Laurent Couturier
email: laurent.couturier@uninv-nantes.fr

Frédéric De Geuser

Alexis Deschamps

A comparative study of Fe-Cr unmixing using differential scanning calorimetry and small-angle scattering

Keywords: unmixing, stainless steel, small-angle scattering (SAS), differential scanning calorimetry

come

A comparative study of Fe-Cr unmixing using differential scanning calorimetry and small-angle scattering

Introduction

The unmixing of Fe-Cr solid solution is a concern in many industrial applications (nuclear power plants, aeronautics…) where stainless steels -ferritic, martensitic and duplex -are employed at medium temperatures -typically in-between 300°C and 500°C, depending on the alloy composition -limiting the exposition time in this temperature range and/or restraining the use of these alloys below 300°C [START_REF] Grobner | The 885° f (475° c) embrittlement of ferritic stainless steels[END_REF][START_REF] Danoix | Atom Probe Studies of the Fe-Cr System and Stainless Steels Aged at Intermediate Temperature: A Review[END_REF][START_REF] Sahu | Effect of 475 °C embrittlement on the mechanical properties of duplex stainless steel[END_REF][START_REF] Hedström | The 475°C embrittlement in Fe-20Cr and Fe-20Cr-X (X=Ni, Cu, Mn) alloys studied by mechanical testing and atom probe tomography[END_REF][START_REF] Takahashi | Influence of spinodal decomposition structures on the strength of Fe-Cr alloys: A dislocation dynamics study[END_REF]. This very slow decomposition of the BCC phase, due to the presence of a miscibility gap in the Fe-Cr binary system, is causing continuous embrittlement of the alloy which is also known as the 475°C (885°F) embrittlement of ferritic stainless steels [START_REF] Grobner | The 885° f (475° c) embrittlement of ferritic stainless steels[END_REF][START_REF] Sahu | Effect of 475 °C embrittlement on the mechanical properties of duplex stainless steel[END_REF][START_REF] Hedström | The 475°C embrittlement in Fe-20Cr and Fe-20Cr-X (X=Ni, Cu, Mn) alloys studied by mechanical testing and atom probe tomography[END_REF]. Due to its very large impact on the industrial use of stainless steels, this Fe-Cr decomposition has been extensively studied over the past decades. Experimental characterization of the degree of unmixing has been mainly carried out using Atom Probe Tomography (APT) [START_REF] Danoix | Atom Probe Studies of the Fe-Cr System and Stainless Steels Aged at Intermediate Temperature: A Review[END_REF][START_REF] Hedström | The 475°C embrittlement in Fe-20Cr and Fe-20Cr-X (X=Ni, Cu, Mn) alloys studied by mechanical testing and atom probe tomography[END_REF][START_REF] Miller | APFIM and AEM investigation of CF8 and CF8M primary coolant pipe steels[END_REF][START_REF] Brown | Atom probe studies of spinodal processes in duplex stainless steels and single-and dual-phase Fe-Cr-Ni alloys[END_REF][START_REF] Danoix | An atom-probe investigation of some correlated phase transformations in Cr, Ni, Mo containing supersaturated ferrites[END_REF][START_REF] Hyde | A critical comparison between experimental results and numerical simulations of phase separation in the Fe-Cr system[END_REF][START_REF] Murayama | Microstructural evolution in a 17-4 PH stainless steel after aging at 400°C[END_REF][START_REF] Zhou | Quantitative Evaluation of Spinodal Decomposition in Fe-Cr by Atom Probe Tomography and Radial Distribution Function Analysis[END_REF][START_REF] Zhou | Initial clustering -a key factor for phase separation kinetics in Fe-Cr-based alloys[END_REF][START_REF] Pareige | Kinetics of G-phase precipitation and spinodal decomposition in very long aged ferrite of a Mo-free duplex stainless steel[END_REF][START_REF] Lach | Co-dependent microstructural evolution pathways in metastable δ-ferrite in cast austenitic stainless steels during thermal aging[END_REF][START_REF] Badyka | Quantification of hardening contribution of G-Phase precipitation and spinodal decomposition in aged duplex stainless steel: APT analysis and micro-hardness measurements[END_REF] and Small-Angle Scattering (using neutrons or X-rays, respectively SANS and SAXS) [START_REF] Lasalle | Further studies of spinodal decomposition in Fe-Cr[END_REF][START_REF] Furusaka | Phase Separation Process in FeCr Alloys Studied by Neutron Small Angle Scattering[END_REF][START_REF] Simon | Phase separation in a Fe-Cr-Co alloy studied by anomalous small angle X-ray scattering[END_REF][START_REF] Bley | Neutron small-angle scattering study of unmixing in Fe-Cr alloys[END_REF][START_REF] Shek | Spatial fractal characteristic of spinodal decomposition in Fe-Cr-Ni duplex stainless steel[END_REF][START_REF] Ujihara | Kinetic analysis of spinodal decomposition process in Fe-Cr alloys by small angle neutron scattering[END_REF][START_REF] Hörnqvist | Early stages of spinodal decomposition in Fe-Cr resolved by in-situ small-angle neutron scattering[END_REF][START_REF] Tissot | Comparison between SANS and APT measurements in a thermally aged Fe-19 at.%Cr alloy[END_REF][START_REF] Couturier | Microstructural evolution during long time aging of 15-5PH stainless steel[END_REF][START_REF] Das | Small-angle neutron scattering quantification of phase separation and the corresponding embrittlement of a super duplex stainless steel after long-term aging at 300°C[END_REF]. These experimental data have extensively served as a basis for establishing the validity of physically-based models, most often using Kinetic Monte Carlo modelling, and for establishing the link between the degree of unmixing and the evolution of mechanical properties [START_REF] Danoix | Atom Probe Studies of the Fe-Cr System and Stainless Steels Aged at Intermediate Temperature: A Review[END_REF][START_REF] Hedström | The 475°C embrittlement in Fe-20Cr and Fe-20Cr-X (X=Ni, Cu, Mn) alloys studied by mechanical testing and atom probe tomography[END_REF][START_REF] Hyde | A critical comparison between experimental results and numerical simulations of phase separation in the Fe-Cr system[END_REF][START_REF] Badyka | Quantification of hardening contribution of G-Phase precipitation and spinodal decomposition in aged duplex stainless steel: APT analysis and micro-hardness measurements[END_REF][START_REF] Couturier | Microstructural evolution during long time aging of 15-5PH stainless steel[END_REF][START_REF] Hyde | Spinodal decomposition in Fe-Cr alloys: Experimental study at the atomic level and comparison with computer models-II. Development of domain size and composition amplitude[END_REF][START_REF] Danoix | Hardening of Aged Duplex Stainless Steels by Spinodal Decomposition[END_REF][START_REF] Pareige | Study of phase transformation and mechanical properties evolution of duplex stainless steels after long term thermal ageing (>20 years)[END_REF][START_REF] Emo | Kinetics of secondary phase precipitation during spinodal decomposition in duplex stainless steels: A kinetic Monte Carlo model -Comparison with atom probe tomography experiments[END_REF][START_REF] Pettersson | Nanostructure evolution and mechanical property changes during aging of a super duplex stainless steel at 300°C[END_REF].

Both APT and SAS aim at characterizing the spatial fluctuations of the concentration in Cr. Results provide characteristic dimensions of these fluctuations and their amplitude, as well as auto-correlation (or radial distribution) functions. It has been recently demonstrated that a common framework could be used to derive such parameters from the two types of measurements [START_REF] Couturier | Direct comparison of Fe-Cr unmixing characterization by atom probe tomography and small angle scattering[END_REF]. However, these experimental techniques suffer from some drawbacks.

APT measurements provide very local information, which may be difficult to generalize in case of a material containing inhomogeneity at the scale of an APT tip or larger -≳ 100 nm. This is often the case in complex stainless steels, which may contain both austenite and ferrite, and a complex distribution of substitutional species in addition to Cr. Additionally, realizing APT measurements is time-consuming and intrinsic limitations exist to detect composition fluctuations of very small dimensions (sub-nanometric) [START_REF] Geuser | Metrology of small particles and solute clusters by atom probe tomography[END_REF].

Small-Angle Scattering, on the other hand, is well suited to measure a large number of samples or to realize in-situ measurements. However, Fe-Cr composition fluctuations are of small magnitude, especially during early stages of unmixing, and the contrast in electron density between Fe and Cr is small, so that the measured signals are generally weak and require the use of large scale facilities, neutron sources or synchrotron X-ray sources, with little availability for systematic routine measurements. Thus, there is a strong need for a versatile, easily accessible measurement method, for assessing the degree of unmixing of the Fe-Cr solid solution in stainless steels.

As any phase transformation, Fe-Cr unmixing results in enthalpy changes that can be measured by calorimetry methods. While the isothermal unmixing kinetics is so slow that enthalpy changes cannot be measured in situ, this is not the case of the reversion of a phase separated microstructure being heated back to a homogeneous solid solution, since this reversion will take place at a temperature higher than that of the unmixing, where the atomic mobility is larger. Differential Scanning Calorimetry (DSC) or Differential Thermal Analysis (DTA) has been occasionally used to assess the degree of phase separation in Fe-Cr based alloys [START_REF] Herny | Study by Differential Thermal Analysis of Reverse Spinodal Transformation in 15-5 PH Alloy[END_REF][START_REF] Capdevila | Reverse α-α´ phase separation in Fe-20Cr-6Al alloy[END_REF]. Being a bulk technique, with easy data analysis, such experiments are promising candidates for establishing a routine analysis tool overcoming most of the drawbacks presented above. However, there is no direct way to link the data from a DSC thermogram to the characteristics of unmixing (characteristic length and amplitude of composition fluctuations).

The present work aims at establishing such links, for the special case of the long-term aging of a precipitation hardening stainless steel 15-5PH, by performing a systematic comparison between DSC experiments and SAXS measurements, realized in anomalous mode at the Cr edge with a synchrotron source to maximize the contrast and thus the data quality. This comparison will be carried out over a wide range of aging times and temperatures, so that very early and relatively late stages of aging, with different combinations of dimension and amplitude, will be available to assess the robustness of the comparison.

2.

Experimental procedure

The present study was carried out on a 15-5PH stainless steel whose nominal composition is given in Table 1. Prior to long-term ageing, the material was subjected to a solutionizing treatment of about 80 min at 1000°C followed by an air quench and a precipitation hardening treatment of 5 h at 505°C performed by the material's supplier, Aubert & Duval. The initial microstructure consists of a martensitic matrix containing Cu precipitates with an average size of 2.6nm and a volume fraction of about 3%, and a small fraction of austenite. More details of the initial microstructure and its evolution during long term ageing can be found in our previous publication [START_REF] Couturier | Evolution of the microstructure of a 15-5PH martensitic stainless steel during precipitation hardening heat treatment[END_REF].

Long-term aging of this material was carried out at six different temperatures ranging from 290°C to 420°C for various durations up to 15,000 h for the lowest aging temperatures. For each aging condition (temperature and duration), the samples used for DSC and SAXS experiments in this work originate from the same piece of aged material and all aged pieces originate from a single material beam.

SAXS samples were cut from the various aged material blocks in the shape of 0.3 mm thin sheets which were then ground down to about 40 µm ± 20 µm using mechanical polishing. SAXS experiments were conducted at the D2AM beamline (BM02) of the European Synchrotron Radiation Facility (ESRF) using X-Ray energy just below the Cr absorption edge (5.983 keV) to benefit from the anomalous effect (contrast increase) for the Fe-Cr unmixing measurements. The detector was a 2D-CCD camera, whose images were corrected for electronic noise, spatial distortion, pixel efficiency and background noise. The measured intensity was normalized using the measurements of the transmission of each specimen and of a glassy carbon reference sample [START_REF] Zhang | Glassy Carbon as an Absolute Intensity Calibration Standard for Small-Angle Scattering[END_REF]. An azimuthal averaging has been computed from all corrected images since the measured scattered intensity for all aging condition is isotropic over a scattering vector range of [3x10 -2 Å -1 , 6x10 -1 Å -1 ]. The advancement of unmixing was characterized by 2 parameters: the amplitude of the composition fluctuations and their correlation length. They were extracted from the SAXS data using a model established in a previous work [START_REF] Couturier | Direct comparison of Fe-Cr unmixing characterization by atom probe tomography and small angle scattering[END_REF]. A detailed presentation of the SAXS data as a function of aging conditions can be found in [START_REF] Couturier | Microstructural evolution during long time aging of 15-5PH stainless steel[END_REF].

DSC samples were cut from the same aged material blocks in shape of approximately 3.5 x 3.5 x 0.8 mm 3 parallelepipeds. All DSC measurements were performed under high purity nitrogen flow. A TA Q200 DSC instrument was used, allowing to cover a scanning range of 200°C to 550°C and the use of modulated heating and cooling ramps. The scanning rate was 5°C.min -1 and the amplitude and period of the temperature's modulation were respectively 2°C and 2 min. The modulation of the temperature during scanning presents two advantages. The first one is the ability to separate the reversing and non-reversing contributions to the heat flux variation. The former is mainly due to the heat capacity of the material and the latter is due to the phase transformations occurring within the material [START_REF] Jones | The origin and interpretation of the signals of MTDSC[END_REF]. In our case, only the nonreversing heat flux was of interest since we are looking for the quantification of the solutionizing of chromium and iron composition fluctuations due to aging. The second interest of the temperature modulation is a higher instantaneous temperature scanning rate which gives rise to a stronger measured signal [START_REF] Verdonck | A discussion of the principles and applications of Modulated Temperature DSC (MTDSC)[END_REF][START_REF] Jiang | An introduction to temperature modulated differential scanning calorimetry (TMDSC): a relatively non-mathematical approach[END_REF] -being of particular importance for the very early stages of decomposition, where the signal is expected to be very low.

The whole thermal history of the material is schematically summarised in Figure 1, from its elaboration until the end of the DSC experiment.

3.

Results

Before the interpretation of the measured heat flow variation during temperature scanning, a correction of the data is needed to isolate the DSC signal originating from the reversion of the Fe-Cr unmixing only. Indeed, the material under study possesses a rather complex microstructure and possible other contributions -originating from other microstructural features -may be present in the measured data. We have to make sure that we only consider the signal contribution of the reversion of the Fe-Cr unmixing before processing the data. We will detail in the two following paragraph the two major complementary corrections we applied on the raw data.

Firstly, we draw attention to the fact that the solutionizing of the Fe and Cr composition fluctuations may be considered as a non-reversible solid state transformation -opposed to more conventional precipitates dissolution during heating and re-precipitation during cooling. Indeed, the solutionizing of the Fe and Cr composition fluctuations is rapid above their temperature of formation (400-600°C) whereas their formation (at 300-400°C) is very slow. Thus we can make the following hypothesis: the solutionizing during the heating ramp will be complete and no unmixing will occur during the cooling ramp.

To verify this hypothesis, we applied two consecutive scanning cycles to both unaged and aged specimens. These measurements showed that for the aged specimen an endothermic peak is observed during the first heating ramp, no corresponding exothermic peak is observed during the consecutive cooling ramp and no endothermic nor exothermic peaks are observed during the consecutive cycle whereas for the unaged specimen no evolution of the heat flux variation during scanning is observed between the first and the second thermic cycle (these measurements are shown in supplementary material). Moreover, the measurement obtained during the second thermal cycle for an aged specimen is similar to the ones obtained for an unaged specimen. Based on these observations, we conclude that the measurement obtained during a second heating ramp can be used as a baseline to correct the measurement obtained during the first heating ramp. We subtracted a straight line so the corrected heat flux is constant -equal to 0 -between 250 and 325°C, to eliminate a possible small deviation of the instrument baseline between the two consecutive heating ramps.

The second correction we applied to the DSC data concerns the high-temperature part of the solutionizing peak. For the most advanced stages of unmixing, the peak is spread beyond the maximum temperature of the measuring range (550°C, limitation of the instrument) and in the case of the very early stages of the unmixing corresponding to very weak signals the end of the peak may be difficult to separate from other weak signals in the high-temperature range.

To overcome these two obstacles, we chose to model the peak shape and to use an extrapolated reconstruction of the high-temperature side of the peak for the calculation of the solutionizing enthalpy. Our methodology to do so was to use the experimental data only until 30°C above the peak temperature of the endothermic reaction, and to use extrapolated data above. To estimate the extrapolated data, the experimental data was fitted in the abovementioned range with a pseudo-Voigt function, whose value was then used instead of the experimental data in the high-temperature range. An example of a complete set of the corrected data for one aging temperature -325°C -is shown in Figure 2. The corrected measured data is displayed as solid lines and the reconstructed data based on the pseudo-Voigt fit is displayed as dotted lines.

From the corrected data, we deduced two characteristics of the solutionizing of Fe and Cr composition fluctuations:

-The peak temperature. If we assume that in all cases the solutionizing of Fe-Cr unmixing is triggered at the same starting temperature, then at a constant heating rate the peak temperature represents the extent of diffusion which is needed to dissolve the Cr-rich regions and thus should be linked to the characteristic length of the Fe and Cr composition fluctuations. -The solutionizing enthalpy -the integrated heat flux, named transformation enthalpy in the following -which is linked to the advancement of the unmixing reaction, and should be related to the amplitude of the composition fluctuations.

As can be observed on the DSC dataset for multiple aging durations at a single temperature in Figure 2, both these DSC characteristics increase with aging time. This evolution of both peak temperature and transformation enthalpy at all the studied aging temperatures is displayed in Figure 3. Considering Figure 2 and Figure 3, we may also point out that DSC is a very sensitive technique if we compare these measurements to previous studies [START_REF] Couturier | Microstructural evolution during long time aging of 15-5PH stainless steel[END_REF][START_REF] Couturier | Direct comparison of Fe-Cr unmixing characterization by atom probe tomography and small angle scattering[END_REF]. Indeed, significant peak temperatures and transformation enthalpies are measured even for extremely short aging durations -down to 10 h -at low temperatures -290°C and 325°C -and a significant increase of both these Fe-Cr unmixing quantifiers may be observed for very early stages.

Discussion

As previously mentioned, all the aging conditions characterised here using DSC have already been characterised using SAXS -the material used for both techniques extracted from the same samples for each aging condition. Several authors have characterised the Fe-Cr compositions fluctuations appearing and developing during unmixing in terms of amplitude and characteristic length using APT (based on 1D autocorrelation function and radial distribution functions or RDFs more recently) [START_REF] Danoix | Atom Probe Studies of the Fe-Cr System and Stainless Steels Aged at Intermediate Temperature: A Review[END_REF][START_REF] Hyde | A critical comparison between experimental results and numerical simulations of phase separation in the Fe-Cr system[END_REF][START_REF] Zhou | Quantitative Evaluation of Spinodal Decomposition in Fe-Cr by Atom Probe Tomography and Radial Distribution Function Analysis[END_REF][START_REF] Pareige | Kinetics of G-phase precipitation and spinodal decomposition in very long aged ferrite of a Mo-free duplex stainless steel[END_REF][START_REF] Das | Small-angle neutron scattering quantification of phase separation and the corresponding embrittlement of a super duplex stainless steel after long-term aging at 300°C[END_REF][START_REF] Emo | Kinetics of secondary phase precipitation during spinodal decomposition in duplex stainless steels: A kinetic Monte Carlo model -Comparison with atom probe tomography experiments[END_REF]. In addition, we already showed that the RDFs extracted from APT datasets and from SAS data can be analysed in a common framework leading to an equivalent characterisation of the Fe-Cr composition fluctuations in terms of amplitude and characteristic length [START_REF] Couturier | Direct comparison of Fe-Cr unmixing characterization by atom probe tomography and small angle scattering[END_REF].Thus, we will use our SAXS measurements as Fe-Cr composition fluctuations reference descriptors to compare with the characteristics obtained from the DSC temperature scans.

We describe the parameters extracted from DSC data by phenomenological models whereby the prefactor of the aging time power-law can be expressed using an Arrhenius equation. For the transformation enthalpy 𝐻, it reads:

where 𝑘 0𝐻 and 𝑎 𝐻 are constants -1.1x10 13 (J. g -1 ) -𝑎 𝐻 . ℎ -1 and 0.31 respectively -, 𝑄 𝐻 is the Arrhenius function's activation energy -155 kJ.mol -1 -and 𝑡 and 𝑇 are the aging duration and temperature respectively. For the peak temperature 𝑇 𝑝 the model has a different form due to the presence of asymptotic behavior for elevated temperatures: where 𝑇 𝑝 0 , ∆𝑇 𝑝 , 𝑘 0𝑇 𝑝 and 𝑎 𝑇 𝑝 are constants -663 K, 151 K, 7.4x10 18 ℎ -1 . and 0.29 respectively -, 𝑄 𝑇 𝑝 is the Arrhenius function's activation energy -263 kJ.mol -1 -and 𝑡 and 𝑇 are the aging duration and temperature respectively.

𝐻 = (𝑘 0𝐻 . 𝑒𝑥𝑝 (- 𝑄 𝐻 𝑅𝑇 ) . 𝑡) 𝑎 𝐻 (1)
As we previously reported for the amplitude and correlation length [START_REF] Couturier | Microstructural evolution during long time aging of 15-5PH stainless steel[END_REF], the activation energies associated with the transformation enthalpy and the peak temperature are significantly different. The one associated with the peak temperature is close to the activation energy of Cr diffusion in body-centered cubic iron (240 kJ.mol -1 [START_REF] Bowen | Diffusion in Bcc iron base alloys[END_REF][41][START_REF] Huntz | Mesure des coefficients de diffusion en volume et intergranulaire du chrome radioactif dans le fer alpha[END_REF]) which was also the case of the activation energy associated with the correlation of Cr composition fluctuations (269 kJ.mol -1 ) [START_REF] Couturier | Microstructural evolution during long time aging of 15-5PH stainless steel[END_REF]. This result is consistent with the hypothesis that the peak temperature measured in DSC for the Cr composition fluctuations solutionizing is controlled by the characteristic spatial extension of these fluctuations, hence the Cr atoms diffusion distance.

On the contrary, the value associated with the transformation enthalpy is significantly smaller than the Cr diffusion activation energy, close to that associated with the Cr composition fluctuations' amplitude -126 kJ.mol -1 [START_REF] Couturier | Microstructural evolution during long time aging of 15-5PH stainless steel[END_REF]. Again, this result is consistent with the hypothesis that the transformation enthalpy is linked to the Fe-Cr unmixing advancement and not to the Cr atoms characteristic diffusion length during solutionizing. Now that we determined the activation energies associated with both peak temperature and transformation enthalpy evolution during aging, we can use an equivalent aging temperature description to represent the DSC data measured at the six aging temperatures for a single arbitrary chosen aging temperature -here 350°C, being the most relevant for the material application. This equivalent aging temperature representation is based on the existence of a single activation energy associated with the unmixing quantifiers' evolution over the whole aging temperature range considered. It is introduced in details in our previous work [START_REF] Couturier | Microstructural evolution during long time aging of 15-5PH stainless steel[END_REF]. On this simplified representation given in Figure 4, we can observe that all data points gather on )) a single master curve that can be modeled using equations ( 1) and ( 2

Conclusions

This work shows that the Fe-Cr unmixing characterization in stainless steels, which normally involves advanced instrumentation such as synchrotrons, neutrons or APT, can be as accurately performed using DSC with a quite easy to implement methodology consisting of two successive temperature scans up to 550°C. The subtraction of the two scans leads to the measurement of the transformation enthalpy and the peak temperature of the endothermic peak corresponding to the Fe and Cr atoms solutionizing. The first parameter is related to the advancement of the unmixing reaction and the second one is related to the characteristic size of the unmixing.

By comparing the characterization of the Cr composition fluctuation occurring during aging obtained by SAXS and DSC, we showed that these two are directly correlated. The phenomenological relationships presented in this work can be used to access the physical descriptors of the Cr composition fluctuations -its amplitude and characteristic length -using a DSC experiment which is much easier to implement compared to a synchrotron SAXS experiment. This may allow studies of the Fe-Cr unmixing including numerous aging conditions and/or the use of this technique to follow the long term ageing of stainless steel components during use in the industry.

Figure 1 :

 1 Figure 1: Schematic temperature evolution versus time of the DSC samples from elaboration of the alloy to the DSC experiment.

Figure 2 :

 2 Figure 2: Complete set of corrected DSC data for the aging at 325°C. The measured data is displayed in solid lines and the corresponding fitted data (high temperature part of the peak) is displayed in dotted lines.

Figure 3 :

 3 Figure 3: Kinetics of evolution of peak temperature and transformation enthalpy of the solutionizing of Fe and Cr composition fluctuautions obtained after aging for different durations at six different temperatures. Solid lines are guides for the eye.

Figure 4 :

 4 Figure 4: Equivalent aging time representation of the peak temperature (left hand side) and the transformation enthalpy (right hand side) for all aging conditions. The solid line in each graph is the master curve fitted to all data points.

  ) at the equivalent aging temperature.As we mentioned, the activation energies associated with the correlation length of Cr composition fluctuations (SAXS) and with the peak temperature (DSC) on one hand and with the amplitude of Cr composition fluctuations (SAXS) and the transformation enthalpy (DSC) on the other hand are quite close from each other. This observation may indicate a correlation between the Fe-Cr unmixing quantifiers measured using SAXS and DSC. The relationships existing between them are illustrated in Figure5by plotting the SAXS correlation length versus the DSC peak temperature (graph on the left-hand side) and the SAXS amplitude versus the DSC transformation enthalpy (graph on the right-hand side). These graphs show phenomenological relationships between the unmixing quantifiers obtained with DSC and the physical descriptors obtained with SAS. The relationships presented in this work between the SAXS and DSC characterizations allow the determination of Cr composition fluctuation's amplitude and correlation length knowing the transformation enthalpy and the peak temperature respectively.

Figure 5 :

 5 Figure 5: Comparison between the SAXS and DSC quantifiers. On the left hand side, correlation length of the Cr composition fluctuations measured in SAXS versus the peak temperature obtained in DSC. On the right hand side, amplitude of the Cr composition fluctuations measured in SAXS versus the transformation enthalpy obtained in DSC.

Table 1 : Nominal chemical composition of the studied stainless steel.

 1

	Cr	Ni	Cu	Mn	Si	Mo	Nb	C	P	S	Fe
	%wt 14.87	4.93	2.99	0.8	0.38	0.27	0.21	0.025	0.019 <0.002	bal.
	%at 15.85	4.65	2.61	0.81	0.75	0.16	0.13	0.115	0.034 <0.004	bal.

Acknowledgments

Dr. J. Hugues of CIRIMAT laboratory is thanked for realizing some of the aging heat treatments of this study. The technical staff of ESRF-D2AM beamline is thanked for technical help during the SAXS experiments. Janine Sziklasi is thanked for her help in the preparation and realization of a large part of the numerous DSC experiments.

Declarations of interest: none

Funding: This work was supported by the Agence Nationale de la Recherche (ANR, France) [grant number ANR-10-RMNP-0017]

Supplementary materials

Figure 6: Graph a) represents the heat flow evolution acquired on an aged sample during a first heating scan (solid line) together with the one acquired during a subsequent heating scan (dotted line) which can be used as a baseline. Graph b) is the result of the subtraction of this baseline from the first heating scan. Graph c) illustrates the slight linear deviation remaining in the subtracted heat flow (black straight line). Graph d) is the result of the subtraction of this slight linear deviation from the subtracted heat flow. Finally graph e) represents this corrected heat flow. In this graph the experimental data are cut off above the temperature of the peak maximum plus 30°C (solid line) the rest (dotted line) is steaming from the fit of the peak with a pseudo-Voigt function.


Dr Frank Schulze

Dr Anke Dienelt

Dr Sven Geissler

Paul Zaslansky

Prof Janosch Schoon

Dr Katja Henzler

Dr Peter Guttmann

Dr Azza Gramoun

Dr Lindsey A Crowe

Dr Lionel Maurizi

Prof Jean-Paul Vallée

Heinrich Hofmann

Georg N Duda
email: georg.duda@charite.de

Andrea Ode

N Georg

Dr Andrea Duda

Ode

Prof Heinrich Hofmann

Amino-polyvinyl Alcohol Coated Superparamagnetic Iron Oxide Nanoparticles are Suitable for Monitoring of Human Mesenchymal Stromal Cells In Vivo

Keywords: mesenchymal stromal cells, superparamagnetic iron oxide nanoparticles, polyvinylalcohol, magnetic resonance imaging, cell based therapies

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Mesenchymal stromal cells (MSCs) have gained much interest as a promising source for cellbased therapies. Their potential to regenerate damaged tissue has been attributed to their ability of self-renewal, differentiation into a variety of specialized cell types (e.g. in bone MSCs are able to differentiate into bone-forming osteoblasts) and migration towards gradients of growth factors secreted by damaged tissue. [1] Experimental cell-therapy approaches in animals using MSCs led to promising results for a number of neurological, myocardial and musculoskeletal disorders (e.g. femoral head necrosis, osteogenesis imperfecta, and osteoarthritis). [2][3][4][5][6][7] Even though numerous clinical trials have been initiated and some revealed a degree of success, a broad clinical application of such therapies is still not available. [8,9] Critical parameters for successfully transferring results from animal experiments to clinical application include the number of transplanted cell and their cultivation and delivery process. Visualizing and monitoring the temporal and spatial distribution of transplanted cells can provide valuable insight into understanding how to optimize cell delivery and/or dosing. Unfortunately, methods for non-invasive tracking of transplanted cells in vivo are still limited.

Visualization of cells in vivo can be achieved by using different molecular imaging modalities such as magnetic resonance imaging (MRI), radionuclide imaging (positron emission tomography (PET), single-photon emission computed tomography (SPECT)) and optical imaging. [10,11] Although none of these imaging techniques is optimal, MRI is still the preferred imaging modality for visualization of exogenously delivered cells, because of its non-destructive and non-invasiveness, deep penetration and high spatial resolution. [12] The most commonly used imaging agents for MRI application are superparamagnetic ironoxide nanoparticles (SPIONs), which were introduced several decades ago and have become a part of daily clinical routine use such as in imaging liver metastasis. SPIONs are nanoscaled (5 -15nm) crystals that consist of the biodegradable iron oxides magnetite (Fe 3 O 4 ) or maghemite (γ-Fe 2 O 3 ) or a mixture of both phases and exhibit magnetism only under the influence of an external magnetic field (superparamagnetism). [13] In MRI, SPIONs exhibit a negative enhancement on T2-and T2* weighted sequences, thus generating a signal change that is several magnitudes stronger compared to other contrast agents (e.g. gadolinium). [START_REF] Gossuin | Wiley interdisciplinary reviews[END_REF] To improve colloidal stability, solubility, and biocompatibility, SPIONs are coated with polymers such as dextran. [15] Most previous studies on cellular tracking of MSCs used commercially available dextran-or carboxydextran-coated SPIONs (Endorem/Feridex or Resovist, respectively). [16][17][18][19][20][21][22] However, manufacturing of both products was discontinued in 2008 and 2009, which prevents their future applications. But more importantly, these nanoparticles were originally developed to be taken up by phagocytic cells from the reticuloendothelial system (e.g. monocytes, macrophages and osteoclasts) but not by non-phagocytic cells such as MSCs. To overcome this limitation, transfection agents (TA) were used. [23] However, some TAs are reported to be toxic under certain circumstances and their influence on MSCs biology is an issue of debate. [19,[24][25][26] In addition, the colloidal stability of dextran-or carboxydextran-coated SPIONs is impaired in cell culture media, making in vitro labeling difficult. [27] Furthermore, the dextran-coating itself raises problems as it is susceptible to lysosomal degradation, resulting in exposure of cellular compartments and the cytosol to uncoated iron oxide nanoparticles and ions causing cytotoxic effects. [27,28] Therefore, it is necessary to develop SPIONs with non-toxic coatings that meet the physiochemical need for efficient cellular uptake by MSCs in vitro.

In recent years, several studies focused on the development of novel SPION-coatings for MSC-labeling. [28][29][30][31][32][33] Unfortunately, most of the previous studies suffer from missing information on either one or more of the following aspects: (1) characterization of the physiochemical properties of SPIONs, (2) exclusion of SPION-interference with the applied methods (especially fluori-and colorimetric toxicity assays), (3) proof of SPIONinternalization, (4) information on the correct dosimetry, which includes not only the administered, but also the delivered and effective cellular dose, (5) analysis of possible secondary effects introduced by SPIONs on MSC beyond their key characteristics and (6) the proof of principle for MRI visualization of SPION-labeled MSCs in vitro and in vivo. It is thus difficult to accurately interpret the results and compare them between different studies.

In this study, we describe a novel approach to address the above-mentioned challenges. Our aim was to label MSCs with amino-polyvinyl alcohol coated SPIONs (A-PVA-SPIONs) and to find a balance between cellular uptake without TAs for MRI visualization and low toxicity/impact on MSC cellular functionality. In particular, we aim (1) to develop an efficient A-PVA-SPION-labeling procedure for MSCs based on particle internalization, (2) to analyze the influence of A-PVA-SPIONs on MSC viability, proliferation, adipogenic, osteogenic and chondrogenic differentiation as well as migration and (3) to provide proof of principle for visualization of A-PVA-SPION-labeled MSCs in MRI in vitro by using MRI-phantoms and in vivo by using animal models. We hypothesize that A-PVA-SPIONs are suitable to label MSCs without provoking cytotoxicity allowing their visualization and monitoring in MRI.

Results

Development of an efficient A-PVA-SPION-labeling procedure for MSCs

Developing an efficient A-PVA-SPION labeling procedure is crucial for subsequent visualization of MSCs in MRI and requires information about the nanoparticles physiochemical properties. For example, the extent to what nanoparticles are internalized by cells is determined by characteristics like size and surface charge, i.e. hydrodynamic diameter and Zeta potential. [15] Furthermore, the size of the nanoparticle is needed for dosimetry calculations and for correct identification when confirming internalization by nanoscaleresolution imaging methods. The physiochemical properties of polymer coated SPIONs can change in response to pH (i.e. osmotic swelling) and protein concentration (i.e. formation of protein corona). [49] It is thus important to thoroughly characterize the A-PVA-SPIONs under conditions that are identical to the read-out experiments. [50] Therefore, we characterized size and surface charge of A-PVA-SPIONs not only in their solvent (HNO 3 10mM, pH 2) but also in cell culture media (physiological pH 7.4) with and without fetal calf serum (FCS) supplementation. The iron oxide crystal mean diameter was 7.2 ± 2.5 nm (Figure S1Figure 1). The SPIONs hydrodynamic diameter measures 14 ± 2 nm for the uncoated and 25 ± 3 nm for the A-PVA-coated SPION in its solvent HNO 3 (10mM, pH2). The Zeta potential of the uncoated SPIONs is at 26 ± 2 mV and slightly decreases to 20 ± 2 mV when the A-PVAcoating is added. When transferred into FCS-free DMEM, the A-PVA-SPIONs hydrodynamic diameter increases to 42 ± 2 nm, in the presence of FCS to 45 ± 2 nm. The addition of FCS to the cell culture media results in a negative shift in the Zeta potential of A-PVA-SPIONs from 21 ± 5 mV to -25 ± 5 mV (summarized in Table S1Table 1). Both, the increased hydrodynamic diameter and the negative zeta potential confirm the adsorption of proteins. [51] In conclusion, we now expect intracellular A-PVA-SPIONs with a diameter of 42 ± 2 nm to determined size of 45 ± 2 nm will be the basis for calculating the A-PVA-SPIONs colloidal behavior that is needed for establishing a dosimetry (details see Table S1).

We then investigated whether A-PVA-SPIONs are internalized by MSCs without any external support such as transfection agents or magnetic fields. To confirm cellular internalization, we used methods beyond Prussian Blue staining that allow resolution in the nanoscale: transmission electron microscopy (TEM) and transmission X-ray microscopy (TXM). For this purpose, MSCs were incubated for four hours with A-PVA-SPIONs (100µg Fe /ml) under serum-deprived conditions, which is known to be beneficial for efficient internalization. [START_REF] Teeguarden | [END_REF] The qualitative assessment of A-PVA-SPION internalization was facilitated by TEM and TXM. TEM revealed that A-PVA-SPIONs are internalized by MSCs and stored in intracellular vesicles (mean vesicle diameter: 357 ± 68.4 nm) (Figure 2). TXM supported these findings (mean vesicle diameter: 387 ±48.4 nm) and provided additional information that A-PVA-SPIONs are also found in smaller high contrast spheres (mean sphere diameter: 52 ± 9.2 nm), clusters of irregular shape, and a micron-sized cluster (length: 2000 nm; width: 291 nm) in the cytoplasm (Figure 3 and see also Video S1).

After having proven that A-PVA-SPIONs are internalized by MSCs, we sought experimental conditions to optimize their cellular dose and define the corresponding dosimetry. Reporting a comprehensive dosimetry that consists of the administered, delivered and cellular dose is crucial for the establishment of a correct dose-response relationship. [START_REF] Teeguarden | [END_REF] The administered dose itself only describes the amount of nanoparticles that was employed at the beginning of the experiment. A more relevant metric is described by the delivered dose that also takes the particles colloidal behavior and the exposure time into account and gives thus information about the amount of particles that reaches the cell monolayer. [34] Finally the cellular dose can be determined experimentally and describes the amount of A-PVA-SPIONs internalized by the cells. For this, MSCs were incubated with varying concentrations of A-PVA-SPIONs (administered dose) and the corresponding cell-bound iron (cellular dose) was determined.

After four hours, the value for cell-bound iron reaches 5.9 ± 2.5 pg Fe /cell at the lowest administered dose (50µg Fe/ml), which does not further increase significantly at higher administered doses. The TA Protamine had no beneficial effect (ANOVA, p=0.126) on this pattern (Figure 4A). However, when incubation time was extended to 24 hours, an increase of cell-bound iron was observed (ANOVA, p=0.014). After 24 hours, the cell-bound iron increases to 8.2 ± 3.6 pg Fe /cell at the lowest administered dose (50µg Fe /ml), which is again not affected by increasing the administered dose (Figure 4B). For accurate interpretation of the results and comparability with other studies, a summary of the particle dosimetry results is given in Table 2. In summary, we found that an optimized cellular dose in MSCs is reached at A-PVA-SPION-labeling for 4h under serum-deprived conditions followed by 20h under standard MSC culture conditions without the need of additional Protamine as TA.

Non-toxic A-PVA-SPIONs stimulate MSCs migration

When using A-PVA-SPIONs for MSC-labeling in cell-based therapy approaches, compromising effects on MSC survival and function have to be avoided. We thus investigated viability, multilineage differentiation and migration of MSCs after A-PVA-SPION-labeling with four different administered doses ranging from 0 to 100µg Fe /ml. Viability and proliferation of A-PVA-SPION-labeled MSCs was assessed after four and eight days and found to be unaffected compared to unlabeled MSCs (Figure 5). Notably, the amount of cell bound iron is below the critical value that leads to interference with these assays (Figure S2 andInformation S3). Differentiation of A-PVA-SPION-labeled MSCs towards the adipogenic, osteogenic, and chondrogenic phenotype was achieved without differences to their respective controls (Figure 6). Migration was analyzed in a modified wound scratch assay. A-PVA-SPION-labeled MSCs exhibit an increase in migration rate compared to unlabeled controls (Figure 7 and Video S2 and S3). Quantitative analysis revealed that this effect reaches statistical significance at the highest analyzed A-PVA-SPION concentration (50µg Fe /ml vs. control: p=0.069; 100µg Fe /ml vs. control: p=0.001; Figure 7).

Our results show that A-PVA-SPION-labeling does not affect differentiation, a key function of MSCs as defined by The International Society for Cellular Therapy (ISCT), but rather stimulates their migratory behavior. [35]

A-PVA-SPION-labeled MSCs can be visualized in MRI in vitro and in vivo

We found that labeling of MSCs with A-PVA-SPIONs had no negative effects on their viability or regenerative and therefore we investigated whether the amount of cell-bound iron was sufficient for visualization of A-PVA-SPION labeled MSCs using MRI in vitro and in vivo. To this end, cell phantoms with different numbers of A-PVA-SPION-labeled MSCs were prepared and scanned by MRI using T2 STIR and T1 VIBE sequences. A small effect could be seen using both sequences where a signal loss due to the A-PVA-SPION labeled MSCs was detected only at the highest cell concentration on the transverse plane of the MR images (Figure 8A). Acquisition of the orthogonal plane showed that the cells were concentrated at the bottom of the wells (data not shown). However, due to the small depth of the gel, which was lower than the minimum slice thickness available, the meniscal 'partial volume' effect precluded any quantification. Cell distribution was not homogenous enough to determine a precise effect of cell number on T1 and T2 star relaxation times. Nonetheless, the phantom results showed a trend in effect on T2 and indicated that MSC labeling was efficient for MRI visualization with the sequences used.

In vivo, A-PVA-SPION induced signal loss was detectable on T1 weighted (VIBE) MR images as a black region superior and anterior to the lower section of the femur 24 hours after the injection of the A-PVA-SPION-labeled MSCs into the right naïve knee joint of Lewis rats

Discussion

Our aim in this study was to develop an efficient labeling procedure for human MSCs with A-PVA-SPIONs bypassing detrimental secondary effects on MSC viability and functions and verifying the feasibility of visualizing A-PVA-SPION-labeled MSCs in MRI.

Development of an efficient labeling procedure

A major advantage of PVA is the fact that it is biocompatible and safe to use in humans as it has been in medical use for several years, such as for cartilage replacements, wound packing and contact lenses. [54] A-PVA-SPIONs are already well characterized for their physiochemical and magnetic properties. [55] In contrast to dextran-or carboxydextran-SPIONs, they exhibit excellent colloidal stability and dispersion in different cell culture media in vitro even in the presence of fetal calf serum (FCS). [START_REF] Petri-Fink | European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e[END_REF] The amine-functionalization promotes A-PVA-SPION internalization by non-phagocytic cells without the need for compromising TAs , which also applies for primary human cells such as MSCs as proven in our study. [38,56,57] Evidence of the internalization of A-PVA-SPIONs by MSCs and their subcellular location was provided by both TEM and TXM approaches. The advantages of the TXM approach over methods used in other studies are artifact-free sample preparation of the MSCs, visualization in the nanometer range and 3D spatial information, i.e. conclusive evidence of cellular internalization. The TEM approach showed A-PVA-SPIONs as high contrast particles that accumulate in intracellular vesicles. The TXM data confirmed this result, but also demonstrated that smaller high contrast spheres and irregular shaped clusters can be found.

The size of these small high contrast spheres analyzed by TXM is similar to the size of A-PVA-SPIONs in DMEM + FCS determined by PCS (TXM: 52.9 ±9 nm vs. PCS: 45 ± 2nm).

We thus assume that single A-PVA-SPIONs are either internalized individually or are a result of endosomal escapes. So far, we cannot distinguish whether the single A-PVA-SPIONs are either vesicle-bound or freely dispersed in the cytosol. Vesicle-bound single particles would indicate that A-PVA-SPIONs enter via a typical endocytosis-exocytosis route by being internalized as individual nanoparticles and further sorted into bigger vesicles like lyso-or exosomes. [58] Freely dispersed A-PVA-SPIONs could directly interact with constituents of the cytosol, i.e. proteins, mRNA, and cellular organelles, which may be other avenues of A-PVA-SPION-induced functional changes. However, further research is needed to provide conclusive evidence for one of those assumptions. Quantitative assessment of the cellular dose revealed that a higher amount of cell-bound iron can be achieved by prolonging the incubation time, but not by increasing the administered dose above 50µg Fe /ml. Similar results were already observed for the internalization of PVA-SPIONs by non-phagocytic cell lines. [38,57] These results point towards an active uptake mechanism, which is energy dependent as recently suggested. [START_REF] Petri-Fink | European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e[END_REF] A more detailed discussion of the dosimetry can be found in Information S4.

Analysis of possible secondary effects

For MSC tracking approaches in vivo, it is important that those A-PVA-SPIONs are not only non-toxic, but also do not interfere with the cells' regenerative functions. Therefore, we first focused on proliferation and multi-lineage differentiation both are key functions of MSCs as defined by The International Society for Cellular Therapy (ISCT). [35] In our study, we observed no signs of A-PVA-SPION-induced toxicity as proliferation and mitochondrial activity were unchanged similar to results observed for other cells. [START_REF] Petri-Fink | European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e[END_REF]57] Next, the MSCs' ability to differentiate into the adipogenic, osteogenic and chondrogenic lineage was investigated and was found to be unchanged. These positive results are noteworthy since a number of studies reported impaired chondrogenesis after SPION-application. [18,19,25,59] Only two of these publications report the corresponding cellular doses that were higher than the one determined in our study; 25.7 ± 0.96 pg Fe /cell and 13 -16 pg Fe /cell. [19,25] The impairment of chondrogenesis might thus be caused by a high intracellular iron load as already hypothesized. [18,25] It can be thus assumed that the cellular dose of 8.2 ± 3.6 pg/cell in our study is below a critical threshold that leads to impaired chondrogenesis. A number of in vivo studies provide evidence that exogenously delivered MSCs migrate and target specific tissues via an active mechanism. For example, when injected into femurs MSCs were later detected in the contralateral bone or MSCs implanted into the tibial bone marrow cavity were detected in the callus of the ulnar fracture site after three weeks. [60,61] Three days after injection into the tail-vein MSCs were detected at the fracture site. [62] Interestingly, in our study, migration of MSCs is increased after labeling with A-PVA-SPIONs. This effect could be advantageous in the context of cell-based therapies as exogenously delivered MSCs might migrate better in vivo. On the other hand, this effect could also be disadvantageous as it indicates cellular changes by A-PVA-SPIONs in MSCs that could influence so-far unknown parameters beyond migration. Future studies are needed to determine if the change in migration upon A-PVA-SPION-labeling has consequences for the outcome of MSC-based therapies.

Conclusion

SPION-labeling in combination with MRI is still the most promising approach for in vivo visualization of exogenously delivered cells and has gained high interest in cell-based therapies using MSCs. In the current study, we characterized the physiochemical properties of A-PVA-SPIONs, investigated their interference with viability assays and their internalization by human MSCs, report a correct dosimetry, found no impact on MSC viability and differentiation, but enhanced migration, and finally provided the proof of principle for MRI visualization of A-PVA-SPION-labeled MSCs in vitro and in vivo. The current study thus provides comprehensive information about the impact of A-PVA-SPIONs on MSCs and the feasibility of MRI visualization. In summary, the A-PVA/PVA copolymer has proven to be a suitable SPION-coating used for MSC labeling. What remains unknown is the particles' longterm fate with respect to MRI visualization of A-PVA-SPION labeled MSCs. For example, the accuracy of MRI data in vivo is compromised by the inability to distinguish signals (1) from viable and dead cells, ( 2

Experimental Section

SPION synthesis and A-PVA surface modification: A-PVA-SPION. SPIONs were synthesized following a co-precipitation protocol. [36,37] Briefly 0.064 moles of iron II from FeCl 2 and 0.128 moles of iron III from FeCl 3 were solved in 1.5 L deionized (DI) water and mixed with 120 mL of an NH 4 OH solution (25%). After 10 min the suspension was sedimented under a magnetic field and washed with DI water until pH 7. SPIONs were redispersed in 400 mL and oxidized with 160 mL HNO 3 (2M) and 240 mL Fe(NO 3 ) 3 (0.35M) under reflux for 1.5h to achieve maghemite (γ-Fe 2 O 3 ). The suspension was washed again with DI water and was dialyzed (with MWCO 12-14 kDa cellulose membrane dialysis tubing) against HNO 3 (10mM) for 3 days by changing the solution every 12h. The suspension was finally centrifuged at 30000 g for 15 min and the supernatant was kept. The final suspension of SPION had a concentration of 10 mg Fe /mL and a pH of approximately 2. Surface modification of the SPION with PVA was done following a protocol described previously. [36,38,39] PVA-OH (10 wt%; Mowiol 3-85, Kuraray Europe GmbH) and A-PVA (2 wt %; M12, Erkol.) solutions were prepared by dissolving dry PVA in ultrapure DI water and the solutions were rapidly heated for 1 hour at 90°C, cooled down, filtered at 0.45 µm with a PTFE filter syringe and stored at 4°C. 10 volumes of naked SPION were mixed with 9 volumes of PVA-OH solution (100 mg PVA OH/mL) and 1 volume of A-PVA solution (20 mg A-PVA/mL). The final A-PVA-SPION suspension (5 mg Fe /mL, pH 3) was stored at least 1 week at 4°C before further use. Human MSC isolation, cultivation and functional analysis: This study was approved by the local ethical committee; all donors gave informed written consent. Primary human MSCs were isolated from bone marrow of human donors (8 male, mean age: 59 ± 9.1 years; 7 female, mean age: 60 ± 16.6 years) undergoing hip surgery as described previously. [40] The "culture medium" was Dulbecco's modified Eagle's medium (DMEM; Low Glucose, Gibco, Grand Island, NY) with 10% fetal calf serum (FCS; Biochrom AG, Berlin, Germany), 5mM L-alanyl-L-glutamine (Gibco, Grand Island, NY), 100 U/mL penicillin plus 100 µg/mL streptomycin. Cells were counted by using CasyTT for standard cell culture (Schärfe Systems, Reutlingen, Germany) or a Neubauer chamber (C-Chip, Peqlab, Erlangen, Germany) when only small volumes of cells were available. All experiments described in this section where performed with cells from n=5 individual donors.

A-PVA-SPION characterization:

Proliferation rates were assessed by using a CyquantNF® Cell Proliferation assay kit (Life Technologies, Carlsbad, CA, United States). Cell viability was assessed using PrestoBlue® (Life Technologies, Carlsbad, CA, United States) as described earlier. [41] Briefly, 2000 MSCs/cm 2 were seeded into 48-well plates and measured after one (d1), four (d4) and eight (d8) days of culture. CyquantNF® values from d4 and d8 were normalized to d1. PrestoBlue® values were normalized to cell number determined by CyquantNF®. All measurements were performed in triplicates using a multimode microplate reader (m200 pro, Tecan). Osteogenic differentiation of 2.4 x 10 4 MSCs per 24-well was induced by supplementing culture media with 200 μM ascorbic acid, 7 mM ß-glycerol phosphate, 0.01 μM dexamethasone for 13 d. The calcified matrix was visualized by Alizarin Red S (AR) and quantified photometrical by dissolving AR in 10% cetylpyridinium chloride (readout wavelength at λ=562nm). Adipogenic differentiation was induced by supplementing culture media with 1 μM dexamethasone, 2 μM insulin, 200 μM indomethacin, 500 μM isobutylmethyl-xanthin for 14 d. Fatty acids were detected by OilRed O staining and quantified photometrical by dissolving in 100% isopropanol (readout wavelength at λ=500nm). Each experiment was conducted in triplicate. Chondrogenesis was induced by stimulating a pellet culture (3 x 10 5 cells/pellet) with FCS-free culture media plus 10 ng/mL TGF-β1, 10-7 M dexamethasone, 50 μg/mL ascorbic acid, 40 μg/mL proline, 100 μg/mL pyruvate, 6.25 μg/mL ITS, 1.25 mg/mL BSA, 5.35 mg/mL linoleic acid) for 21 d and detected by Alcian Blue staining and quantification of proteoglycan as described elsewhere with the modification for pellet cultures and optimized read out wavelength (λ= 516nm). [42] Migration was analyzed in culture inserts for self-insertion (IBIDI, Munich, Germany) in duplicate. 8x10³ cells were allowed to attach for 5h in each cavity of the insert prior to insert removal and addition of culture media with 5µg/ml Mitomycin C (Sigma-Aldrich, St. Louis, USA). Migration into the defined cell free gap (500 μm) was observed for 24h under an inverted microscope (DMI6000B, Leica, Germany) with a live cell imaging system, taking images every 20min. Assay analysis (area covered by cells) was performed with Tscratch. [43] A-PVA-SPION-labeling of MSCs and A-PVA-SPION dosimetry: Prior use, A-PVA-SPIONs underwent sonication for 1 min and pH-adjustment to neutral range (7.2 -7.6). Adherent MSCs (80-90% confluence) were washed with PBS and incubated with A-PVA-SPION containing FCS-free culture media for 4h (n=4) with fixed media height (1. taking additive transport by diffusion and sedimentation into account. [44] The error compared to the ISDD model is reasonably small compared to all the uncertainty arising from the in vitro agglomeration and formation of the protein corona, both influencing the diameter and density of the particles. The characteristic properties of the particles used for the calculation of the dose delivered to the cell surface are summarized in Table S2.

Determination of cell-bound iron: After centrifugation at 400xg, the cell pellet was dried overnight at 50°C, re-suspended in 125µl 6N HCL followed by a second overnight incubation step at 50°C. 25µl sample was then mixed with 25µl 6N HCL followed by adding 50µl of 5% K 4 [Fe(CN) 6 ] (Merck, Darmstadt, Germany). After 20min, absorbance was read at 690nm (m200 pro, Tecan, Männedorf, Switzerland) against a standard curve using FeCl 3 . Each measurement was carried out in quadruplicate. To obtain cell bound iron, iron (pg Fe /cell) was normalized to total cell number (average of 2 x 10 6 cells).

Transmission electron microscopy (TEM) and Transmission X-ray microscopy (TXM):. For TEM, 5x10 5 MSCs were trypsinized and further processed as described previously [45] . After fixation and prior embedding, fixed cells were centrifuged for pellet formation. The mean vesicle size was determined by measuring the diameter of n=4 vesicles from one representative TEM micrograph using ImageJ Software. [46] For TXM, MSC were cultivated for 24 h on gold grids (type HZB-2, Gilder Grids, Grantham, UK) coated with a perforated carbon film (Quantifoil Micro Tools GmbH, Jena, Germany) prior to A-PVA-SPION-labeling. Samples were then plunge frozen in liquid ethane and transferred into liquid nitrogen. Data acquisition using the HZB TXM at the undulator beamline U41-FSGM, electron storage ring BESSY II, Berlin, was performed as described with resolution of 0.35mm and slice thickness 0.2mm,TR/TE 22/9.5ms, Flip angle 10° and FOV 160*80mm.

For in vivo visualization of labelled cells, the A-PVA-SPION-labeled MSCs and the nonlabelled MSCs were injected intra-articularly into the right and left naïve knee joints of Lewis rats (Janvier Labs, Cedex, France) respectively. The following day, MR imaging of rat knee joints in vivo was conducted using a Siemens Magnetom® Trio 3T clinical scanner. A standard 4cm loop coil and respiratory monitoring with a pressure pad were used during the imaging session. The imaging protocol begins with a standard low-resolution localization sequence and the isotropic resolution 3D Ultra-short Echo time (UTE) double echo MR sequence fixed orthogonal and at the magnet centre. This was subsequently used to localize the correct plane for the 2D or thinner slab 3D images as well as for quantitative analysis. The protocol parameters of the sequences used were as follows:

(

  Figure 8B I, III). No signal could be seen at the region on VIBE MR images of the left knee joint where non-labeled MSCs were injected (Figure 8C I and III). These findings were confirmed using corresponding dUTE MR images where A-PVA-SPION-labeled MSCs result in positive MR enhancement and can be seen as a white region at the same position which is absent in controls (Figure 8B II, IV and 8C II, IV). Post mortem histology of the animal's knee joints confirmed the presence of A-PVA-SPION labeled MSCs (Figure S3).

  ) from internalized and excreted SPIONs and (3) from SPIONs and MSCs engulfed by macrophages. Another concern is the A-PVA-SPIONs metabolism within the body that is determined by its stability in vivo. Future work should therefore focus on research addressing 1) the A-PVA-SPIONs' retention time in the cell and elucidation of the involved endo-and exocytosis mechanisms and 2) whether the A-PVA-coating separates from the iron core resulting in renal excretion of A-PVA and integration of the SPION's iron in the body's iron metabolism. Taken together, these data help to develop A-PVA-SPIONbased MRI-tracking of MSCs towards a reliable research tool where non-invasiveness, deep penetration, and high spatial resolution are needed. Thereby, it might be possible to gain further insight into the spatial and temporal distribution of transplanted MSCs in tissue repair and thus to optimize cell-based therapies.

  Crystallite's size was measured by counting of 400 crystallites sizes on Transmission Electron Microscopy (TEM CM12; FEI Co. Philips Electron Optics, Zürich, Switzerland) pictures. Hydrodynamic diameters and Zeta potential of A-PVA-SPIONs were measured on a Photon Correlation Spectroscopy apparatus (PCS ZetaPals from Brookhaven: Laborchemie GES.M.B.H., Vienna, Austria). Uncoated SPIONs and A-PVA-SPION suspension were investigated by Fourier Transformation InfraRed spectroscopy (FTIR) showing the characteristic vibration bands for γ-Fe2O3 and A-PVA (Figure S1 and Information S2).

  3 mm) throughout different culture vessels to prevent variations in the administered dose. Protamine was used at a final concentration of 5 µg/ml (n=2). If MSCs were labeled for 24h (n=2), 10% FCS was added after 4h for sufficient cell nutrition. Finally, A-PVA-labeled MSCs were washed 6x with PBS before further use. For dosimetry calculations, we used a simplified model based on In vitro Sedimentation, Diffusion and Dosimetry model (ISDD) developed by Hinterliter et al.,

3D

  T1 gradient echo was used to detect and visualize A-PVA-SPIONs by signal loss. Parameters: TR/TE 14.3/5.9ms, flip angle 12°, fat suppression, isotropic resolution 0.31mm, and FOV 100mm, acquisition time 4 minutes 54 seconds. Difference Ultra-short Echo time imaging (dUTE) was used for A-PVA-SPION positive contrast detection and quantification. Parameters: 3D isotropic matrix 448 and 80mm FOV, giving 180 µm in all three dimensions, 50000 radial projections, UTE/TE(2) 0.07ms/2.46ms (for in-phase fat/water image), TR 9.6ms (in vivo 100 segments), flip angle 10° , acquisition time 16 minutes 54 seconds.Statistics: When performing multiple pair-wise comparisons, one-way or two-way analysis of variance (ANOVA) were performed, and p-values were adjusted using Bonferroni's p-value adjustment multiple comparison procedure. Results are presented as mean ± standard deviation (SD). P-values < 0.05 were considered statistically significant.

Figure 1 . 3 crystalsFigure 2 .

 132 Figure 1. TEM pictures of A-PVA-SPIONs. (A+B) TEM micrographs show iron oxide

Figure 3 .

 3 Figure 3. Internalized A-PVA-SPIONs are differently distributed in cytoplasm. The

Figure 4 .

 4 Figure 4. Efficient labeling of MSCs with A-PVA-SPIONs can be achieved at low

Figure 5 .

 5 Figure 5. Proliferation and viability of MSCs are not affected by A-PVA-SPION-

Figure 6 .

 6 Figure 6. Differentiation capacity of MSCs is not influenced by A-PVA-SPIONs. A-PVA-

Figure 7 .

 7 Figure 7. Migration of MSCs is stimulated by A-PVA-SPION-labeling. (A) Migration of

Figure 8 .

 8 Figure 8. Visualization of A-PVA-labeled MSCs in MRI in vitro and in vivo. (A) A

Table 1 . Physiochemical properties of A-PVA-SPIONs in different solvents Particles Medium Concentration (mg Fe /mL) ɣ Fe 2 O 3 chrystal (nm) Hydrodynamic diameter (nm) Zeta potential (mV) PVA/Fe ratio (mg PVA /mg Fe )

 1

	SPION	HNO 3 10mM	10	7.2 ± 2.5	14 ± 2	+26 ± 2	0
	PVA-SPION HNO 3 10mM	5	7.2 ± 2.5	25 ± 3	+20 ± 2	9
	PVA-SPION	DMEM	5	7.2 ± 2.5	42 ± 2	+21 ± 5	9
	PVA-SPION DMEM+FCS	5	7.2 ± 2.5	45 ± 2	-25 ± 5	9

Table 2 .

 2 Dosimetry

	administered dose
	iron per volume [μg Fe /ml]		0	50	100	200	400
	iron per area [μg Fe /cm²]		0	6.6	13.2	26.4	52.8
	delivered dose
	iron per area [μg Fe /cm²]	4h	0	0.9	1.6	3.1	6.2
		24h	0	2.3	4.1	7.9	15.3
	iron per cell [pg Fe /cell]	4h	0	3	6	12	24
		24h	0	7.5	15	30	60
	cellular dose
	iron per cell [pg Fe /cell]	4h	0.4 ± 0.3 5.9 ± 2.5 6.5 ± 3.0 7.4 ± 2.2	6.6 ± 1.9
		24h	0.7 ± 0.5 8.2 ± 3.6 9.5 ± 3.3 9.6 ± 5.7 10.8 ± 4.2

Acknowledgements

The research leading to these results has received funding from the European Union Seventh Framework Programme NMP-2008-4.0-1, GRANT AGREEMENT No 228929. We gratefully acknowledge Prof. Petra Knaus and P. Paarmann at the Institute for Chemistry and Biochemistry, Freie Universität Berlin for fruitful discussions and Petra Schrade at the Institute of Vegetative Anatomy, Charité -Universitätsmedizin, Berlin for performing the Transmission electron microscopy analysis. We thank A. Blankenstein, D. Jacobi, L. Schumann, M. Textor and Marie-Gabrielle Beuzelin for excellent technical assistance. MSCs were generously provided by the BCRT Core Unit Cell Harvesting. We thank HZB for the allocation of synchrotron radiation beamtime. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Received: ((will be filled in by the editorial staff)) Revised: ((will be filled in by the editorial staff)) Published online: ((will be filled in by the editorial staff))

previously. [47] For imaging, a zone plate objective with 25 nm outermost zone width was used.

The tilt range of the sample was from -60° to +60°. For tomographic reconstruction of the acquired Tilt series eTomo was used and visualized using CTvox (CTvox 2.6, Bruker CT, Kontich, Belgium) for 3D remodeling of the volumetric data. [48] The mean diameter of vesicles and high contrast spheres was determined from seven representative images of the tomograms z-stack using ImageJ Software (vesicles measured: n=9; high contrast spheres measured: n=80).

Visualization by MRI in vitro (phantoms) and in vivo (animals): Female Lewis rats were obtained from Janvier Labs (Cedex, France). The rats weighed between 150 and 175g and were 6-8 weeks old on arrival. They were housed in the animal facility at the University of Geneva under pathogen-free conditions in standard cages and were fed standard diet and water ad libitum. Animal handling was in accordance with guidelines of the Swiss Committee of Animal Experiments. The experimental protocol was approved by the Animal Care Committee at the University of Geneva (authorization no.1049/3580/3). MSC labeling was performed as described. A-PVA-SPION-labeled (100µg Fe /ml) MSCs were trypsinized, counted, and fixed in 4% formaldehyde (Roth, Karlsruhe, Germany). Increasing numbers of fixed cells were spun down and embedded in 200µl 3% (w/v) gelatine on a 48-well plate. The cell phantoms were scanned using the same sequences as optimized for in vivo imaging and a 15cm surface for homogeneous signal response. The experiment was carried out on 1.5T scanner. Longer scan times (10 signal averages) were needed to regain the SNR lost going to a larger coil and lower field.

The MR imaging parameters for the phantoms are as followed: A 'T2-weighted' 2D acquisition with TR/TE/TI 8640/44/160ms, Flip angle 160°, Resolution 0.26 mm, FOV 200*100 mm and slice thickness 1mm. The 'T1-weighted' gradient echo is a 3D acquisition

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

Table of content

Amino-polyvinyl alcohol coated superparamagnetic iron oxide nanoparticles (A-PVA-