Santa Barbara, CA, USA) controlled by Nanoscope 5.30r2 software. The offset and tilt background of all images were removed by subtraction of a 1 st and 2 nd polynomial background due to scanner movement. The size of immersed TiO 2 particles (without polymer matrix) and of the TiO 2 /(PEO)MA-PDMS-MA(PEO) was assigned using a field emission SEM (LEO 1530 "Gemini"). The acceleration voltage was 1 kV.

Results and Discussion

Theoretical discussion

GISAXS geometry

In a typical GISAXS experiment the primary beam is directed in a certain incident angle α i onto the sample's surface. The incident beam is reflected and scattered at the samples surface, with an exit angle α f perpendicular to the surface and an angle 2θ parallel to the surface (Figure 1). The scattering wave vector q is composed of its single components q x , q y and q z , related to the experimental angles by (1) When absorption in the samples medium cannot be neglected, q z becomes complex and is related to the imaginary part, β, of the refractive index n = 1δ + i . β and the materials critical angle by 23,24 (

Throughout the rest of this work we use the and reciprocal planes to describe scattering, reflection and refraction perpendicular and parallel to the specimen's surface.

Approximation of diffuse scattering by BA and intrinsic limits

In order to reduce GISAXS analysis to lateral density fluctuations, detector scans in q || are commonly performed at the Yoneda's maximum at α f or α i equal to α c of one film component 5 . When analyzing transverse detector scans with conventional BA, the averaged intensity in the DWBA, which describes the four major scattering effects (curly brackets in Eq. ( 3)) has to be considered 9 .

(3) Sinha et.al. 8 derived the differential cross-section of diffuse scattering as (4) where A is the irradiated sample surface area, k c 2 is the critical impulse and T i , T f the Fresnel transmission coefficient of the incoming and outcoming wave, respectively, given by (5) Here and are the wave vectors perpendicular to the sample surface in vacuum and in the film medium, respectively. Γ(q) can be related to density autocorrelation functions, which describe the form of idealized scattering objects. To deconvolute Γ(q) Rauscher et.al. 9 used an infinitely thin and perfectly flat δ-layer with interior density fluctuations of a lateral distance R || and obtained (6) where is the Fourier Transform of the density autocorrelation function of the lateral surface structures within this δ-layer, and t is the thickness of the δ-layer. In the case of non correlated density fluctuations, can be substituted with an idealized particle form factor. It has to be noted that the lateral approximation in Eq. ( 6) is only valid for infinitely thin cylindrically symmetric scattering objects. From of spherical particle islands 13 (7) one can see that GISAXS in the q || detector plane will have a non constant q ┴ scattering proportion. Nevertheless, it is shown later on that a lateral approximation for leads to deviations below 10% for monodisperse particle samples. For polydisperse film systems mean deviations are not exceeding σ or usual experimental error intervals, which is sufficient for the analysis of typical polydispers colloidal particles.

Ψ (0)
At this point an expression is needed, which includes scattering from rough interfaces coupled with scattering from objects with different scattering length densities. Apart from height-height correlation functions , which return root mean square (rms) values, lateral roughness correlations at q || ≠ 0 can be studied. Characteristic Porod decays, I ~ q -3-h , is used to describe the fractality or self affinity of the rough surface or interface. The Hurst parameter h is equal to unity for smooth surfaces or very small for jagged surfaces 8,26-28 . These surfaces exhibit a lateral cut-off length ζ. Considering a self-affine rough particle island of an arbitrary shape, which can be supported on a film or buried in a film (e.g.

polymer) matrix, it becomes appropriate to associate the cut-off length of this rough structure with its radius of gyration R g .

Unified Exponential/Power-Law Fit model

At this point an expression is needed, which is capable of describing simultaneously scattering from rough interfaces and density fluctuations. In this context density fluctuations shall be described with different idealized shapes. Beaucage introduced in a set of publications [START_REF] Beaucage | [END_REF]17,29 a general unified fit model, which is able to describe scattering over several orders of magnitude for spherical averaged particles in transmission geometry using BA. This model describes material microstructures in terms of structural levels. Thus it can be applied to model the system's structural features starting from the smallest structural level, such as a nanoparticle towards clusters of particles up to the macro-scale. It was applied successfully in transmission scattering geometry to several particle systems 30,31,32 . In addition, it was also applied to scattering from soft matter systems 29,33,34 . Nevertheless, this global model was originally developed for analysis of scattering results in transmission geometry using BA.

Therefore we have to show that it is (within certain limitations) also applicable to the analysis of scattering problems in grazing incidence geometry.

For one structural level the scattered intensity in the unified fit approach is given by ( 8)

The first term corresponds to Guinier's law and describes the size of spherical averaged particles. It is related to the radius of a sphere by . For upstanding particles with cylindrical symmetries, q can be substituted with q || and term one in Eq. ( 8) with

. R C is defined as the radius of gyration of the cross-sectional area of the particle and can be related to the radius of the cross-sectional area by .

The second term in Eq. ( 8) corresponds to the structural limited Porod regime, with the Porod prefactor B. The cubed error function limits the fractal regime of the structure's surface at low q over three possible orientations with its radius of gyration R g . For smooth spherical particles q -4 dependence is obtained, while for surface scattering from cylindrically symmetrical structures q -3 dependence is obtained [START_REF] Higgins | Polymers and Neutron Scattering[END_REF] .

For q || scattering from cylindrically symmetric particles Eq. ( 8) can be rewritten with a squared error function allowing a power law cut-off over two orientations (9) When more than one structural level is present in a sample, the unified scattering intensity described in Eq. ( 8) can be extended to 16 :

(10)

Eq. ( 10) is a sum of scattered intensities over n structural levels. When there are correlations between two structural levels, as in particle aggregates, a second term, which limits Porod scattering for n > 1 at R g(i-1) has to be introduced. For q || scattering of cylindrically symmetric structures an analogue expression can be written (11) In SAXS analysis the Guinier and Porod prefactors G and B, can be related to the Polydispersity Index (PDI) of spherical averaged particles in SAXS by [START_REF] Beaucage | [END_REF] .

However, simulations presented later show that such PDI approaches are not valid when applied to q || scans in GISAXS at α i > α c , because of the non constant scattered q ┴ portion to (Eq.7). Therefore we do not recommend to use such kind of BA based PDI approaches to estimate particle polydispersity in GISAXS for α i > α c .

In case of weakly correlated structures the unified intensity can be correlated with a common structure factor S(q) (12) is a function, which describes the correlation between two structures with a centre to centre distance and a packing factor : [START_REF] Guinier | Small-Angle Scattering of X-rays[END_REF][START_REF] Porod | Small-Angle X-ray Scattering[END_REF]

The packing factor κ describes the degree of correlation. For a perfect three dimensional crystal one yields κ max = 5.92, while for a perfect two dimensional crystal κ max = 6.24 is obtained. is the 'form factor' for structural correlations occurring at an average distance 15 (14) Eqs. (12-14) can be used only in the case of weakly correlated systems, when I (q,R g ) can be decoupled from S(q) and S(q) is not dependent on R g . This is usually the case for low correlations at κ < 4.

Comparison with Simulations

In this chapter the adaptability of the unified fit approach to q || dependent GISAXS is tested. Simulated q || scans were fitted with the unified fit approach and deviations from DWBA to BA theory are discussed. To test the pictured BA approach for common particle types GISAXS from spherical and cylindrical particle shapes were simulated following Gaussian distributions with σ = 0 -0.6. IsGISAXS simulation results using BA and DWBA are compared with calculated intensity from the unified fit model using the free accessible Irena software 37,b for Igor Pro, Wavemetrics Inc.

As a first step simulations, which mimic diffuse surface scattering from monodisperse, smooth particle islands supported on homogenous surfaces were performed. Such a model is used in the experimental section to describe the lateral correlations of rough Au films.

Isotropic spherical particles were simulated with R sim = R x = R y = R z = 5 nm (Figure 2a,b).

Particle islands of cylindrical symmetries were simulated with R || sim = R x = R y = 5 (Figure 2c,d). R g Sim and R c Sim were calculated according to and . Details of the parameters used in IsGISAXS simulations are given in the supplementary information. For BA simulated q || scans (similar to transmission SAXS geometry) incident and exit angles α i and α f were set to 0°, respectively.

For DWBA simulated GISAXS the incident angle was set to α i = 0.7°, equal to the performed GISAXS experiments on the studied Au film, while the exit angle was set to α f = α c (Au) = 0.50°. Simulated q || scans of spherical particle islands (Figure 2a,b) were compared with three dimensional averaged intensities from Eq. ( 8), while simulated q || scans of cylindrical particles (Figure 2c,d) were compared with radial averaged intensities from Eq. ( 9).

The observed power-law decays of q -4 for spherical particle islands and of q -3 for radial averaged cylindrical particle islands are addressed to Porod surface scattering as described in the theoretical discussion. Oscillations, resulting from the monodisperse form factor and (in the DWBA) a coherent interference of reflected, refracted and scattered waves, vary from BA to DWBA. However, results of R g Fit and R c Fit from Guinier fits ( Especially for spherical particles one may await deviations of R g , when DWBA theory is approximated with BA. As expected, the discrepancies of the unified fit results from simulated values are dependent on α i , R and σ (Figure 3). For monodisperse particle islands the quality of the applied fit is in the range of 10% over a large area in the contour diagram.

However, for α i ≥ 1.0 the fit quality reduces especially for R ≥ 7 nm. This observation can be best explained by dominating refraction effects at high α i . For the simulated conditions the penetration depth into the film material was calculated to be 11 nm 38,39 . For small particle sizes of R < 7 nm the beam's pathway in the particle is small and deviations occurring refraction are small. For R ≥ 7 nm the beam's pathway in the Au particles becomes longer, corresponding refraction effects are enhanced and deviations from BA, disregarding beam refraction, become larger.

Contour diagrams for polydisperse particle islands show enhanced fit discrepancies compared to monodisperse scatterers. Positive deviations towards higher R g Fit values indicate higher refraction contributions of big particles to GISAXS. Especially for σ = 0.6 (Figure 3c) the observed deviations seem to be high. However, in highly polydisperse particle samples defined spherical symmetries are usually difficult to achieve and knowledge of particle dimensions with mean deviations in the order of the system's polydispersity is in many cases sufficient. Concluding simulation results from Figure 3, acceptable unified fit results for particle island systems are obtained for GISAXS experiments, conducted in the range of 0.7° ≤ α i ≤ 0.85°, corresponding to 1.4 . α c (Au) ≤ α i ≤ 1.7 . α c (Au). Within the GISAXS sample to detector distances used in our experimental set-up, incident angles of α i << 0.7° are usually not recommended, due to overlap of specular scattering and off specular scattering in the Yoneda peak region. However for higher sample to detector distances as used in GIUSAXS, α i << 0.7° could be used for better fit results.

Similar to simulations from particle islands, diffuse GISAXS of TiO 2 particles buried in a polymer film matrix was simulated in dependence of α i , R and σ. For the simulated two component film system, q || scans were performed at α f = α c (TiO 2 ) = 0.26° and at the Yoneda's maximum (α f = 0.15°), which is near α c of the matrix polymer (α c = 0.15°).

Transverse q || scans at α f = 0.26°, show fit discrepancies up to 80 % from the expected value.

Fitting errors were only found to be acceptable for R < 4 nm or α i < 0.26°. Using arguments from particle island systems, refraction effects, which are not included in BA, become more important for high α i and increasing R. Within the simulated conditions the polymer matrix of 50 nm is totally transparent and the possible penetration depth into the particle phase is 30 nm. Thus refracted and reflected waves at all film interfaces interfere with particle scattered waves. These effects are less pronounced for small particles or small α i , where the beam's pathway in the particle phase is small.

Smaller deviations from DWBA simulations were obtained for q || scans at the Yoneda's maximum at α f = 0.15° (Figure 5). At these conditions the polymer matrix is still transparent for the incoming beam, while the penetration depth into the particle phase drops to 6 nm. For particles bigger than the penetration depth, the beam is not transmitted to the polymer/substrate interface. Consequently the superposition of polymer/substrate reflected wave and scattered wave is minimized. Similar to Au particle island systems deviations become larger due to enhanced refraction for increasing particle sizes and incident angles.

Polydisperse systems show deviations towards higher R g Fit values comparable to particle island systems. In contrast, fit deviations for buried particle systems are most distinctive for small particles. However, deviations in the range of 2 nm for such particle dimensions are not crucial in many experimental cases, since the usual low signal to noise ratio at high 2θ angles do not allow fits more precise than 1 nm.

Defining optimum experimental conditions we suggest incident angles in the range of 0.35° ≤ α i ≤ 0.55° corresponding to 2.5 α c (polymer) ≤ α i ≤ 3.9 α c (polymer).

Experimental Discussion

Unified analysis from rough Au films

Figure 6 shows experimental q || GISAXS data at α f ≈ α c (Au) = 0.5° obtained from the Au film measured at α i = 0.7°, which is in the proposed angular range. Studying experimental data transverse scattering from two structural levels, which are separated by a correlation peak, can be observed. The found Porod decay of ~ 4 at high q suggests the presence of particle islands, which can be approximated with the form of a hemisphere. Therefore, the use of the three dimensional averaged Eq. 10 can be used. From fit results of the 1 st structural level, one can assign the average lateral dimension of the Au particles of R g 1 = 10.9 nm.

Results from the correlation functions (Eqs. 12-14) included in the applied fit suggest that the covering particle islands are randomly arranged -as deduced from the low packing factor κ 1 = 1.8 -with a mean centre to centre distance of ξ 1 = 33 nm. In our experimental q || range we were only able to record few data-points, which account for Porod scattering of level 2.

However, the found power law decay of P 2 ≈ 1.5 can give evidence to a mass fractal, which is related to a two dimensional arrangement of Au particle islands. GISAXS q || scans from the studied Au film and corresponding unified fit results were compared with comparative SPM data from randomly distributed Au islands (Figure 7). Modelling them as hemispherical islands, mean particle island sizes of R = 13 nm corresponding to R g = 10 nm with a σ = 0.27 can be assigned. Accordingly a fit discrepancy towards SPM results of 10% can be assigned, which is even better than the error estimated by simulations. For islands centre to centre correlation lengths mean values of ξ = 32 nm ± 10 nm were obtained. The average particle island distance of 32 nm with the calculated standard deviation of σ = 0.32 is in good agreement with the average distance and the low degree of correlation κ, found by GISAXS experiments.

Unified analysis from TiO 2 /PMMA films

To test the formalism for GISAXS on buried particle systems experimentally, q || scans deduced from GISAXS in combination with comparative SEM results from spin-coated TiO 2 /PMMA hybrid material films are studied. Figure 8 shows a q || detector scan at the Yoneda's maximum at α f = 0.15°, which is near α c (PMMA) = 0.14° obtained from a GISAXS pattern taken at α i =3.9 . α c = 0.55°. At high q || a power law decay with P 1 ≈ 4 for diffuse Porod scattering of the first structural level, meaning the immersed TiO 2 particles, with R g 1 = 8.7 nm can be found. Immersed TiO 2 particles seem to be uncorrelated and far separated in the PMMA matrix. This led to negligibly small contributions of the interference function S(q || ) to the unified intensity and was therefore approximated as unity. From the 2 nd level Porod approach a power law decay in the range of 2 < P 2 < 3 was found. This finding proposes a particle arrangement in an arbitrary three dimensional mass fractal. From comparison with SEM analysis (Figure 9) a mean particle size of R = 9.2 nm, corresponding to R g = 7.1 nm with σ = 0.17 was assigned. The corresponding deviation between SEM and BA approximated GISAXS is 23%, which is in the order of the error range as proposed from simulations. To summarize GISAXS, SPM and SEM results: when transverse q || detector scans at α f = α c are performed, predictions on particle island sizes, forms and arrangements including mass fractal dimensions can be made with the unified fit approach within a reasonable accuracy.

Unified analysis from novel TiO 2 /(PEO)MA-PDMS-MA(PEO) films

In this last experimental study the usefulness of the unified fit approach for clarifying morphologies in functional films containing polydisperse colloidal particles is demonstrated.

Using conductive SPM it was suggested that electrical charges are transported through partly percolating TiO 2 /(PEO)MA-PDMS-MA(PEO) networks. This allows for applications in solar cells [START_REF] Memesa | [END_REF] . A ceramized isolating PDMS shell prevents lateral shortcuts. For this material, high resolution SEM (Figure 10) or SPM studies are unable to unravel the morphology of the film. Thus no clear conclusion on the charge transport mechanism can be made without additional experimental proof. In Figure 11 the analysed experimental GISAXS intensity of a 20 nm thick TiO 2 /(PEO)MA-PDMS-MA(PEO) film taken at α i = 0.7°, is shown. The average R g of the primary uncorrelated TiO 2 particles is found to be 4.0 nm. Despite not being in the optimum angular range the dependence of the fit accuracy on α i is not crucial for such small particles, as proposed by the presented simulations (Figure 5). From P 2 = 2.2 can be concluded that the small particles are not two dimensionally arranged in the film matrix, but in a three dimensional mass fractal. Therefore the mechanism of charge transport can be specified to electrons percolating perpendicular to the film surface through a network of TiO 2 particles. Particles are not correlated laterally, facilitating charge carrier transport perpendicular to the interface.

Summary

We have shown that the application of the unified fit equations, which were used over ten years for data analysis in transmission SAXS, USAXS and SLS, to transverse q || detector scans in GISAXS experiments is allowed within certain limits. Theoretical consideration and simulations show that the unified formalism can be used for particles with spherical or cylindrical symmetry, either placed at the free surface or buried in a film matrix. Theoretical deviations in the range of the particles polydispersity are not critical for the analysis of typical colloidal particles. We concluded from simulations that experimental incident angles should be chosen carefully before conducting the GISAXS experiment. For our Au particle island film system best results are obtained for incident angles in the range of 1.4 . α c (Au) ≤ α i ≤ 1.7 . α c (Au), while for TiO 2 containing buried film systems best results are expected in the range of 2.5 α c (Polymer) ≤ α i ≤ 3.9 α c (Polymer) with α f at the Yoneda's maximum to guarantee acceptable results. However, this range may vary for different refractive indices. It was further shown that a BA approximation for small particles of R ≤ 6 nm is less dependent on experimental angles.

Comparing experimental GISAXS results with particle analysis from SPM and SEM studies, we were able to verify mean particle island sizes and centre to centre correlation lengths. In addition, further considerations on particle arrangements, described by packing factors and mass-fractal dimensions, were shown to be useful. A clear limit to the presented approach is that polydispersity approaches used in transmission SAXS [START_REF] Beaucage | [END_REF] , can be misleading for analysis of GISAXS at α i > α c However, using the unified fit approach for GISAXS on novel functional films can help to unravel physical mechanisms where microscopic techniques like SEM or SPM would not be sufficient.

Yoneda's maximum

  at α f ≈ α c (Au) = 0.50 for the measured Au film, α f ≈ α c (PMMA) = 0.14 for the TiO 2 /PMMA film and α f ≈ α c ((PEO)MA-PDMS-MA(PEO)) = 0.14 for the TiO 2 /(PEO)MA-PDMS-MA(PEO).

  denotes the specular reflected amplitude from a smooth film surface without interior density fluctuations. The subscript S denotes the surface, while d denotes density fluctuations of the film's interior. describes the scattered intensity at the specular beam position, describes diffuse scattering from the surface roughness, can be correlated to diffuse scattering from density fluctuations within the medium. The last term includes possible correlations between scattered waves from surface roughness and density fluctuations and can in many cases be approximated to 0. With proper selection of α i and α f , minimization of refraction effects and separation of scattering at the specular beam position and non-specular, diffuse scattering at the Yoneda peak can be achieved. Thus it becomes possible to treat term one independently from term two and three 9,25 . However, the non-specular scattered intensity is still a sum of scattered intensities resulting from surface roughness and interior density fluctuations.

Figure 2 usingFigure 1 Figure 2 Figure 3

 2123 Figure 1 GISAXS geometry

Figure 4

 4 Figure 4 Contour plot of unified fit results from DWBA simulated q || scans at α f = α c (TiO 2 ) in dependence of R g and α i Contour lines represent the deviation of R g Fit from R g Sim in

Figure 5

 5 Figure 5 Contour plots of unified fit results from DWBA simulated q || scans at α f equal the

Figure 6

 6 Figure 6 Experimental GISAXS data of low correlated Au film prepared by chemical vapour

Figure 7

 7 Figure 7 SPM image of the 20 nm thick Au film.

Figure 8

 8 Figure 8 Experimental GISAXS data of uncorrelated spin coated TiO 2 /PMMA film fitted

Figure 9 Figure 10

 910 Figure 9 SEM picture of the TiO 2 particles (Solaronix T)

Figure 11

 11 Figure 11 Experimental GISAXS data of spin coated TiO 2 /(PEO)MA-PDMS-MA(PEO)

Figure 1 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10

 1678910 Figure 1

Figure 11

 11 Figure 11

Table 1

 1 Consequently in GISAXS analysis at α i > α c one should avoid BA based PDI approaches, which are commonly used in SAXS analysis.

	) follow the employed

DWBA simulated GISAXS. In contrast to R g and R c the obtained ratios of B/G appear to be highly dependent on the scattering geometry and the applied perturbation theory (e.g. BA and b www.usaxs.xor.aps.anl.gov/staff/ilavsky/irena.html DWBA).

Table 1

 1 information 2 contains a set of parameters for GISAXS of monodisperse spherical particles buried in a typical polymer matrix. H. Dosch, B. W. Batterman, and D. C. Wack, Physical Review Letters 56, 1144-1147 (1986). Unified fit results for particle types simulated in

39 H. Dosch, Physical Review B 35, 2137-2143 (1987).

Table 1

 1

			Full Sphere					Cylinder
		BA		DWBA	BA		DWBA
	G	42.7		23.4		17.0		11.3
	R g [Å]	41.5		38.0
	R c [Å]							37.7		37.8
	B	5.6	. 10	-5	3.0	. 10	-5	3.5	. 10	-4	2.2	. 10	-4
	P	4		4		3		3

Acknowledgements This research was supported by the Max Planck Society (Institutsübergreifende Forschungsinitiative FRM II), the DFG (GU771/2, MU1487/5) CSGI, MiUR and CNR-FUSINT. We gratefully thank the HASYLAB for provision of beamtime, Martin Dommach, and Ralph Döhrmann for technical support at the beam line BW4, Dr.

Rüdiger Berger for support with SPM and Maren Müller for support with SEM.

Supporting Information

IsGISAXS simulations presented in section 3 are based on input data files, which contain all the specific parameters. Supporting information 1 contains a set of parameters on which


PhD Giuseppe M Campo
email: gcampo@unime.it

Angela Avenoso

Salvatore Campo

Angela D'ascola

Giancarlo Nastasi

SMALL HYALURONAN OLIGOSACCHARIDES INDUCE INFLAMMATION BY ENGAGING BOTH TOLL-LIKE-4 AND CD44 RECEPTORS IN HUMAN CHONDROCYTES

Keywords: Hyaluronan, cytokines, chondrocytes, NF-kB, TLR-4

Small degradation fragments of hyaluronan (HA) may stimulate an inflammatory response in a variety of tissues at the injury site. HA oligosaccharides are endogenous ligands for the CD44 receptor as well as for toll-like receptor 4 (TLR-4). Previous data have shown that HA fragments may induce pro-inflammatory cytokine expression by interacting with both the CD44 receptor and TLR-4. CD44 and TLR-4 stimulation activates different inflammatory pathways that culminate with the activation of the transcriptional nuclear factor kappaB (NF-kB) which is responsible for the expression of inflammation mediators such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and interleukin-1 beta (IL-1β). The aim of this study was to investigate the inflammatory effects of very small HA oligosaccharides on both TLR-4 and CD44 involvement in normal human articular chondrocytes.

Adding HA fragments to chondrocyte cultures up-regulated CD44 and TLR-4 expression, activated NF-kB translocation and increased the pro-inflammatory cytokines TNF-α, IL-6 and IL-1β.

The addition of a specific CD44 blocking antibody reduced CD44 and all inflammatory cytokine expression as well as protein production. However, cytokine expression remained significantly higher than in untreated chondrocytes. TLR-4 expression was not affected. The treatment with TLR-4 blocking antibody decreased TLR-4 and inflammatory cytokine expression, although cytokine expression was significantly higher than in control cells. CD44 expression was unaffected.

The addition of both CD44 and TLR-4 blocking antibodies significantly reduced CD44, TLR-4 and inflammatory cytokine expression.

Introduction

Toll-like receptors are a class of proteins that play a key role in the innate immune system. They recognize structurally conserved molecules derived from microbes. Once these microbes cross physical barriers such as the skin or intestinal tract mucosa, they are recognized by TLRs which activates the innate immune response [START_REF] Beutler | Genetic analysis of host resistance: Toll-like receptor signalling and immunity at large[END_REF]. Although each TLR recognizes specific ligands, they may trigger other different molecules. Recently, the glycosaminoglycan (GAG) hyaluronan (HA) has also been identified as an inducer of TLR-4 activation, capable of causing the release of proinflammatory cytokines [START_REF] Taylor | Hyaluronan fragments stimulate endothelial recognition of injury though TLR4[END_REF][START_REF] Jiang | Regulation of lung injury and repair by Toll-like receptors and hyaluronan[END_REF].

ECM is a complex mixture of proteins, glycoproteins, proteoglycans (PGs) and GAGs. ECM plays several biological roles, in particular mechanical strength and also provides signals affecting cell adhesion, shape, migration, proliferation, survival and differentiation [START_REF] Maquart | Matrikines in the regulation of extracellular matrix degradation[END_REF][START_REF] Senior | Fragments of extracellular matrix as mediators of inflammation[END_REF]. ECM presents many domains that become active after proteolytic or glycolytic cleavage. These active ECM fragments are called matrikines and they may play different roles, in particular they may act as potent inflammatory mediators including collagen type one and four, elastin, fibronectin, laminins, entactin/nidogen, thrombospondin, HA [START_REF] Senior | Fragments of extracellular matrix as mediators of inflammation[END_REF]. Among these, HA is the most widely studied because of its varying activity depending on the different state of aggregation. HA may exist both as a soluble polymer as well as in complexes with non-covalently linked proteins called HA-binding proteins or hyaladherins. HA exists as a high-molecular-weight polymer (10 6 D) under physiological conditions. However, under inflammatory conditions HA has been shown to have greater polydispersity in size, with a preponderance of low molecular weight forms, especially after tissue injury [START_REF] Noble | Hyaluronan and its catabolic products in tissue injury and repair[END_REF]. HA functions are determined in part by the size of the molecule, in addition to the structure and its interaction with HA binding proteins.

The cluster determinant 44 (CD44) is the best-known receptor of HA [START_REF] Aruffo | CD44 is the principal cell surface receptor for hyaluronate[END_REF]. CD44 is a transmembrane glycoprotein that is widely distributed on leukocytes and several other cell types.

CD44 stimulation with HA plays a role in various physiological functions, e.g. cell adhesion, cellsubstrate interaction, and lymphocyte recruitment, as well as in pathological processes such as A c c e p t e d M a n u s c r i p t 4 inflammation and metastasis [START_REF] Heldin | Importance of hyaluronan-CD44 interactions in inflammation and tumorigenesis[END_REF]. Previous studies have reported that the separate stimulation of TLR4 and CD44 receptors may prime pro-inflammatory intermediates through NF-kB activation [START_REF] Loniewski | Sensitivity of TLR4-and -7-induced NF kappa B1 p105-TPL2-ERK pathway to TNF-receptor-associated-factor-6 revealed by RNAi in mouse macrophages[END_REF][START_REF] Hutas | CD44-specific antibody treatment and CD44 deficiency exert distinct effects on leukocytes recruitment in experimental arthritis[END_REF].

It has also been reported that fragments of HA or HA at low molecular weight are able to interact with TLR-4 and CD44 receptors, thereby stimulating inflammation or increasing the inflammatory mechanism previously induced by other agents in different cell types [START_REF] Jiang | Hyaluronan in tissue injury and repair[END_REF][START_REF] Yamawaki | Hyaluronan receptors involved in cytokine induction in monocytes[END_REF][START_REF] Fitzgerald | Ras, protein Kinase Czeta and IkB kinase 1 and 2 are downstream effectors of CD44 during the activation of NF.kB by hyaluronic acid fragments in T-24 carcinoma cells[END_REF][START_REF] Taylor | Recognition of hyaluronan released in sterile injury involves a unique receptor complex dependent on toll-like receptor 4, CD44, and MD-2[END_REF][START_REF] Campo | Differential effect of molecular size HA in mouse chondrocytes stimulated with PMA[END_REF][START_REF] Campo | Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes[END_REF]. Hence, the generation of lower molecular weight HA in pathologies may act as an endogenous danger signal, leading to the activation of both innate and acquired immunity.

As, in two separated experiments, we previously reported that low molecular weight HA (50 kD) significantly activated NF-kB-induced transcription of pro-inflammatory mediators [START_REF] Campo | Differential effect of molecular size HA in mouse chondrocytes stimulated with PMA[END_REF][START_REF] Campo | Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes[END_REF] by stimulating TLR-4 or CD44 receptors, the aim of the present study was to evaluate whether the inflammation stimulated by low molecular weight HA or other small HA fragments could be the result of the activation of both TLR-4 and CD44 receptors. For this purpose we treated normal human chondrocytes with a specific HA oligosaccharide containing only six monosaccharides (6mer) since previous reports [START_REF] Termeer | Oligosaccharides of hyaluronan are potent activators of dendritic cells[END_REF][START_REF] Nishida | Osteogenic protein-1 inhibits matrix depletion in a hyaluronan hexasaccharide-induced model of osteoarthritis[END_REF] had shown that HA at this size was able to stimulate inflammation by interacting with both receptors TLR-4 and CD44. We also evaluated the degree of inflammation by using specific selective blocking antibodies.

Materials and methods

2.1.Materials

HA 6-mer oligosaccharides as sodium salt were obtained from Cosmo Bio Co., Ltd (Tokyo, Japan).

The product was endotoxin free (endotoxin content <0.1 ng/mg). Antibodies against the TLR-4 receptor to evaluate TLR-4 protein levels and to block its activity were supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against NF-kB p50/p65 subunits and antibodies against the CD44 receptor to evaluate CD44 protein levels and to block its activity were supplied by Cell Signalling Technology (Danvers, MA, USA). Human TNF-α (cat. DTA00C), IL-6 (cat. (Minneapolis, MN, USA). Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum (FBS), L-glutamine, penicillin/streptomycin, trypsin-EDTA solution and phosphate buffered saline (PBS) were obtained from Gibco Brl (Grand Island, NY, USA). All cell culture plastics were obtained from Falcon (Oxnard, CA, USA). RNase, proteinase K, protease inhibitor cocktail, sodium dodecylsulphate (SDS) and all other general laboratory chemicals were obtained from Sigma-Aldrich S.r.l. (Milan, Italy).

Cell Cultures

Primary human chondrocytes from articular cartilage (cod. 06090702), provided by ECACC-Health Protection Agency -Cell Applications (Salisbury, UK), were cultured in 75 cm 2 plastic flasks containing 15 ml of DMEM to which 10% FBS, L-glutamine (2.0 mM) and penicillin/streptomycin (100 U/ml, 100 µg/ml) were added and then incubated at 37° C in humidified air with 5% CO2. Experiments were performed using chondrocyte cultures between the third and the fifth passage.

HA treatment

Chondrocytes were cultured in six-well culture plates at a density of 1.3 x 10 5 cells/well. Twelve hours after plating (time 0) 6-mer HA was added using three different doses of 10, 20 and 40 µg/ml.

A separate set of plates were also first treated with the specific antibody against either TLR-4 or CD44 receptors alone or with both antibodies, or/and NF-kB p50/p65 subunits and HA was added 60 minutes after the antibody treatment. Finally, the cells and medium underwent biochemical evaluation 24 hours later. Being for experimental use, 6-mer HA was diluted to a final concentration of 10-40 µg/ml, the maximum estimated endotoxin content of 0.004 ng/ml for a 40 µg/ml concentration. The study therefore included the following groups of cells: a first set of five groups (control, HA vehicle, HA 10 µg/ml, HA 20 µg/ml and HA 40 µg/ml) untreated with any A c c e p t e d M a n u s c r i p t 6 anti-TLR-4 or CD44 antibodies; a second set of the same five groups treated 60 minutes before HA addition with a specific antibody against the CD44 receptor; a third set of the same five groups again treated 60 minutes before HA addition with a specific antibody, this one against the TLR-4 receptor, a final set of the same five groups treated 60 minutes before HA addition with a specific antibodies against both the TLR-4 and CD44 receptors. The mRNA expression and the related protein production of TLR-4, CD44, TNF-α, IL-6 and IL-1β were evaluated for each set of these five groups.

RNA isolation, cDNA synthesis and real-time quantitative PCR amplification

Total RNA was isolated from chondrocytes for reverse-PCR real time analysis of TLR-4, CD44, TNF-α, IL-6 and IL-1β (RealTime PCR system, Mod. 7500, Applied Biosystems, USA) using an Omnizol Reagent Kit (Euroclone, West York, UK). The first strand of cDNA was synthesized from 1.0 µg total RNA using a high capacity cDNA Archive kit (Applied Biosystems, USA). β-actin mRNA was used as an endogenous control to allow the relative quantification of TLR-4, CD44, TNF-α, IL-6 and IL-1β. PCR RealTime was performed by means of ready-to-use assays (Assays on demand, Applied Biosystems) on both targets and endogenous controls. The amplified PCR products were quantified by measuring the calculated cycle thresholds (C T ) of TLR-4, CD44, TNFα, IL-6, IL-1β, and β-Actin mRNA. The C T values were plotted against the log input RNA concentration in serially diluted total RNA of chondrocyte samples and used to generate standard curves for all mRNAs analysed. The amounts of specific mRNA in samples were calculated from the standard curve and normalized with β-actin mRNA. After normalization, the mean value of normal cartilage cell target levels became the calibrator (one per sample) and the results are expressed as the n-fold difference relative to normal controls (relative expression levels).

A c c e p t e d M a n u s c r i p t 7

Western blot assay of TLR-4 and CD44 proteins

For SDS-PAGE and Western blotting, chondrocytes were washed twice in ice-cold PBS and subsequently dissolved in SDS sample buffer (62.5 mM Tris/HCl, pH 6.8, 2% w/v SDS, 10% glycerol, 50 mM dithiothreitol, 0.01% w/v bromophenol blue). β-actin protein was used as an endogenous control to allow the normalization of TLR-4 and CD44 proteins. Aliquots of the protein extracted (10-25 µl/well) were separated on a mini gel (10%). The proteins were blotted onto polyvinylidene difluoride membranes (Amersham Biosciences) using a semi-dry apparatus (Bio-Rad). The blots were flushed with double distilled H 2 O, dipped into methanol, and dried for 20 min before proceeding on to the next steps. Subsequently, the blots were transferred to a blocking buffer solution (1x PBS, 0.1% Tween 20, 5% w/v non-fat dried milk) and incubated for 1 h. The membranes were then incubated with the specific diluted (1:1) primary antibody in 5% bovine serum albumin, 1x PBS, and 0.1% Tween 20 and stored in a roller bottle at 4 °C overnight After being washed in three stages in wash buffer (1x PBS, 0.1% Tween 20), the blots were incubated with the diluted (1:5,000) secondary polyclonal antibody (goat anti-rabbit conjugated with peroxidase, Santa Cruz Biotechnology, Santa Cruz, CA, USA) in TBS/Tween-20 buffer containing 5% non-fat dried milk. After 45 min of gentle shaking, the blots were washed five times in wash buffer and the proteins were made visible using a UV/visible transilluminator (EuroClone, Milan, Italy) and Kodak BioMax MR films. A densitometric analysis was also run in order to quantify each band.

NF-kB p50/65 transcription factor assay

NF-kB p50/65 DNA binding activity in nuclear extracts of chondrocytes was evaluated in order to measure the degree of NF-kB activation. The analysis was carried out following the manufacturer's protocol for a commercial kit (NF-kB p50/65 Transcription Factor Assay Colorimetric, cat. n°SGT510, Chemicon International, USA). In brief, cytosolic and nuclear extraction was performed by lysing the cell membrane with an apposite hypotonic lysis buffer containing protease inhibitor A c c e p t e d M a n u s c r i p t 8 cocktail and tributylphosphine (TBP) as reducing agent. After centrifugation at 8,000 x g, the supernatant containing the cytosolic fraction was stored at -80 °C, while the pellet containing the nuclear portion was then re-suspended in the apposite extraction buffer and the nuclei were disrupted by a series of drawing and ejecting actions. The nuclei suspension was then centrifuged at 16,000 x g. The supernatant fraction was the nuclear extract. After determining protein concentration and adjustment to a final concentration of approximately 4.0 mg/ml, this extract was stored in aliquots at -80° C for the subsequent NF-kB assay. After incubation with primary and secondary antibodies, colour development was observed following the addition of the substrate TMB/E. Lastly, the absorbance of the samples was measured using a spectrophotometric microplate reader set at λ 450 nm. Values are expressed as relative optical density (OD) per mg protein.

TNF-α, IL-6 and IL-1β ELISA assay

Samples of cell-secreted protein extracted from the culture media in the presence of 1 nM PMSF and protease inhibitor cocktail were centrifuged at 13,000 rpm at 4°C for 10 min,. The analysis of TNF-α, IL-6 and IL-1β was carried out using a specific commercial kit. Briefly, 100 µ of assay diluent was added to each well of the coated microplate followed by the addition of 100 µl of standards, samples and controls to each well. After two hours of incubation at room temperature, the supernatant of each well was aspirated and the plate was washed four times. Wells were filled with 200 µl of each specific biotin-conjugate antibody. After two hours of incubation at room temperature, the liquid from the wells was discarded and the plate washed four times to which 200 µl of substrate solution was then added. After further incubation for 20 min (protected from light) 50 µl of a stop solution was added. The absorbance of each well was read spectrophotometrically, within 30 minutes, at λ 450 nm. TNF-α, IL-6 and IL-1β values are expressed as pg/ml. The amount of protein was determined using the Bio-Rad protein assay system (Bio-Rad Lab., Richmond, CA, USA) with bovine serum albumin as a standard, in accordance with the published method [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF].

Statistical analysis

Data are expressed as the mean ± S.D. values of at least seven experiments for each test. All assays were repeated three times to ensure reproducibility. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test. The statistical significance of differences was set at p<0.05.

Results

TLR-4 and CD44 mRNA expression and Western blot analysis.

TLR-4 and CD44 mRNA evaluation (Figs 1 and 2, panels A, B, C and D of each Figure) and

Western blot analysis with densitometric evaluation (Figs. 1 and2, panels E, F, G and H of each Figure) were assayed in order to estimate the degree of TLR-4 and CD44 activation in the presence/absence of 6-mer HA with/without the specific antibodies against TLR-4 (panel C), CD44 (panel B) or against both receptors (panel D). The results showed a marked dose-dependent increase in the expression and protein synthesis of the TLR-4 receptor (Fig. 1) in chondrocytes treated with 6-mer HA and those untreated with any TLR-4 and CD44 antibodies. The same results were also obtained in chondrocytes pre-treated with the antibody against the CD44 receptor. These results mean that 6-mer HA, by interacting with TLR-4, was able to increase TLR-4 expression and protein synthesis and the CD44 blocking antibody did not interfere with the activation. However, pretreatment of chondrocytes with the specific antibody against the TLR-4 produced no effect in cells exposed to 6-mer HA. This finding means that TLR-4 was the target for 6-mer HA and the pretreatment with the specific anti-TLR-4 antibody prevented TLR-4-HA interaction. Similar results

A c c e p t e d

M a n u s c r i p t 10 were obtained by pre-treating chondrocytes with both anti-TLR-4 and anti CD44 antibodies.

Analogous results were seen on evaluating CD44 mRNA expression and the related protein levels (Fig. 2). In this case 6-mer HA was also able to interact with the CD44 receptor, increasing its expression and protein levels in a dose-dependent manner. These results confirm that the CD44 receptor also functions as target for 6-mer HA Blocking this receptor prevented CD44-HA interaction and the increase of its expression. Once again, the pre-treatment of chondrocytes with the antibody against the TLR-4 receptors did not interfere with CD44-HA interaction (Figs. 1 and 2). No effects were observed in 6-mer HA un-stimulated cells, as well as no changes were seen in 6mer stimulated chondrocytes and pre-treated with the NF-kB antibodies (data not shown) .

NF-kB activation.

Fig. 3 shows the changes in the NF-kB p50/p65 heterodimer translocation over the course of the experiment, in the absence (panel A) and the presence (panels B, C and D) of antibodies. This assay was also carried out in order to estimate the initiation of inflammatory process, since the NF-kB factor can be activated by both the TLR and CD44 pathways that in turn may converge to stimulate the expression of several genes that prime/amplify inflammation. The results obtained by evaluating the NF-kB factor confirmed that 6-mer HA was able to activate NF-kB expression in a significant dose-dependent manner via both TLR-4 and CD44 receptors. Interestingly, the chondrocytes pretreated with the specific antibody against CD44 (panel B) showed a significant reduction in NF-kB activation, although activation remained significant and dose-dependent. A similar result was obtained by pre-treating chondrocytes with the specific antibody against the TLR-4 receptor (panel C). Pre-blocking both TLR4 and CD44 receptors drastically reduced NF-kB activation induced by 6-mer HA (panel D). while NF-kB activation was not completely suppressed by this pre-blocking of the two receptors, thus demonstrating that 6-mer HA may also activate NF-kB through the stimulation of other pathways that differ from TLR-4 and CD44 receptors. No significant effect was

3.3.Pro-inflammatory cytokines mRNA expression and protein levels.

TNF-α (Fig. 4), IL-6 (Fig. 5) and IL-1β (Fig. 6) mRNA evaluation (panels A, B, C, and D of each obtained by evaluating the NF-kB factor. In fact, the results showed a marked dose-dependent increase in the expression and protein synthesis of all inflammatory cytokines in chondrocytes treated with 6-mer HA but not treated with any anti-TLR-4 and anti-CD44 antibodies (panels A and E). This increase is a direct consequence of NF-kB activation by 6-mer HA-induced stimulation of TLR-4 and CD44 receptors. The pre-treatment of chondrocytes with the specific antibody against TLR-4 (panels C and G) or the specific antibody against CD44 (panels B and F) was able to significantly decrease these inflammatory cytokines as a consequence of the reduction in NF-kB activation. The pre-treatment of cells with both blocking antibodies (panels D and H) produced a further significant decrease in these inflammatory mediators although, once more, their expression was not completely suppressed, therefore suggesting other possible targets for 6-mer HA or activation of other pathways not mediated by NF-kB. The block of NF-kB translocation in chondrocytes stimulated with 6-mer HA produced a slight significant effect on the production of all pro-inflammatory cytokines. This effect was significant for TNF-α levels both in cells untreated with TLR-4 and CD44 antibodies and cells treated with TLR-4 antibodies. It was not effective in chondrocytes treated with CD44 and CD44 plus TLR-4 antibodies. The same trend was observed for IL-6 and IL-1β levels, except the fact that in this case the increment in the cytokine expression and protein production was at limit of significance. This pro-inflammatory cytokine activation, not mediated by NF-kB, but mediated by CD44 receptor could be the result of PKC activation due to 6mer HA-induced CD44 stimulation that may directly activate TNF-α which in turn may activate other pro-inflammatory cytokines such as IL-6 and IL-1β. These results taken together could mean

Hypothetical mechanism from the reformulation of the results

The data of the present study concur with results previously obtained [START_REF] Campo | Differential effect of molecular size HA in mouse chondrocytes stimulated with PMA[END_REF][START_REF] Campo | Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes[END_REF] and allow us to hypothesize the following integrated mechanism (Scheme 1). In normal conditions, native HA is produced on the inner surface of the plasma membrane and extruded into the extracellular environment. It binds TLR-4 and CD44 receptors and it is able to maintain the homeostasis of the cell. CD44 deals with normal HA degradation in small fragments and internalization and the successive passage first to endosomes and then to lysosomes where hyaluronidases, especially hyaluronidase-1 (HYAL-1), look after its further degradation (panel A). Following inflammatory stimuli, generated by different pathologies, such as arthritis, diabetes, cirrhosis, etc., native HA is massively degraded by reactive oxygen species and hydrolytic enzymes, such as hyaluronidases, chondroitinases and exosaminidases, whose expression increase dramatically during tissue injury, into small fragments, which are able to stimulate TLR-4 and CD44 receptors (panel B). The TLR-4 and CD44 stimulation exerted by these HA fragments, in turn primes inflammation by two different pathways that finally both converge in NF-kB activation. Activated NF-kB moves to the nucleus where it primes the transcription of several pro-inflammatory mediators, such as TNF-α, IL-1β and IL-6. Pro-inflammatory mediators, in turn, are responsible for HA synthase induction, particularly HA synthase-3, that leads to increased HA production, at not very high molecular weight, which is in part deposited in tissues and in part degraded via the mechanism illustrated above. The further degradation of the newly produced HA continues to stimulate TLR-4 and CD44 receptors and therefore the outcome is the amplification and the perpetuation of inflammation (panel C). High molecular weight HA production by HA synthases, especially HAS-1, by interacting with TLR-4 receptors, displaces small fragments from TLR-4 receptors and at the same time blocks TLR-4

A c c e p t e d M a n u s c r i p t 13 activity [START_REF] Campo | Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes[END_REF] by preventing TLR-4 interaction with HA fragments derived from HA degradation while CD44 provides for their removal from the environment. At the same time, high molecular weight HA may also inhibit PKC, thereby limiting NF-kB activation [START_REF] Campo | Differential effect of molecular size HA in mouse chondrocytes stimulated with PMA[END_REF] with the consequent contribution to inflammation resolution and tissue repair (panel D).

Discussion

The mechanisms that regulate the host response to non-infectious tissue injury are little understood, but increased ECM turnover is a hallmark of tissue injury. HA fragmentation during ECM degradation after acute tissue injury serves the important function of initiating the host innate immune response by providing an essential signal to macrophages and other cell types to produce chemokines that recruit other leukocyte subsets required to achieve tissue injury and to begin restoring tissue integrity [START_REF] Termeer | Oligosaccharides of hyaluronan are potent activators of dendritic cells[END_REF][START_REF] Noble | Hyaluronan and its catabolic products in tissue injury and repair[END_REF][START_REF] Stern | Hyaluronan catabolism: a new metabolic pathway[END_REF][START_REF] Noble | Matrix regulation of lung injury, inflammation, and repair[END_REF]. HA can be depolymerised into small fragments via oxygen radicals and enzymatic degradation by hyaluronidase, β-glucuronidase and hexosaminidase [23][START_REF] Deguine | Free radical depolymerization of hyaluronan by Maillard reaction products: role in liquefaction of aging vitreous[END_REF].

HA oligosaccharides may modulate immunocompetent cell types, such as dendritic cells and macrophages or in malignant cells through the TLR-4 receptor [2-3, 6, 11-12]. Recent data have demonstrated the involvement of low molecular weight HA and other GAGs in the modulation of inflammation by interacting with TLR-4 in mouse cartilage chondrocytes [START_REF] Campo | Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes[END_REF][START_REF] Campo | Glycosaminoglycans reduced inflammatory response by modulatine toll-like receptor-4 in LPS-stimulated chondrocytes[END_REF]. Articular cartilage homeostasis is in fact a result of an intricate interplay between anabolic and catabolic, antiand pro-inflammatory, anti-and pro-apoptotic mediators [START_REF] Schulze-Tanzil | Activation and dedifferentiation of chondrocytes: implications in cartilage injury and repair[END_REF], and chondrocytes are the versatile regulators of cartilage equilibrium.

The interaction of HA degradation products with CD44 also provides signals to initiate inflammation [START_REF] Heldin | Importance of hyaluronan-CD44 interactions in inflammation and tumorigenesis[END_REF][START_REF] Hutas | CD44-specific antibody treatment and CD44 deficiency exert distinct effects on leukocytes recruitment in experimental arthritis[END_REF][START_REF] Weiss | Induction of resistance to diabetes in non-obese diabetic mice by targeting CD44 with a specific monoclonal antibody[END_REF]. Other data support the hypothesis that a balance between low molecular weight HA and high molecular weight HA may control the activation of inflammation [START_REF] Steeber | A new twist to the leukocyte adhesion cascade: intimate cooperation is key[END_REF].

Therefore we suggest that high molecular weight HA was inactive on CD44 modulation, while the low molecular weight HA found at the inflammation site was active. This mechanism may be explained by the fact that, in the event of tissue destruction, high molecular weight HA is broken and destructive enzymes such as MMPs and also by facilitating the recruitment of CD44 + leukocytes [START_REF] Senior | Fragments of extracellular matrix as mediators of inflammation[END_REF][START_REF] Eberlein | Anti-oxidant inhibition of hyaluronan fragment-induced inflammatory gene expression[END_REF]. While high molecular weight HA maintains homeostasis and potentially downregulates inflammation, the generation of lower molecular weight HA may act as an endogenous danger signal leading to the activation of both innate and acquired immunity. This hypothesis, is supported by evidence that when clearance of lower molecular weight HA is lacking this leads to excess damage whereas the over-expression of high molecular weight HA is protective in the noninfectious lung injury model [START_REF] Noble | Hyaluronan and its catabolic products in tissue injury and repair[END_REF][START_REF] Jiang | Hyaluronan in tissue injury and repair[END_REF].

The identification of the TLR-4 and CD44 receptors as the target for 6-mer HA had previously been demonstrated by several studies [START_REF] Taylor | Hyaluronan fragments stimulate endothelial recognition of injury though TLR4[END_REF][START_REF] Termeer | Oligosaccharides of hyaluronan are potent activators of dendritic cells[END_REF][START_REF] Nishida | Osteogenic protein-1 inhibits matrix depletion in a hyaluronan hexasaccharide-induced model of osteoarthritis[END_REF][START_REF] De La Motte | Mononuclear leukocytes bind to specific hyaluronan structures on colon mucosal smooth muscle cells treated with polyinosinic acid:polycytidylic acid: inter-alpha-trypsin inhibitor is crucial to structure and function[END_REF][START_REF] Knudson | Hyaluronan and CD44: modulators of chondrocyte metabolism[END_REF][32][33]. We recently reported that low molecular weight HA was able to stimulate inflammation in normal articular mouse chondrocytes [START_REF] Campo | Differential effect of molecular size HA in mouse chondrocytes stimulated with PMA[END_REF][START_REF] Campo | Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes[END_REF]. In these studies it was shown that cytokines and other pro-inflammatory mediators were produced as the consequence of the interaction either between HA and TLR-4 or between HA and CD44 receptors.

Although HA stimulation of TLR-4 and CD44 receptors activated two distinct pathways, in the end both converged in NF-kB activation [START_REF] Campo | Differential effect of molecular size HA in mouse chondrocytes stimulated with PMA[END_REF][START_REF] Campo | Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes[END_REF]. However, in normal chondrocytes the effect exerted by low molecular HA was not found to be so high, although significant [START_REF] Campo | Differential effect of molecular size HA in mouse chondrocytes stimulated with PMA[END_REF][START_REF] Campo | Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes[END_REF], compared with the effect obtained by other Authors in different cell types [START_REF] Taylor | Hyaluronan fragments stimulate endothelial recognition of injury though TLR4[END_REF][START_REF] Termeer | Oligosaccharides of hyaluronan are potent activators of dendritic cells[END_REF][32][33][34], and this was probably due to the different size of the HA fragment used. In addition, the studies using chondrocytes separately evaluated TLR-4 or CD44 receptor involvement [START_REF] Campo | Differential effect of molecular size HA in mouse chondrocytes stimulated with PMA[END_REF][START_REF] Campo | Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes[END_REF] and there was no information available on the evaluation of both these receptors by HA fragment stimulation in chondrocytes. The aim of this study was therefore to evaluate at the same time both TLR-4 and CD44 involvement in human articular chondrocytes following stimulation with well-defined very small HA fragments which had been shown in previous results to have a great capacity to prime inflammation through these receptors.

A c c e p t e d M a n u s c r i p t 15

In this study, we examined the effects of 6-mer HA, at different concentrations, on both the TLR-4 and CD44 receptor modulation in human chondrocytes, compared with a series of cell groups blocked with the specific TLR-4 and CD44 receptors either separately or combined. This study suggests that 6-mer HA may interact with both receptors by stimulating a dose-dependent effect. In addition, the block of one captor at a time reduced the inflammatory effect exerted by 6-mer HA.

The block of both receptors dramatically decreased the inflammatory effect, although a significantly low effect persisted. These results prompt the following considerations: on the one hand, the inflammatory effect exerted by 6-mer HA is the sum of the inflammatory effect individually stimulated by both receptors; on the other hand, both TLR-4 and CD44 receptors contributed equally to the production of the inflammatory cytokines. Furthermore, since the blocking of both receptors did not totally suppress inflammatory cytokine production, other different inflammatory pathways are presumed to be activated by small HA fragments. For instance, pathways not NF-kB dependent could be stimulate by TLR-4 or CD44 activation. In fact, the data obtained using the antibodies against NF-kB leads to the hypothesis that mediators comprises between CD44 activation and NF-kB could be responsible of pro-inflammatory cytokine level increment. The activation of NF-kB following CD44 stimulation is mediated by PKC. As PKC may, in part, directly activate TNF-α expression (35), it is plausible that the increment in the pro-inflammatory cytokine levels, in cells in which NF-kB activity was blocked, may be due to the activation of this pathway.

In conclusion, we believe that the HA pathways should be carefully considered for future antiinflammatory strategies although further studies are needed to fully confirm these hypotheses.

6.Figure legends

  and IL-1β (cat. DLB50) commercial ELISA kits were provided by R&D Systems Inc.

A c c e p t e d M a n u s c r i p t 9 2. 8 .

 98 Protein determination.

  A c c e p t e d M a n u s c r i p t 11 seen in 6-mer HA un-stimulated cells and in 6-mer HA stimulated cells previous treated with the specific NF-kB antibodies and TLR-4/CD44 antibodies.

Figure

  Figure), and ELISA assay (panels E, F, G and H of each Figure) confirmed the previous data

A

  c c e p t e d M a n u s c r i p t 12 that small fragments of HA prime inflammation in part by interacting with both TLR-4 and CD44 receptors, since the partial block or the total block of these receptors may change the production of the inflammation mediators TNF-α, IL-6 and IL-1β.

  molecular weight HA species which have the ability to promote inflammation by inducing the release of reactive oxygen species such as NO, cytokines such as TNF-α and IL-1β

  Fieber C, Baumann P, Vallon R, Termeer C, Simon JC, Hofmann M, et al. Hyaluronanoligosaccharide-induced transcription of metalloproteases. J Cell Sci 2004; 117: 359-67. [33] Voelcker V, Gebhardt C, Averbeck M, Saalbach A, Wolf V, Weih F. Hyaluronan fragments induce cytokine and metalloprotease upregulation in human melanoma cells in part by signalling via TLR4. Exp Dermatol 2007;17:100-7 [34] Termeer C, Benedix F, Sleeman J, Fieber C, Voith U, Ahrens T, et al. Oligosaccharides of Hyaluronan activate dendritic cells via toll-like receptor 4. J Exp Med 2002;195: 99-111. [35] Mitchell PG, Cheung HS. Protein kinase regulation of tumor necrosis factor alpha stimulated collagenase and stromelysin message levels in chondrocytes. Biochem Biophys Res Commun 1993; 196:1133-1141.

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Acknowledgements

This study was supported by a PRA grant (Research Athenaeum Project 2008) from the University of Messina, Italy.

*Graphical Abstract