How can a high level of synchrony can be reached in natural ecosystems? Need of coordination ➢ Synchrony requires the coordination of many plant-soil processes Review of latest advances in ecology, biogeochemistry & agronomy Identification of 4 systems of synchrony (coordination of processes) 1 2 3 4 Soil organic nutrient reserves Nutrient retrieved from atmosphere and soil minerals Common nutrient market Review of latest advances in ecology, biogeochemistry & agronomy Identification of 4 systems of synchrony (coordination of processes) 1 2 3 4 Soil organic nutrient reserves Nutrient retrieved from Atmosphere and soil minerals Common nutrient market Synchrony based on organic nutrient reserve 1. MAOM-based synchrony (Sync-MAOM)

Current intensive cropping systems are not sustainable on many aspects • Semi-natural forests and grasslands used as models • Which aspects should be copied ? How to translate in practices?

• Current focus on:

• General ecosystem attributes such as high diversity, root biomass, fungal biomass… • Improvement of specific functions such as N retention by plant roots, symbiotic N 2 fixation, C input to soil.

CONTEXT

Current difficulties/limits for agroecology development

• Diversity and traits of organisms (plant, soil) are extremely variables between ecosystems

• Ecosystem functioning depends on the coupling of many plant-soil processes

• Improvement of one process does not necessarily improve the sustainability of the whole ecosystem (e.g. legumes)

Need of a more systemic approach considering:

• the interactions of multiple co-occurring processes

•

••

  Continuous degradation of soil assets: • -40% of initial soil organic matter (SOM) stock within 10 years • Loss of « soil fertility » • Dependency of humankind to mineral fertilizer • Half of world population directly depends on mineral fertilizers • Peak of phosphorus extraction planned for 2050 • C-cost of ammonia production: 1,5-4 kg CO 2 /kg NH 3 Damage ecosystem health, water resource and climate • Stream, lake and coastal eutrophication • Agriculture contributes to 17% of global GHG Li et al 2019 Ornes 2022 NiD France Rapport 2020 FAO Report « Emissions due to agriculture 2000-2018) Lal 2003 CONTEXT How to develop efficient agroecological strategies ?

  the adaptation of organisms/processes to pedoclimatic contexts Introduction of the plant-soil synchrony concept breeding of acquisitive species with strong capacity of stimulating nutrient mineralization/immobilization (e.g., high C rhizodeposition) -The carbon:nutrient ratio of plant species or organic residues must be high enough to induce nutrient immobilization by I-Microbes. Ideally, the different plant species have contrasting carbon:nutrient ratios (a, c, I, j, k) -Maintaining a continuous cover of active plants fueling microbes in energy-rich C (all pictures but e) -Recycling organic nutrients at local scale (farm-watershed) to preserve soil organic reserve on the long-term (d, ebreeding of conservative species producing recalcitrant litter with reactive compounds fixing organic nutrients (e, f)* -Or/and amendment of recalcitrant organic residues harboring reactive compounds more or less charged in organic nutrients (e) -Recycling organic nutrients at local scale (farm-watershed) to preserve soil organic reserve on the long-term (d, e, f) Sync-Inorganic -Plant symbiosis with mycorrhizal fungi & N 2 fixing bacteria -Nutrients stored in bedrock, soil minerals and/or precipitates -Insertion/breeding of species with strong capacity of mobilizing nutrients from rock and soil minerals (e.g., mycorrhized roots exerting strong mechanic pressure on minerals, secreting high amount of organic acids & ligands) -Insertion of plant with deep roots colonizing bedrock (g, h, i) -Insertion of legumes (a, c, k) -Inoculation with mixed mycorrhizal fungi & N 2 fixing bacteria in highly degraded soils Sync-Market -Plant species with complementary nutritional needs -Common mycorrhizal networks -Mixing plant species with different nutrient acquisition strategies and carbon:nutrient ratios (a, c, I, j, k) -Promoting perennial plants (f, g, h, I, k) and/or permanent plant cover (all pictures but e) to fuel mycorrhizae in energy-rich carbon -No or limited use of soil tillage (b) and pesticides to preserve mycorrhizae networks -Inoculation with mixed mycorrhizal fungi in highly degraded soils Increasing overall synchrony -Synchrony systems adapted to pedoclimatic context -Complementary synchrony systems -Plant plasticity & reserve -Analyzing the soil profile and climate, defining the most adapted synchrony systems -Mixing plant species with different nutrient acquisition strategies (a, c, I, j, k) -Breeding crops species on their suitability to association -Promoting perennial plants with high reserve and organ plasticity (f, g, h, I, j, k) Thank you for your attention Let's continue to play the synchrony's doctor

•

  As productive as high-input annual crops • Accumulate soil C, retain nutrients, low GHG emissions

Joswig et al, 2022

High level of synchrony promotes -biomass production by N limitation -excess of soluble N, N losses (<5%) -building of SOM

Microbes


Max Langer
email: max.langer@creatis.insa-lyon.fr

In-Line X-Ray Phase Tomography of Bone and Biomaterials for Regenerative Medicine

The aim of this chapter is to present recent developments in X-ray tomography using in-line phase contrast and their applications to mineralised tissue, whether bone or artificial biomaterials, at micro-and nanoscale. Recently, the main efforts in reconstruction algorithms for in-line X-ray phase contrast imaging have been to push resolution towards the nanoscale and extend the possibilities for quantitative imaging to more general objects. The first is made possible by the use of X-ray optics and the second by the introduction of more advanced priors in the reconstruction. We summarise here these developments and outline recent applications of these techniques, namely, nano-tomography of the ultrastructure of bone and microtomography of bone formation in artificial bone grafts as well as in healthy growing mice. While still relatively little used in the field of regenerative medicine, we hope that these examples will stimulate further studies in this field.

Introduction

X-ray computed tomography on the micrometric scale (μCT) is nowadays a standard technique in the characterisation of biological materials such as bone [START_REF] Bonse | Developments in X-ray tomography II[END_REF][START_REF] Bouxsein | Guidelines for assessment of bone microstructure in rodents using micro-computed tomography[END_REF][START_REF] Engelke | Micro-CT. Technology and application for assessing bone structure[END_REF][START_REF] Hildebrand | Direct three-dimensional morphometric analysis of human cancellous bone: microstructural data from spine, femur, iliac crest, and calcaneus[END_REF][START_REF] Salomé | A synchrotron radiation microtomography system for the analysis of trabecular bone samples[END_REF]. Due to the penetrative nature of X-rays, it is ideally suited to study hard, opaque tissues. It is a 3D technique, which allows the precise study of certain structures such as trabecular bone, vessels, collagen texture and osteocyte lacunae without the need to resort to stereology. Further, if a sufficiently monochromatic beam is used, it is a quantitative technique permitting densitometric imaging [START_REF] Nuzzo | Quantification of the degree of mineralization of bone in three dimensions using synchrotron radiation microtomography[END_REF]. This is of special interest in the study of regenerative medicine, since characterisation of, for example, bone healing is tantamount to evaluating the progression of mineralisation in the newly formed bone tissue.

These properties make X-ray μCT a prime candidate as a characterisation tool in biomaterial research and regenerative medicine, especially in the case of mineralised tissue. The specific challenges of imaging such samples have proven somewhat limiting, however, due to insufficient sensitivity of X-ray μCT. Often, the structures of interest are lowly contrasted, for example, small mineralisation gradients in forming bone, or vessels, fat and other soft tissues embedded in hard tissues such as bone or mineralised scaffolds. A concrete example is the imaging of 3D cultures of bone cells on a mineralised substrate, where ideally newly formed bone, pre-bone matrix, bone cells, vessels as well as mineralisation of bone and substrate should be quantified. This application will be discussed more in detail below.

While certain structures, e.g. vessels, can be enhanced by the use of contrast agents [START_REF] Langer | Simultaneous 3D imaging of bone and vessel microstructure in a rat model[END_REF][START_REF] Prisby | Intermittent PTH 1-84 is osteoanabolic but not osteoangiogenic and relocates bone marrow blood vessels closer to bone forming sites[END_REF], this often poses the risk of altering the structure to be imaged. One way to increase the sensitivity of X-ray μCT is to use phase contrast [START_REF] Momose | Phase-contrast X-ray computed tomography for observing biological soft tissues[END_REF]. While several ways of achieving X-ray phase contrast exist [START_REF] Zanette | Holotomography versus X-ray grating interferometry: a comparative study[END_REF][START_REF] Lang | Experimental comparison of grating-and propagation-based hard X-ray phase tomography of soft tissue[END_REF][START_REF] Varga | Synchrotron X-ray phase nanotomography for bone tissue characterization[END_REF][START_REF] Langer | X-ray in-line phase microtomography for biomedical applications[END_REF][START_REF] Hagen | High contrast microstructural visualization of natural acellular matrices by means of phase-based x-ray tomography[END_REF][START_REF] Bravin | X-ray phase-contrast imaging: from pre-clinical applications towards clinics[END_REF], if a sufficiently coherent source, such as a synchrotron, is used, X-ray phase contrast can be achieved simply by placing the detector a small distance away from the sample [START_REF] Snigirev | On the possibilities of x-ray phase contrast microimaging by coherent high-energy synchrotron radiation[END_REF]. The contrast thus achieved is due to Fresnel diffraction. The resulting imaging modality can be several orders of magnitude more sensitive than standard (attenuation-based) X-ray imaging depending on the material (particularly in soft tissue, the contrast is enhanced). To achieve a quantitative imaging modality, an additional reconstruction step has to be performed, however: to reconstruct the phase shift in the object from the Fresnel diffraction phase contrast images through a process known as phase retrieval [START_REF] Cloetens | Holotomography: quantitative phase tomography with micrometer resolution using hard synchrotron radiation x rays[END_REF]. The resulting phase images can then be used in conjunction with tomographic imaging to create 3D reconstructions of the refractive index and by extension the mass density distribution in the object.

Here, we will show the latest developments of X-ray phase contrast μCT relevant to imaging in regenerative medicine and recent applications. Nano-tomography of bone ultrastructure shows great promise for the analysis of cellular structure as well as collagen and mineral distribution at the ultrastructural level. So far only the proof of concept has been reported, but the technique should provide plenty of opportunity for studies, e.g. of bone healing and pathology [START_REF] Langer | X-ray phase nanotomography resolves the 3D human bone ultrastructure[END_REF][START_REF] Varga | Investigation of the 3D orientation of mineralized collagen fibrils in human lamellar bone using synchrotron X-ray phase nano-tomography[END_REF][START_REF] Hesse | Canalicular network morphology is the major determinant of the spatial distribution of mass density in human bone tissue -evidence by means of synchrotron radiation phase-contrast nano-CT[END_REF][START_REF] Peyrin | Micro-and Nano-CT for the study of bone ultrastructure[END_REF][START_REF] Hesse | Accessing osteocyte lacunar geometrical properties in human jaw bone on the submicron length scale using synchrotron radiation μCT[END_REF][START_REF] Langer | 3D X-ray ultra-microscopy of bone tissue[END_REF][START_REF] Giuliani | Three years after transplants in human mandibles, histological and inline holotomography revealed that stem cells regenerated a compact rather than a spongy bone: biological and clinical implications[END_REF]. In micro-tomographic imaging, newly developed reconstruction algorithms have provided unprecedented insight in bone formation in healthy growing mice [START_REF] Bortel | Combining coherent hard X-ray tomographies with phase retrieval to generate three-dimensional models of forming bone[END_REF] and in bone cell culture in mineralised 3D substrates [START_REF] Weber | Quantitative evaluation of regularized phase retrieval algorithms on bone scaffolds seeded with bone cells[END_REF].

Method

When an X-ray beam passes through an object, it is mainly affected in two ways: it might be absorbed in the object (through the photoelectric effect), which changes its amplitude, and it might be retarded in the object, which changes its phase. For X-rays we can consider the object completely described by the complex refractive index. Both the attenuation and the phase shift induced by the object can be described as projections through the absorption and refractive index distributions. This means that both parts of the complex refractive index can be reconstructed by tomographic reconstruction if amplitude and phase can be measured or reconstructed for different angular settings of the sample.

For the relatively short propagation distances we are interested in here, usually up to 1 m, the effect of propagation on the exit wave field can be described in the framework of Fresnel diffraction. Such an image is referred to as a Fresnel diffraction pattern or a phase contrast image. Intensity is measurable in essentially every plane downstream of the object, but the phase shift is lost and has to be reconstructed from intensity images. There is a quantitative but non-linear relationship between the induced phase and the contrast.

We can distinguish two distinct imaging setups: one for phase contrast μCT and one for nano-CT.

In-line X-ray phase contrast μCT uses essentially the same imaging setup as μCT but with the detector mounted on a translation stage, or fixed at a position downstream of the sample stage, to permit the free space propagation necessary for the phase contrast fringes to become visible [START_REF] Cloetens | Observation of microstructure and damage in materials by phase sensitive radiography and tomography[END_REF] (Fig. 6.1a). This setup is limited in resolution by the scintillating screen used to convert X-rays to visible light and the visible light microscope used to image the scintillating screen onto the CCD camera. This gives a diffraction-limited resolution of ~400 nm depending on the wavelength of the light emitted by the scintillator. This is the same resolution limit as in visible light microscopy-based techniques such as histology and histomorphometry. In X-ray microscopy, X-rays are taken from an insertion device and then optionally monochromatised depending on the application. The long source to sample distance yields a high degree of spatial coherence necessary for good phase contrast fringe visibility. The sample is mounted on a translation-rotation stage (standard SR-μCT setup). The detector, consisting of a scintillator, light microscope optics and a CCD, is mounted on a translation stage to allow for free space propagation of the beam after the sample. (b) In nanoscopic imaging, the incoming (parallel) X-ray beam is first focused into a focal spot using reflective optics. The sample is placed between the focal spot and the detector in the resulting divergent beam to create geometric magnification. The sample is placed on a translation stage (with the detector position kept fixed) to vary the propagation distance and magnification [START_REF] Langer | X-ray phase nanotomography resolves the 3D human bone ultrastructure[END_REF] X-ray in-line nano-tomography uses X-ray reflective (Kirkpatrick-Baez) optics to focus the X-ray beam [START_REF] Mokso | Nanoscale zoom tomography with hard x rays using Kirkpatrick-Baez optics[END_REF]. The sample is then placed behind the focus in the resulting divergent beam (Fig. 6.1b). This setup achieves geometric magnification (projection microscopy) on the X-ray side. The image formed on the scintillator is thus already magnified and is then further magnified by the visible light optics. This setup is in theory diffraction limited by the wavelength of the X-rays (0.1-1 nm for hard X-rays) and in practice limited by the quality of the X-ray optics and the stability of the imaging setup.

In the microscopic case, the recorded images can be used directly as input to a tomographic reconstruction algorithm [START_REF] Cloetens | Phase objects in synchrotron radiation hard x-ray imaging[END_REF]. This gives rise to images that are enhanced at the interfaces between different materials in the sample. This is roughly a superposition of the standard (attenuation) tomogram and the second derivative of the real part of the 3D complex refractive index distribution and is often called edge-enhancement tomography or sometimes simply phase contrast tomography. In nano-tomography, due to the high resolution and the resulting relatively long propagation distance, the phase contrast fringes are much too delocalised to be useful in direct reconstruction [START_REF] Mokso | Nanoscale zoom tomography with hard x rays using Kirkpatrick-Baez optics[END_REF]. Here, phase retrieval becomes a mandatory step to achieve useful images.

While edge-enhanced imaging can be an interesting technique in its own right, it is often more interesting to consider the quantitative (but non-linear) relationship between phase shift induced on the beam by the object and the resulting phase contrast. This relationship can be used to reconstruct the phase shift at the exit of the sample through a process usually called phase retrieval. Phase retrieval is a nontrivial, non-linear inverse problem and is still the subject of active research [START_REF] Langer | Priors for X-ray in-line phase tomography of heterogeneous objects[END_REF][START_REF] Davidoiu | In-line phase tomography using nonlinear phase retrieval[END_REF][START_REF] Sixou | Absorption and phase retrieval with Tikhonov and joint sparsity regularizations[END_REF][START_REF] Davidoiu | Non-linear phase tomography based on Fréchet derivative[END_REF][START_REF] Ruhlandt | Three-dimensional phase retrieval in propagation-based phase-contrast imaging[END_REF][START_REF] Kostenko | Phase retrieval in in-line x-ray phase contrast imaging based on total variation minimization[END_REF][START_REF] Langer | Fourier-wavelet regularization of phase retrieval in x-ray in-line phase tomography[END_REF][START_REF] Hehn | Nonlinear statistical iterative reconstruction for propagation-based phasecontrast tomography[END_REF][START_REF] Rositi | Computer vision tools to optimize reconstruction parameters in x-ray inline phase tomography[END_REF]. We focus here on two recent developments of particular interest for imaging in regenerative medicine, namely, extension of linear phase retrieval algorithms to include multi-material and heterogeneous objects [START_REF] Langer | Priors for X-ray in-line phase tomography of heterogeneous objects[END_REF][START_REF] Langer | X-ray in-line phase tomography of multimaterial objects[END_REF] and the use of a non-linear conjugate gradient method to improve the resolution in the reconstruction [START_REF] Langer | X-ray phase nanotomography resolves the 3D human bone ultrastructure[END_REF].

Due to the physics of the image formation, the transfer of information from phase to contrast in the low spatial frequency range is low (this can be understood, e.g. through observing that the phase contrast is approximately proportional to the second derivative of the phase shift, as mentioned above). Because of this, the reconstructed refractive index can be affected by strong low-frequency noise [START_REF] Langer | Quantitative comparison of direct phase retrieval algorithms in in-line phase tomography[END_REF]. This can strongly inhibit the possibility to quantitatively analyse the reconstructed samples. This kind of artefact has been handled by introducing information in the low frequency range from the measured attenuation [START_REF] Paganin | Simultaneous phase and amplitude extraction from a single defocused image of a homogeneous object[END_REF][START_REF] Langer | Regularization of phase retrieval with phase-attenuation duality prior for 3-D holotomography[END_REF].

By assuming that the object is homogeneous, the phase (and equivalently the attenuation, considering strict proportionality between attenuation and phase) can be estimated from a single phase contrast image [START_REF] Paganin | Simultaneous phase and amplitude extraction from a single defocused image of a homogeneous object[END_REF]. This has proven to be a very successful method due to the facility of using a single phase contrast image. Great care must be taken when imaging heterogeneous objects, however, especially if quantitative (densitometric) results are required: the ratio between the real (related to phase) and imaginary (related to attenuation) part of the refractive index must vary within a narrow range, and a careful calibration must be performed to correct for the bias introduced by the homogeneity assumption [START_REF] Marinescu | Synchrotron radiation X-ray phase micro-computed tomography as a new method to detect Iron oxide nanoparticles in the brain[END_REF].

A more recent development seeks to overcome the limitation of homogenous objects by using an attenuation tomogram as prior knowledge on the low spatial frequencies of the phase [START_REF] Langer | Priors for X-ray in-line phase tomography of heterogeneous objects[END_REF][START_REF] Langer | X-ray in-line phase tomography of multimaterial objects[END_REF]. A tomographic scan at a short distance (depending on the resolution; short enough that phase contrast fringes are not too visible) is reconstructed and then processed in various ways to achieve an estimate of the low spatial frequencies of the real part of the refractive index. This new volume is then forward projected and used as a prior in a least square minimisation problem to retrieve the phase. This approach has some advantages, namely, it is quantitative even for non-homogeneous objects (not for all objects, however; it is somewhat dependent on the composition. Some materials, for example, bone, show a convenient quantitative, non-linear functional relationship between attenuation and phase). It also permits (somewhat paradoxically) to reduce the imaging dose by recording images at several distances, fractioning the dose between the images [START_REF] Frachon | Dose fractionation in synchrotron radiation x-ray phase micro-tomography[END_REF]. The disadvantages, on the other hand, are the increased complexity of using several distances. Each sample requires somewhat longer total acquisition time (due to camera displacements; the exposure time can actually be reduced for a given final image quality) and more storage space, and the images at each projection angle have to be realigned through image registration (albeit this problem has been thoroughly addressed in literature) [START_REF] Weber | Registration of phase-contrast images in propagation-based X-ray phase tomography[END_REF]. Also, this method can clearly not be used in in situ and high frame rate imaging due to the requirement of images at several distances.

The two approaches mentioned above are based on linearised versions of the forward problem, however. In high-resolution imaging (approximately <1 μm in practice), the non-linear contributions start to be important. This presents itself mainly as a loss of resolution. Several methods to overcome this problem have been suggested [START_REF] Langer | X-ray phase nanotomography resolves the 3D human bone ultrastructure[END_REF][START_REF] Davidoiu | Non-linear phase tomography based on Fréchet derivative[END_REF][START_REF] Ruhlandt | Three-dimensional phase retrieval in propagation-based phase-contrast imaging[END_REF][START_REF] Moosmann | Nonlinear phase retrieval from single-distance radiograph[END_REF][START_REF] Davidoiu | Nonlinear approaches for the single-distance phase retrieval problem involving regularizations with sparsity constraints[END_REF]. The most successful so far seems to be the method based on a non-linear conjugate gradient algorithm [START_REF] Langer | X-ray phase nanotomography resolves the 3D human bone ultrastructure[END_REF]. This algorithm must be initialised with a reconstruction sufficiently close to the desired reconstruction, usually with a reconstruction using a linear algorithm, to yield good results. It has been shown to improve resolution of the reconstructions in the nano-tomography case, although there is no reason to believe it should not improve image quality in the microscopic case as well. This would be achieved at the cost of a substantially increased computation time (albeit fully acceptable using current computational resources: currently around 10 min per projection, fully parallelisable over the projections due to the independence of the projections in tomographic imaging). It should be noted that this type of algorithm does not seem to improve the low spatial frequency content of the images. Regularisation-based methods such as the ones mentioned above seem to be mandatory to resolve this problem.

Applications

Nano-tomography of Bone

Nano-tomography has proven to be a very interesting imaging modality for bone imaging. This is due to the ability of X-rays to image the otherwise opaque material in 3D coupled with the ability to reach resolutions below 100 nm, which is important for the imaging of the osteocyte network, which presents structures down to approximately 100 nm [START_REF] Pacureanu | Nanoscale imaging of the bone cell network with synchrotron X-ray tomography: optimization of acquisition setup[END_REF]. While imaging of the osteocyte network was the first intended application, the first in-line nano-tomography study of bone revealed that the high sensitivity of the technique made possible to also study the collagen structure of bone, as well as mineralisation on the <100 nm length scale [START_REF] Hesse | Canalicular network morphology is the major determinant of the spatial distribution of mass density in human bone tissue -evidence by means of synchrotron radiation phase-contrast nano-CT[END_REF][START_REF] Pacureanu | Nanoscale imaging of the bone cell network with synchrotron X-ray tomography: optimization of acquisition setup[END_REF].

Analysis of Collagen Structure

Effectively, the lacuno-canalicular network (LCN) can be imaged over a relatively large field of view (Fig. 6.2) with very good spatial resolution. The bone matrix appears strongly textured, showing an arching structure (Fig. 6. 2a-c). This arching structure can be identified, by comparison to qBEI [START_REF] Kingsmill | Mineralisation density of human mandibular bone: quantitative backscattered electron image analysis[END_REF] and TEM [START_REF] Besseau | Liquid crystalline assemblies of collagen in bone and in vitro systems[END_REF] images, to be due to the oblique virtual cutting of the mineralised collagen fibrils. The texture due to the collagen fibrils is sufficiently contrasted to be studied directly in 3D using texture analysis [START_REF] Varga | Investigation of the 3D orientation of mineralized collagen fibrils in human lamellar bone using synchrotron X-ray phase nano-tomography[END_REF].

Using this technique, it can be seen that the mineralised collagen fibrils in this sample are organised in a plywood structure, consisting of parallel quasi-planes, where the fibrils have the same alignment. The collagen structure in bone has been hypothesised to act as a crack arresting boundary and thus protect vessels and nerves embedded in canals within the bone [START_REF] Fratzl | Nature's hierarchical materials[END_REF]. A qualitative validation of the texture analysis can be performed by constructing a virtual 3D fibril model, where the local orientation is represented by cylindrical stream lines. This model compares well with the corresponding sub-volume of the original greyscale image. The arch pattern is clearly reproduced (Fig. 6.3). This illustrates the necessity of 3D analysis: the complex arrangement of the collagen fibrils cannot be retrieved from 2D sections.

Finite Element Modelling of Osteocytes

The porosity in bone is at least as important as the tissue matrix itself, both biologically and mechanically. In cortical bone, on the micrometric scale, the pore network is formed by the osteocyte lacunae hosting the cell bodies and the canaliculi (small tunnels connecting lacunae of neighbouring cells [START_REF] Schneider | Towards quantitative 3D imaging of the osteocyte lacuno-canalicular network[END_REF][START_REF] Currey | Cavities in the compact bone in tetrapods and fish and their effect on mechanical properties[END_REF]. Given the presumed importance of the osteocyte in the coordination of other bone cells and in remodelling [START_REF] Bonewald | The amazing osteocyte[END_REF][START_REF] Qing | Demonstration of osteocytic perilacunar/canalicular remodeling in mice during lactation[END_REF], the LCN has recently received increasing attention. Imaging of this pore network is an important, but not yet fully solved challenge.

An unresolved issue with the LCN was the difference in strain affecting bone in vivo (thousands microstrains) [START_REF] Group | In vivo measurement of human tibial strains during vigorous activity[END_REF] and the strain required to stimulate osteocytes in vitro (tens of thousands of microstrains) [START_REF] You | A model for strain amplification in the actin cytoskeleton of osteocytes due to fluid drag on pericellular matrix[END_REF]. One hypothesis is that there is a "strain amplification mechanism" affecting the osteocytes for them to sense deformation, for example, through the morphology of the LCN.

To test this hypothesis, in-line phase tomography was used to image human femoral cortical bone. Imaging provided volumes with 102 μm field of view and 50 nm voxel size. Rectangular volumes of interest (VOIs) were selected from the images to analyse the LCN and deformations of osteocytes. Each VOI contained a single lacuna with its canaliculi (Fig. 6.4a). The geometry of the pores was extracted using an ad hoc segmentation algorithm using thresholding, morphological operations and a connectivity enhancement algorithm [START_REF] Varga | Strains experienced by osteocytes in situ as predicted by case specific finite element analysis[END_REF]. In each VOI, the lacuna and canaliculi were separated into two distinct compartments, and several morphological parameters of both were quantified (Fig. 6.4b). These included volumes, surfaces, descriptors of shape as well as number, diameter and spacing of canaliculi.

Case-specific finite element models of the VOIs were created. The models included the extracellular matrix surrounding the pores, the pericellular soft tissue matrix in the lacuna and the canaliculi, as well as the cell body and processes of the osteocyte (Fig. 6.4c). The osteocytes are not directly visible in the images. Therefore, the extent of the cells was extrapolated based on observations using electron microscopy [START_REF] Anderson | Idealization of pericellular fluid space geometry and dimension results in a profound underprediction of nano-microscale stresses imparted by fluid drag on osteocytes[END_REF][START_REF] Mcnamara | Attachment of osteocyte cell processes to the bone matrix[END_REF]. Material properties were modelled as linear elastic and isotropic, with parameters assigned based on literature data [START_REF] Verbruggen | Strain amplification in bone mechanobiology: a computational investigation of the in vivo mechanics of osteocytes[END_REF]. A confined uniaxial compressive deformation along the direction the longest lacunar dimension was simulated in the models, with magnitude set to the physiologically relevant 1000 microstrains [START_REF] Group | In vivo measurement of human tibial strains during vigorous activity[END_REF].

The finite element analysis showed that the magnitude of the strain at the dendrite/cell body junction reached up to 70 times the externally applied deformations, and further local peaks were observed in the dendrites (Fig. 6.4d). Resulting strain magnitudes are in the range reported to stimulate osteocytes in vitro [START_REF] You | A model for strain amplification in the actin cytoskeleton of osteocytes due to fluid drag on pericellular matrix[END_REF]. This means that the shape of the osteocytes contribute to a strain amplification effect. The case-specific models predicted higher strain amplification factors compared not only to idealised ones [START_REF] Mccreadie | Osteocyte lacuna size and shape in women with and without osteoporotic fracture[END_REF][START_REF] Bonivtch | Tissue strain amplification at the osteocyte lacuna: a microstructural finite element analysis[END_REF][START_REF] Deligianni | Multilevel finite element modeling for the prediction of local cellular deformation in bone[END_REF] but also to previously reported case-specific simulations using either a different image source [START_REF] Verbruggen | Strain amplification in bone mechanobiology: a computational investigation of the in vivo mechanics of osteocytes[END_REF] or a less fine modelling approach [START_REF] Schneider | Image-based finite element models for the investigation of osteocyte mechanotransduction[END_REF].

In-Line Phase Micro-tomographic Imaging of Bone Formation

A very interesting application of in-line X-ray phase contrast tomography is the imaging of mineralised three-dimensional porous biomaterials used as scaffolds in bone tissue engineering and more generally for 3D bone cell culture [START_REF] Dorozhkin | Calcium orthophosphates as bioceramics: state of the art[END_REF][START_REF] Eliaz | Calcium phosphate bioceramics: a review of their history, structure, properties, coating technologies and biomedical applications[END_REF]. The desired properties of these biomaterials are a surface favourable for cell attachment, that they stimulate bone cell precursors to differentiate into mature bone cells and that they can contribute to bone regeneration [START_REF] Glazer | In vivo evaluation of calcium sulfate as a bone graft substitute for lumbar spinal fusion[END_REF]. They should also be resorbable by the receiving body and be replaced by new, living bone. While bone formation and resorption can, to a certain extent, be quantified by SR-μCT and microdiffraction [START_REF] Komlev | Kinetics of in vivo bone deposition by bone marrow stromal cells into porous calcium phosphate scaffolds: an X-ray computed microtomography study[END_REF][START_REF] Cancedda | Bulk and interface investigations of scaffolds and tissue-engineered bones by X-ray microtomography and X-ray microdiffraction[END_REF], these techniques lack the sensitivity to resolve soft and weakly mineralised tissue. Imaging of soft tissue in mineralised scaffolds is challenging due to the opacity of the structure to visible light and the necessary trade-off between sufficient penetration and attenuation of X-rays to achieve the necessary sensitivity using standard μCT. Quantification of soft tissue formation is important for the characterisation of such biomaterials, since it constitutes the sites where new bone formation is possible. The increased sensitivity of phase tomography, however, has enabled the analysis of soft issues in mineralised scaffolds [START_REF] Langer | Regularized phase tomography enables study of mineralized and unmineralized tissue in porous bone scaffold[END_REF]. Further, different soft tissues can be differentiated in the images, such as collagen, fat, vessels and cells [START_REF] Weber | Quantitative evaluation of regularized phase retrieval algorithms on bone scaffolds seeded with bone cells[END_REF].

Imaging of Mineralised Bone Scaffolds Cultured with Bone Cells In Vitro

As an illustration, we show the imaging of Skelite bone scaffolds seeded with human osteoblasts. Skelite is a porous biomaterial consisting of 67% silicon-stabilised tricalcium phosphate (Si-TCP) and 33% hydroxylapatite (HA). It has an open pore structure similar to human cancellous bone, with a pore size between 200 and 500 μm and a porosity level around 60% [START_REF] Sayer | Structure and composition of silicon-stabilized tricalcium phosphate[END_REF][START_REF] Reid | Phase formation and evolution in the silicon substituted tricalcium phosphate/apatite system[END_REF]. Disks of Skelite measuring 9 mm in diameter and 1.2 mm in thickness were seeded with osteoblasts and cultured for 8 weeks. Samples were washed in phosphate-buffered saline, fixed in paraformaldehyde, stored in 70% ethanol and then dried before imaging. The samples were mounted three and three in a Perspex cylinder, separated by fibre tissue paper and lightly compressed with a foam material to take advantage of the 7 mm vertical field of view. Imaging was performed using undulator radiation monochromatised to 30 keV using a single Si crystal monochromator. A 10 μm Gadox scintillating screen, standard light microscope optics and a FreLoN CCD camera were used for detection, set to yield a pixel size of 5 μm. Tomographic data sets comprising 2000 angles of view over a 180° rotation were recorded over 3 distances (0.01, 0.33 and 0.99 m), with an exposure time of 0.4 s. Phase retrieval was performed using a multi-material prior. The two δ/β-ratios were calculated using the XOP software and set to 335 for Skelite and 2000 for soft tissue.

The reconstruction is shown in (Fig. 6.5). Attenuation and phase tomograms are shown to illustrate the increased visibility of the soft tissue in the phase tomogram. This enables segmentation, visualisation (Fig. 6.5d) and quantitative analysis (such as volume, volume filling fraction and local thickness) of the soft tissue phase. Additionally, sensitivity was sufficiently high to permit segmentation of the osteoblasts. These types of measurements can be used to evaluate the osteoconductive and osteoinductive properties of different scaffold types, as well as the impact of different culture conditions, for example, different biochemical environments or microgravity.

Imaging of Forming Bone in Young Mice

The sensitivity of X-ray phase contrast imaging permits to reveal the main characteristics of the important tissue compartments in forming bones, including the rapidly changing soft tissue and the partially or fully mineralised bone regions, while revealing subtle density differences in 3D. Growing femur bone midshafts of healthy mice at 1, 3, 7, 10 and 14 days postpartum were imaged. It was possible to create exquisitely detailed maps of the juxtaposed soft, partially mineralised and highly mineralised bone, giving insight into the environment in which bone cells create and shape the bone matrix [START_REF] Bortel | Combining coherent hard X-ray tomographies with phase retrieval to generate three-dimensional models of forming bone[END_REF].

Growing bone exhibits a complex architecture in 3D, which undergoes extensive reorganisation and restructuring, from the initially deposited bone material to the mature bone. Consequently, these forming tissues exhibit a range of very dif-ferent morphologies. Different morphologies can be observed in the same skeleton during growth or healing, for example, following fracture or biomaterial implantation. When non-mineralised osteoid matrix starts to mineralise, bone material in both formation and healing conditions appears as a highly porous structure, where mineralised and soft tissues are intermixed. Such early formed bone tissues gradually transform and rearrange into solid cortical bone that eventually takes on the appearance of the mature tissue [START_REF] Miller | Accretion of bone quantity and quality in the developing mouse skeleton[END_REF][START_REF] Manjubala | Spatial and temporal variations of mechanical properties and mineral content of the external callus during bone healing[END_REF][START_REF] Lange | Fetal and postnatal mouse bone tissue contains more calcium than is present in hydroxyapatite[END_REF][START_REF] Preininger | Spatial-temporal mapping of bone structural and elastic properties in a sheep model following osteotomy[END_REF][START_REF] Sharir | Muscle force regulates bone shaping for optimal load-bearing capacity during embryogenesis[END_REF][START_REF] Vetter | The mechanical heterogeneity of the hard callus influences local tissue strains during bone healing: a finite element study based on sheep experiments[END_REF][START_REF] Rohrbach | The early phases of bone healing can be differentiated in a rat osteotomy model by focused transverse-transmission ultrasound[END_REF][START_REF] Bortel | Long bone maturation is driven by pore closing: a quantitative tomography investigation of structural formation in young C57BL/6 mice[END_REF]. The initial tissue morphology is transient and contains zones of markedly different morphologies. Different degrees of mineralisation can be observed in adjacent sites at the same time. Due to the dynamically changing tissue geometry, mechanical properties are difficult to define and measure. Detailed information about both architecture and density is thus necessary for understanding and possibly predicting bone growth and tissue repair. In-line phase tomography was used for the quantification and characterisation of the temporal evolution of morphology and mineralisation during bone genesis in healthy mice. The left femur was dissected from female wild-type C57BL/6 mice aged 1, 3, 7, 10 and 14 days after birth. Skin and muscle was partially removed, while the bones remained intact. Samples were fixed and stored in 70 wt% ethanol at 4 °C. The samples were imaged in an ethanol atmosphere, mounted within small polypropylene cylindrical vials. A filtered "pink" X-ray beam at 26 keV was used to record tomographic scans of 3999 images at five sample-to-detector distances with an exposure time of 100 ms per image. The detector was a custommade indirect system consisting of a 10×/0.3NA Olympus objective combined with a thin-single crystal scintillator and a pco.edge sCMOS camera [START_REF] Douissard | A versatile indirect detector design for hard X-ray microimaging[END_REF]. Phase retrieval was performed using the multi-material algorithm outlined in Sect. 2, with a delta-to-beta ratio of 429.9 in mineralised bone and 1857 elsewhere. The local mass density can be accessed through a simple scaling of the resulting phase tomograms [START_REF] Langer | X-ray phase nanotomography resolves the 3D human bone ultrastructure[END_REF].

Histogram analysis was used to calibrate slight offsets in the background (air) in density data [START_REF] Hesse | Canalicular network morphology is the major determinant of the spatial distribution of mass density in human bone tissue -evidence by means of synchrotron radiation phase-contrast nano-CT[END_REF]. The images were segmented into bone and soft tissue compartments using standard thresholding [START_REF] Bortel | Long bone maturation is driven by pore closing: a quantitative tomography investigation of structural formation in young C57BL/6 mice[END_REF]. 3D renderings were performed using CTVox (Bruker-microCT, Kontich, Belgium) by mapping colour and transparency in the transfer functions to tissue density.

Renderings are shown in Fig. 6.6, where blue represents soft tissue, green represents bone and red represents very high density within the bone (presumably mineralised cartilage). Cutouts are shown to reveal what would be masked by the encasing soft tissue. During development, the 3D distribution of mineralised material changes from a loosely connected foam-like structure (1-7 days) into a condensed bone cortex, sandwiched between soft tissue layers. High-density islands (possibly mineralised cartilage) seem to be encapsulated within the bone compartment.

The mineralised tissue only is shown in Fig. 6.7. The cortical bone develops from a highly porous scaffold consisting of mineralised struts into the mature solid ring seen in the samples obtained from 14-day-old animals. It can be seen in the high-resolution cutouts that the bone morphology changes extensively during maturation, through a redistribution of high-density regions located within the lowerdensity bone. In addition to the qualitative morphological information, the phase tomograms permit quantification of 3D local mass density. The soft tissue was measured to have a density of 1.1-1.4 g/cm 3 at all ages. These values are slightly higher than published values for soft (skeletal) tissue (1.0-1.06 g/cm 3 ) [START_REF] Bortel | Combining coherent hard X-ray tomographies with phase retrieval to generate three-dimensional models of forming bone[END_REF]. The observed higher values could be due to higher concentrations of mineral precursor phases in the soft tissue. Higher density has similarly been revealed by other methods in forming bone tissue [START_REF] Langer | X-ray phase nanotomography resolves the 3D human bone ultrastructure[END_REF][START_REF] Langer | Regularized phase tomography enables study of mineralized and unmineralized tissue in porous bone scaffold[END_REF][START_REF] Lange | Fetal and postnatal mouse bone tissue contains more calcium than is present in hydroxyapatite[END_REF][START_REF] Golub | Role of matrix vesicles in biomineralization[END_REF] The amount of bone increases with increasing age and contains a high-density component, presumably mineralised cartilage [START_REF] Vanleene | Relationships between density and Young's modulus with microporosity and physico-chemical properties of Wistar rat cortical bone from growth to senescence[END_REF][START_REF] Bach-Gansmo | Calcified cartilage islands in rat cortical bone[END_REF][START_REF] Shipov | Unremodeled endochondral bone is a major architectural component of the cortical bone of the rat (Rattus norvegicus)[END_REF], much of which is later replaced with less dense material (bone).

Perspectives

There is a growing interest in phase sensitive X-ray imaging methods in regenerative medicine. This is mainly motivated by the high resolution and sensitivity offered by the technique. We presented here examples of imaging studies using phase tomography: nano-tomography of bone and micro-tomography of growing bone in healthy growing mice and in artificial bone constructs. Similar approaches could be used to study bone tissue growth and maturation in adult animals, during healing. Indeed, much remains unknown about the similarities and differences in the 3D bone tissue dynamics when comparing bone growth/genesis and fracture and tissue healing, for example, in response to implantation of biomaterials. The ability to resolve fine density variations in different tissues might allow the elucidation of the bone formation process in the tissue engineering scenario.

Fig. 6 . 1

 61 Fig. 6.1 Imaging setups in synchrotron radiation propagation-based phase contrast imaging. (a)In X-ray microscopy, X-rays are taken from an insertion device and then optionally monochromatised depending on the application. The long source to sample distance yields a high degree of spatial coherence necessary for good phase contrast fringe visibility. The sample is mounted on a translation-rotation stage (standard SR-μCT setup). The detector, consisting of a scintillator, light microscope optics and a CCD, is mounted on a translation stage to allow for free space propagation of the beam after the sample. (b) In nanoscopic imaging, the incoming (parallel) X-ray beam is first focused into a focal spot using reflective optics. The sample is placed between the focal spot and the detector in the resulting divergent beam to create geometric magnification. The sample is placed on a translation stage (with the detector position kept fixed) to vary the propagation distance and magnification[START_REF] Langer | X-ray phase nanotomography resolves the 3D human bone ultrastructure[END_REF]

Fig. 6 . 2

 62 Fig. 6.2 Nano-tomography of bone. (a-c) Virtual cuts through human cortical bone. The LCN is exceptionally well contrasted. The strongly contrasted arching structure in the matrix presumably due to oblique cutting of the mineralised collagen fibres, as well as well-resolved cement lines. (d) Volume rendering of all LCN porosity in a tomogram showing a relatively large number of lacunae. (e) Zoom on one lacuna and its canaliculi (pink) and the cement line (green). The LCN is rendered in unprecedented detail. The spatial relationship to structures such as the cement line can be studied

Fig. 6 . 3

 63 Fig. 6.3 Qualitative validation of a texture analysis approach to quantify mineralised collagen fibril orientation. Left: crop of a nano-CT image of a human femoral cortical bone. Grey values represent the inverted mass density in arbitrary units. Scale bar: 10 μm. Middle: cylinders representing the measured fibril orientation if 600 μm thick sections along one line. Right: 3D image generated based on extracted orientations with the fibrils shown in blue and the matrix in white. The arc pattern in the oblique cut corresponds well to that in the original image (left)

Fig. 6 . 4

 64 Fig. 6.4 Quantification of lacunar-canalicular morphology and simulation of strains on the osteocyte. (a) One lacuna and associated canaliculi cropped from the nano-CT image. (b) Surface rendering of the segmented lacuna (yellow) and canaliculi (red). (c) Finite element mesh of the mineralised extracellular matrix (purple and blue), the pericellular soft tissue (yellow) and the canaliculi (red), the cell body (light green) and processes (turquoise). The latter two were inferred based on literature data. Parts of the model are transparent to allow visualisation of all compartments. The direction of the compression of 1000 microstrains is also shown. (d) Minimum (compressive) principal strain on the osteocyte by FEM, showing peaks in the deformation (red: >10,000 microstrains, a strain amplification of at least 10 times) at the junctions of the dendrites and cell body as well as at certain locations of the dendrites further away from the lacuna

Fig. 6 . 5

 65 Fig. 6.5 Phase μCT imaging of mineralised scaffolds. (a) Standard μCT image of a Skelite disk seeded with osteoblasts. (b) Phase tomogram of the same disk, showing clearly the deposited soft tissue. It can be seen that soft tissue is formed mainly inside the scaffold construct and that the volume filling fraction is high. (c) Zoom on the marked area in (b). There is sufficient contrast to segment not only scaffold and soft tissue but the osteocytes as well. (d) Rendering of the scaffold and the segmented osteocytes

Fig. 6 . 6

 66 Fig. 6.6 Renderings of the mass density distribution in growing mouse femurs at different time points. (a) Soft tissue (blue) surrounds the mineralised bone (green). Islands of high mineral density (presumably mineralised cartilage) exceeding 1.9 g/cm 3 (red) can be seen. (b) The denser islands can be better observed on magnified cutouts

Fig. 6 . 7

 67 Fig. 6.7 3D renderings of the mineralised tissue only from Fig. 6.6. Bone (green) and high-density zones (red) with increasing magnifications (a-c) are shown. The high mass density (exceeding 1.9 g/cm 3 ) is always found within regions of lower density. Note the drastic architectural changes from 1 to 14 days, changing from a foam-like open network to a solid tube-like cortical structure


How to develop efficient agroecological strategies to reduce the carbon footprint of agrosystems?

• Semi-natural forests and grasslands as models • As productive as high-input annual crops • Accumulate soil C, retain nutrients, low GHG emissions

• Which aspects should be copied ? How to translate in practices?

• Current focus : ecosystem attributes such as high diversity, high root and fungal biomass… • Improvement of specific functions

• Need of a more systemic approach considering:

• the interactions of multiple co-occurring processes and organisms, • the adaptation of organisms/processes to pedoclimatic contexts

➢

  Ecosystem productivity & sustainability linked to the level of synchrony between plant N demand and soil supply Natural ecosystems : high synchrony -biomass production by N limitation -excess of soluble N, N losses (<5%) -building of SOM ➢ N recycling and BNF account for 90 and 10% of plant N uptake Cleveland et al 2013; Cameron et al 2013 The strong C footprint of annual crops due to a low demand/supply synchrony Microbes Dependence to fertilizer ➢ N losses -> water pollution, GHG ➢ SOM depletion -> C emissions Strong C footprint + Robertson et al., 2012; Fowler et al., 2017; Zhang et al., 2021 How can a high level of synchrony can be reached in natural ecosystems? Need of coordination ➢ Synchrony requires the coordination of many plant-soil processes Review of latest advances in ecology and biogeochemistry 1. Identification of 4 systems of synchrony (coordination of processes) 2. Dominance of these different systems change according to pedoclimatic context -> different agroecological practices to adapt to local conditions 3. Plants partly drive their own nutrition -> Challenges the classical view of fertility as an intrinsic property of soil -> Ecological intensification of production C'est extra Example of system of coordination based on nutrient storage in mineral-associated organic matter (MAOM) Implications of MAOM-based synchrony for the design of sustainable/productive agrosystems Synchrony Biological conditions of synchrony Combination of practices to set up for promoting the targeted synchrony Sync-MAOM -Acquisitive plant species -Continuous activity of microbes M & I -Insertion/selection of acquisitive species with strong capacity of stimulating nutrient mineralization/immobilization (e.g., high C rhizodeposition) -The carbon:nutrient ratio of plant species or organic residues must be high enough to induce nutrient immobilization by I-Microbes. Ideally, the different plant species have contrasting carbon:nutrient ratios (a, c, I, j, k) -Maintaining a continuous cover of active plants fueling microbes in energy-rich C (all pictures but e) -Recycling organic nutrients at local scale (farm-watershed) to preserve soil organic reserve on the long-term (d, e, f) 2) System of synchrony based on FreeOM ➢ Ecosystems with thick organic layer (Heathlands, alpine grasslands…) 3) System of synchrony based on nutrient retrieved from atmosphere and soil minerals (of synchrony based on a common nutrient market emerging from mycorrhizal networks Dominance of 3 systems of synchrony according to pedoclimatic context and plant communities selection of acquisitive species with strong capacity of stimulating nutrient mineralization/immobilization (e.g., high C rhizodeposition) -The carbon:nutrient ratio of plant species or organic residues must be high enough to induce nutrient immobilization by I-Microbes. Ideally, the different plant species have contrasting carbon:nutrient ratios (a, c, I, j, k) -Maintaining a continuous cover of active plants fueling microbes in energy-rich C (all pictures but e) -Recycling organic nutrients at local scale (farm-watershed) to preserve soil organic reserve on the long-term (d, e, fselection of conservative species producing recalcitrant litter with reactive compounds fixing organic nutrients (e, f)* -Or/and amendment of recalcitrant organic residues harboring reactive compounds more or less charged in organic nutrients (e) -Recycling organic nutrients at local scale (farm-watershed) to preserve soil organic reserve on the long-term (d, e, f) Sync-Inorganic -Plant symbiosis with mycorrhizal fungi & N 2 fixing bacteria -Insertion/selection of species with strong capacity of mobilizing nutrients from rock and soil minerals (e.g., mycorrhized roots exerting strong mechanic pressure on minerals, secreting high amount of organic acids & ligands) -Insertion of plant with deep roots colonizing bedrock (g, h, i) -Insertion of legumes (a, c, k) -Inoculation with mixed mycorrhizal fungi & N 2 fixing bacteria in highly degraded species with different nutrient acquisition strategies and nutrient requirements (a, c, I, j, k) -Promoting perennial plants (f, g, h, I, k) and/or permanent plant cover (all pictures but e) to fuel the CMN in energy-rich carbon -No or limited use of soil tillage (b) and pesticides to preserve the CMC -Inoculation with mixed mycorrhizal fungi in highly degraded soils Overall synchrony -High plant functional diversity -Plant plasticity & reserve -Mixing plant species with contrasted functional traits (e.g. phenology) & nutrient acquisition strategies (a, c, I, j, k) -Promoting perennial plants with high reserve and organ plasticity (f, g, h, I, j, k)


Sébastien Fontaine

Luc Abbadie

Gaël Alvarez

Michaël Aubert

Sébastien Barot

Juliette Bloor

Delphine Derrien

Olivier Duchene

Nicolas Gross

Ludovic Henneron

Gaël

Designing sustainable agrosystems by copying the biogeochemical organization of natural ecosystems

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

Designing sustainable agrosystems by copying the biogeochemical organization of natural ecosystems

  kg N ha -1 ) Study of pair sites (Kansas)

Designing productive, sustainable ecosystems requires to improve the efficiency of the system as a whole

(not specific functions)

Step 1 : characterizing the local pedoclimatic content Step 2 : installing the organization systems in agrosystems