Dr Giuseppe Alonci

Prof Federica L Fiorini

Prof. S Pietro Riva

Francisco Monroy

Ivan López-Montero

Silvana Perretta

Luisa De

Luisa De Cola
email: decola@unistra.fr

De Cola

Perretta

Prof I F Monroy

López-Montero

Injectable Hybrid Hydrogels, with Cell-Responsive

Keywords: Degradation, for Tumor Resection Injectable hydrogels, breakable nanocapsules, polyamidoamines, endoscopic submucosal dissection, tumor resection

stimulus triggers the breaking of the silica nanocapsules; therefore, the entire hybrid material can be completely degraded and its decomposition depends entirely on the presence of cells.

Interestingly the hydrogel precursor solution showed rapid gelation when injected in vivo and afforded a long-lasting high mucosal elevation, keeping the cushion volume constant during the dissection. This novel material can provide a solution to ESD limitations and promote healing of tissues after surgery

Introduction

Gastrointestinal (GI) tract cancers are amongst the most aggressive tumors, with an estimated worldwide mortality per year of over 1.75 million people. [START_REF] Ferlay | Estimates of Worldwide Burden of Cancer in 2008: Globocan 2008[END_REF] Endoscopic early detection and surgical treatment could potentially lead to a reduction of mortality, especially for early gastrointestinal neoplasms or precancerous lesions. Nowadays, endoscopic dissection is the first-line treatment granting lower morbidity and mortality rates than surgery with equal oncological results and better quality of life.

Endoscopic submucosal dissection (ESD) is a minimally invasive procedure now accepted worldwide as the preferred treatment modality for the removal of large and early gastric epithelial lesions. [START_REF] Coda | Endoscopic Mucosal Resection and Endoscopic Submucosal Dissection as Treatments for Early Gastrointestinal Cancers in Western Countries[END_REF] ESD, developed in Japan in the mid-1990s, [START_REF] Ono | Guidelines for Endoscopic Submucosal Dissection and Endoscopic Mucosal Resection for Early Gastric Cancer[END_REF][START_REF] Hosokawa | Recent Advances in Endoscopic Mucosal Resection for early Gastric Cancer[END_REF][START_REF] Hirao | Endoscopic Resection of Early Gastric Cancer and other Tumors with local injection of Hypertonic Saline-Epinephrine[END_REF] consists in the injection of a solution or a gel between the mucosal lesion and the muscularis, [START_REF] Ishihara | Application of Hydrogels as Submucosal Fluid Cushions for Endoscopic Mucosal Resection and Submucosal Dissection[END_REF][START_REF] Yu | Poly(lactic acid-co-glycolic acid)poly(ethylene glycol)-poly(lactic acid-co-glycolic acid) Thermogel as a Novel Submucosal Cushion for Endoscopic Submucosal Dissection[END_REF][START_REF] Fujishiro | Comparison of various Submucosal Injection Solutions for maintaining Mucosal Elevation during Endoscopic Mucosal Resection[END_REF] in order to obtain submucosal tissue elevation by generating a submucosal fluid cushion (SFC). The "cushioned" submucosa forms a safety margin to the underlying muscularis, thus minimizing the risk of perforation during ESD. [START_REF] Oka | Advantage of Endoscopic Submucosal Dissection Compared with EMR for early Gastric Cancer[END_REF][START_REF] Gotoda | Endoscopic Submucosal Dissection of Early Gastric Cancer[END_REF] Additionally, this procedure allows an accurate histological assessment, therefore lowering the possibility of neoplastic recurrence. [START_REF] Committee | Endoscopic Submucosal Dissection[END_REF] For gastrointestinal (GI) tumors especially, due to the low thickness of the GI wall and the use of electrocautery, there is a high risk of perforation. [START_REF] Fujishiro | Tissue Damage of Different Submucosal Injection Solutions for EMR[END_REF][START_REF] Cho | Worldwide Experiences of Endoscopic Submucosal Dissection: Not just Eastern acrobatics[END_REF] ESD is indeed a complex procedure requiring a high level of technical skills and associated with high complication rates and long operative time. To improve the efficacy and safety of endoscopic submucosal dissection techniques, the quality and duration of the submucosal lift are crucial. [START_REF] Yu | Poly(lactic acid-co-glycolic acid)poly(ethylene glycol)-poly(lactic acid-co-glycolic acid) Thermogel as a Novel Submucosal Cushion for Endoscopic Submucosal Dissection[END_REF][START_REF] Fujishiro | Comparison of various Submucosal Injection Solutions for maintaining Mucosal Elevation during Endoscopic Mucosal Resection[END_REF] Several injection solutions have been developed for this purpose, but normal saline solution (NS) is the most commonly used, because of its low cost and ease of use. However, simple NS injection suffers from low mucosal elevation, fast diffusion of the solution in the tissues, requiring multiple injections, often resulting in longer operative time, multiple instrument exchange and poor visualization, which could led to bleeding and perforation. [START_REF] Tran | Injectable drug-eluting Elastomeric Polymer: a novel Submucosal Injection Material[END_REF] Viscous solutions such as glycerol, [START_REF] Uraoka | Effectiveness of Glycerol as a Submucosal Injection for EMR[END_REF][START_REF] Fujishiro | Appropriate mixture of Hyaluronic Acid, Glucose and Glycerin for a Submucosal Fluid Cushion during Endoscopic Submucosal Dissection in the Dog Stomach[END_REF] hydroxypropyl methylcellulose, [START_REF] Feitoza | Hydroxypropyl Methylcellulose: A better Submucosal Fluid Cushion for Endoscopic Mucosal Resection[END_REF] chitosan, [START_REF] Hattori | Characterization of a water-soluble Chitosan derivative and its potential for Submucosal Injection in Endoscopic Techniques[END_REF] and hyaluronic acid [START_REF] Yoshida | Efficacy of Hyaluronic Acid in Endoscopic Mucosal Resection of Colorectal Tumors[END_REF] have been proposed to achieve sustained mucosal elevation and avoid injuries to the muscularis.

Although hyaluronic acid solution is one of the best options, it has been shown to stimulate the growth of residual tumors in animal models. [START_REF] Matsui | Hyaluronic Acid stimulates Tumor-cell Proliferation at wound sites[END_REF] Moreover, a large amount of hyaluronic acid is necessary to create a SFC and its use is associated with high costs and a general lack of availability. [START_REF] Committee | Endoscopic Submucosal Dissection[END_REF] To be clinically applicable, the lifting solution should be biocompatible, easily injectable, able to provide a prolonged and thick SFC to make ESD procedure safer, more cost-effective, and preserve tissue specimens for precise pathologic staging. [START_REF] Gotoda | Endoscopic Submucosal Dissection of Early Gastric Cancer[END_REF] Additionally, desirable features are controlled and tunable biodegradability and the possibility of releasing active components to assist the mucosal healing process after the resection, avoiding scarring and stenosis.

Finally, injectable biomaterial systems with a liquid-to-gel transition, such as hydrogels, are more promising in providing extended and thicker submucosal lift and easiness of handling. Using a hydrogel with tunable mechanical and degradation properties, which remains in place even after the procedure is completed, can provide protection for the stomach walls and promote a better healing of the lesions.

In the last years, injectable hydrogels have brought a shift in the search for the optimal SFC material towards the development of solutions that rely on in situ gel formation. For example, a photo-crosslinked chitosan hydrogel has been recently reported as a submucosal injection agent: [START_REF] Hayashi | Usefulness of Photocrosslinkable Chitosan for Endoscopic Cancer Treatment in Alimentary Tract[END_REF] mucosal elevation was created after the injection of the chitosan viscous solution, which was crosslinked in situ via UV irradiation, resulting in an insoluble hydrogel. However, the use of UV light for the photoinitiated radical polymerization may be difficult to perform in certain organs and for large areas. [START_REF] Kumano | Endoscopic Submucosal Dissection for Pig Esophagus by using Photocrosslinkable Chitosan Hydrogel as Submucosal Fluid Cushion[END_REF] Moreover, as the authors mentioned, UV irradiation may be associated with inflammation of the residual tissue.

To replace light with heat, thermoresponsive polymers, or thermogels, have been investigated, such as the recently proposed water solution of a PEG/PLGA-based temperature-sensitive polymers. [START_REF] Cao | Safe and Efficient Colonic Endoscopic Submucosal Dissection Using an Injectable Hydrogel[END_REF] However, many of these materials have been shown to clog inside long delivery tools at normal body temperature. [START_REF] Ifkovits | Review: Photopolymerizable and Degradable Biomaterials for Tissue Engineering Applications[END_REF] Until now, several of the in situ forming hydrogels proposed are hindered by administration difficulties, and adequate materials for ESD are still missing. [START_REF] Yu | Poly(lactic acid-co-glycolic acid)poly(ethylene glycol)-poly(lactic acid-co-glycolic acid) Thermogel as a Novel Submucosal Cushion for Endoscopic Submucosal Dissection[END_REF] An ideal hydrogel for in vivo applications should have a liquid-solid transition at room temperature, must solidify in few seconds without any catalyst or heat and be biodegradable. Herein we report on an injectable hybrid hydrogel, based on degradable polyamidoamines, dPAA, with the aim of meeting all the requirements for an ESD material, in order to address the limitations previously listed. This class of hydrogels, pioneered by Ferruti et al., [START_REF] Ferruti | Poly(amidoamine)s: Past, present, and perspectives[END_REF] has very attractive characteristics: they solidify in water at room temperature, without the need of a catalyst, their mechanical and chemical properties are easily tunable and their high porosity is compatible with cellular colonization and nutrients diffusion.

We have already demonstrated that polyamidoamines-based hydrogel have excellent biocompatibility and even stem cells can proliferate into the porous scaffold. [START_REF] Fiorini | Nanocomposite Hydrogels as Platform for Cells Growth, Proliferation, and Chemotaxis[END_REF] A similar scaffold, developed and optimized for tissue engineering applications, was also applied for the first time as adsorbent material for wastewater treatment, widening the biomedical application to environmental technology. [START_REF] Rizzi | Polyamidoamine-Based Hydrogel for Removal of Blue and Red Dyes from Wastewater[END_REF] The hydrogel developed for this work, namely dPAA, is composed of amino-functionalized breakable [START_REF] Maggini | Breakable Mesoporous Silica Nanoparticles for targeted Drug Delivery[END_REF] silica-shell nanocapsules, BNCs, covalently bound to the polyamidoamines-based hydrogel network. Such network contains cross-linkers bearing a disulfide bond, to impart breakability to the material upon exposure to cell-released glutathione. Therefore, the hydrogel degradation is promoted by the proliferation of cells onto the scaffolds. By focusing on the reduction-triggered degradation at low glutathione level, which mimics the rarely investigated extra-cellular reducing conditions, [START_REF] Yang | PEG-based bioresponsive Hydrogels with Redox-mediated Formation and Degradation[END_REF][START_REF] Kar | Poly(ethylene glycol) Hydrogels with Cell Cleavable Groups for autonomous Cell Delivery[END_REF][START_REF] Huang | Biomimetic ECM Coatings for Controlled Release of rhBMP-2: Construction and Biological Evaluation[END_REF] we present here a cell-responsive degradable hydrogel for in vivo applications.

Moreover, by injecting the developed hydrogel in vivo when still in its fluid phase, the gelation and formation of a stable and long-lasting submucosal fluid cushion was achieved in situ in less than three minutes. This allowed performing ESD with a single injection of material, thus greatly improving the procedure outcome.

Results and discussion

Materials synthesis and characterization

A degradable nanocomposite hydrogel, dPAA, was synthetized using the general procedure shown in Figure 1. The hybrid hydrogel is composed of a polyamidoamines-based network with covalently linked breakable silica nanocapsules, BNCs. The hybrid material is formed in water at room temperature using a catalyst-free Michael-type addition reaction.

To achieve a degradation that could be triggered by cells proliferating onto the material, without the need of any additional stimulus, we have incorporated a disulfide crosslinker in the polymeric network of the hydrogel, i.e. cystamine. Disulfide bonds are susceptible of thiol exchange in the presence of reducing agents, such as glutathione (GSH), which is a cell metabolite. Redox-responsive breakable hybrid nanocapsules, BNCs, and in particular disulfide containing organosilica have been reported by our group. [START_REF] Prasetyanto | Breakable Hybrid Organosilica Nanocapsules for Protein Delivery[END_REF] This material is composed of a silica shell able to encapsulate functional proteins in their active folding and it is engineered to break when a reducing agent, such as GSH, is present, with a complete release of the loading. Thus, we decided to exploit these BNCs to construct hybrid hydrogels able to release active biomolecules during the degradation of the material. The BNCs were functionalized with 3aminopropyltriethoxysilane, to be able to covalently link them to the polymeric hydrogel network. The surface functionalization was confirmed by the shift from negative to positive values of the ζ-potential, from -10.5 mV of the pristine nanocapsules to + 2.2 mV. A scheme of the synthesis and functionalization, as well as the SEM of the pristine and functionalized BNCs and of the degradation via GSH is displayed in Figure 2.

The amino groups on BNCs were then reacted with the unsaturated moiety of methylene bisacrylamide (MBA) through a one-pot Michael poly-addition in water, conducted at room temperature without any additional catalyst, and the reaction resulted in the formation of transparent hydrogel in 48 h (Figure 2a,b). An initial water suspension of 1 mg/ml of BCNs was found to be the optimal particles concentration in order to obtain a homogeneous material, as confirmed by rheological analysis where there is no indication of phase separation. A higher concentration (e.g. more than 2 mg/ml) led indeed to visible aggregates in the initial water dispersion and thus was discarded.

Gelation was confirmed by the absence of gravitational flow when the test tubes containing the hydrogels were inverted, through the so called "inverted test tube method". A full rhelogical characterization is reported below. The formation of a crosslinked network was further established by Fourier-transformed infrared spectroscopy (FTIR), and the resulting spectrum is shown in Figure 2c. Peaks at 1640 and 1530 cm -1 are typical for the absorption of the amides carbonyl (st and  of C=O), while the 3260 cm-1 band is attributed to the N-H stretching (st NH2). Furthermore, the absorption band at 1039 cm -1 , which was ascribed to the vibration of C-S-S-C bond, confirmed the successful incorporation of the disulfide cross-linker in the polymeric network. Since the samples were lyophilized to allow a correct peak determination, the presence of adsorbed water cannot be seen from FTIR spectrum, apart from a weak residual shoulder at 3430 cm -1 , which is related to the OH angular deformation of water.

The morphological analysis of the obtained hydrogel was assessed via scanning electron microscopy (SEM) of the lyophilized scaffolds. SEM images showed a highly porous structure, with pore diameter in the range of 40 to 100 m, as can be seen in Figure 2d.

The swelling behavior of the hybrid hydrogel was analyzed and suggests that the matrix can contain up to 93% of water, and in the absence of reducing agent is stable for weeks.

dPAA degradation in presence of GSH

For ESD application, hydrogels should maintain the required mucosal elevation for the whole time of the procedure (i.e. 1 hour) and degrade, in few days, into small fragments in order to have a complete clearance from the body.

The potential degradation mechanism of the network is shown in Figure S1.

The degradation kinetics of the dPAA was examined by measuring swelling ratio variations as function of time in the presence of a low concentrated GSH solution (i.e. 10 M GSH solution in PBS), mimicking the extracellular environment. Hydrogels, incubated in PBS in the absence of GSH, were used as control.

The swelling ratio curve of dPAA showed an initial phase where a clear increase in sample mass was observed, followed by a rapid downward phase (Figure 3a). The imbibing of the solvent into the hydrogel caused the initial increasing phase. This was then quickly outweighed by the cleavage of the disulfide bonds, leading to the complete degradation of the hydrogel.

The dPAA equilibrated in PBS showed instead a first phase of swelling followed by a plateau that was reached after 24 hours, demonstrating that the material is stable in absence of reducing agents. The swelling was followed for 6 days.

To evaluate the effect of the disulfide crosslinker density on the degradation kinetics of the hydrogel nanocomposite, other two samples were synthetized, containing lower and a higher amount of cystamine, As previously mentioned, the degradable hydrogel is decorated with breakable nanocapsules able to degrade with the same mechanism of the hydrogel, through the reduction of the disulfide bonds, and able to release their content, as already reported by our group. [START_REF] Huang | Biomimetic ECM Coatings for Controlled Release of rhBMP-2: Construction and Biological Evaluation[END_REF] The fragmentation of the BNCs in presence of the reducing GSH after 72 hours was further confirmed by scanning transmission electron microscope (Figure 3b).

In vitro and ex-vivo analyses

Cell-mediated degradation

Since the degradation of dPAA could be achieved at low GSH concentration, such as the extra-cellular one, this prompted us to test the degradation of the scaffold in the presence of cells. In particular, Human Dermal Fibroblast (HDFa) cells were chosen for this study because fibroblasts residing within the extracellular matrix in the body are critical for matrix synthesis and repair. Upon injury or wound formation, these cells migrate to the wound site to repair the damaged tissue. [START_REF] Smithmyer | Hydrogel Scaffolds as in vitro Models to study Fibroblast Activation in Wound Healing and Disease[END_REF] Thus, to simulate the cell-mediated degrading conditions in vivo, we selected HDFa.

The dPAA hydrogels for this test were synthetized in an 8 mm diameter and ~1 mm height disc shape and. 2.5 x 10 5 HDFa cells were seeded onto the hydrogels and cultured in the corresponding growth medium; hydrogels without cells, acellular, incubated in growth medium were used as control.

AlamarBlue assay indicated that the majority of the encapsulated cells were viable and proliferating onto the scaffold (Figure S3a) up to 4 days. This is consistent to what has been observed for similar polyamidoamines-based scaffolds and it confirmed that dPAA containing disulfide moieties supported cell encapsulation and viability. Erreur ! Source du renvoi introuvable. S3b displays an image of the 3D proliferation of the cells stained in red (Vibrant DiD stain) for better visualization, indicating that they permeate in the depth of the scaffold. A 3-channel visualization of the surface of the dPAA is reported in Figure S3c and is indicative of the growth of the cells onto the scaffold.

As hypothesized, the hydrogel underwent degradation responding to cell-secreted GSH, in the absence of any external stimulus. In addition, many cell surface molecules contain thiol groups and thus could contribute to the cleavage of the disulfide bonds of the network. [START_REF] Kagatani | Oxidation of Cell Surface Thiol groups by Contact Sensitizers triggers the Maturation of Dendritic Cells[END_REF][START_REF] Sahaf | Lymphocyte Surface Thiol levels[END_REF][START_REF] Donoghue | Presence of Closely Spaced Protein Thiols on the Surface of Mammalian Cells[END_REF] The scaffold resulted largely reduced in size and weight after 72 hours and the complete degradation was achieved after 96 hours (Figure 4a The acellular hydrogels used as control displayed minimal degradation during the course of the studies (Figure 4a, dashed line). This small degradation (17 %) is probably due to the presence of fetal bovine serum in the culture medium, which contains various proteins and amino acids with thiol groups.

The results obtained confirmed that the dPAA hydrogels were prone to HDFa-mediated degradation through thiol reductive exchange, therefore showing potential for in vivo applications requiring degradation of the scaffold.

Mechanical properties of dPAA and mechanical degradation in presence of GSH

The mechanical properties of dPAA were characterized in terms of shear rheological behavior (Figure 5 and Supplementary Information). Specifically, dPAA is a quite soft hydrogel characterized by a shear modulus ca. 6 kPa, in agreement with other injectable hydrogels for drug delivery, allowing their mechanical properties to be matched with different soft tissues in the human body. The soft character of dPAA is particularly evident as regards the relative high fluidity of the hybrid hydrogel, characterized by a small value of the loss modulus G'' ≈ 100 Pa. Although this parameter corresponds to a high viscosity as compared to a typical fluid, the fact that the viscous losses of the material are much lower than its effective rigidity (G'' << G') converts the system in a quite resilient material with a relatively high mechanical compliance. In effect, the soft mechanical nature of dPAA (a soft hydrogel with a pastylike fluidity) makes it especially well-suited to mimic the mechanical response of muscular tissue, being structurally resilient but compliant to deformation.

first hour of incubation. However, both G' and G'' values decreased down to 25% and 1% of their initial values respectively and eventually stabilize after 5 hours of GSH addition. The mechanical degradation causes a shear softening of dPAA and a concomitant shear fluidization of the hydrogel. Thus, dPPA hydrogel can simultaneously maintain the structural integrity but facilitate the content release of BNCs through a less viscous media under reducing conditions provided by the extracellular environment, here mimicked by the GSH solution.

dPAA: injectability and formation of SFC

Since the ESD procedure requires the injection of the hydrogel solution underneath the mucosa, we first optimized the synthetic procedure of the hydrogel to obtain a material that could be delivered in vivo via injection, and then rapidly jellify inside the body.

Polyamidoamine-based hydrogels have the great advantage of allowing network formation under physiological conditions. [START_REF] Peng | Scarless Wound Closure by a Mussel-Inspired Poly(amidoamine) Tissue Adhesive with Tunable Degradability[END_REF][START_REF] Wang | In Situ-Forming Polyamidoamine Dendrimer Hydrogels with Tunable Properties Prepared via Aza-Michael Addition Reaction[END_REF][START_REF] Feng | Elastic Light Tunable Tissue Adhesive Dendrimers[END_REF] This approach has been already investigated in the past years for various injectable hydrogel preparations. [START_REF] Lutolf | Synthesis and Physicochemical Characterization of End-Linked Poly(ethylene glycol)-co-peptide Hydrogels Formed by Michael-Type Addition[END_REF] This mechanism has been shown to be applicable to a wide range of commercially available precursors, forming polymer networks with minimal structural deficiencies within a surgically relevant timeframe, while avoiding the production of free radicals. [START_REF] Pritchard | An Injectable Thiol-Acrylate Poly(ethylene glycol) Hydrogel for sustained release of Methylprednisolone sodium succinate[END_REF] When mixed all the components start to react after 30 minutes as indicated by the formation of a clear solution.

The ability of this hydrogel solution to flow through a disposable 23-gauge catheter injection needle was then examined. The dPAA solution was able to flow under hand pressure and the maximum needle injection pressure was found to be comparable to saline solution (Figure S4a). At the temperature to 37 °C, we observed the formation of a self-standing hydrogel in 18 hours. Such long time is not suitable for injectable hydrogels but knowing that the submucosa is rich in type I collagen and that this presents amino and hydroxyl groups as side groups, we postulated the possible formation of hydrogen bonds that could lead to a faster gelation kinetic in vivo.

We therefore tested this hypothesis by injecting the dPAA solution, colored in blue with methylene blue, in the submucosa of a porcine stomach, ex vivo. The injection was performed on the tissue at 37 °C and the gelation, with formation of a SFC, was immediately observed (Figure S4b). There was no leakage of the solution from the injected site and, 5-10 min after injection the tissue was cut revealing the formation of the solid phase of the dPAA (Figure S4c).

Interestingly the nanocomposite hydrogel was found completely adherent to the submucosal layer and it had to be removed with scissors and tweezers. Moreover, it was confirmed that there had not been diffusion of the solution into the surrounding tissues.

The investigation of injectability and gelation time was then performed in vivo. The dPAA showed a gelation time of approximately 3 minutes when injected in the submucosal layer in a living pig.

It is undeniable that the physiological conditions of temperature (37 °C) and pH (7.4), contribute to the faster gelation than what observed in vitro, as already reported for similar systems. [START_REF] Nguyen | Injectable Poly(amidoamine)-poly(ethylene glycol)poly(amidoamine) Triblock Copolymer Hydrogel with Dual Sensitivities: pH and Temperature[END_REF] However, tests done in vitro at 37 °C and pH 7.4 did not give the same results. This suggests the contribution of additional factors related to the intermolecular interactions between the hydroxyl and amino groups of the collagen side chains with amide groups and side chains of dPAA that further crosslink the polymer network and facilitate the covalent polymerization. The formation of mechanical entanglements between the collagen fibers and the dPAA backbone could also contribute to the formation of an interpenetrated hydrogel network, favoring the faster formation and adhesion of a stable and elastic hydrogel in situ. This morphological change was observed in the SEM of the explanted tissues, which indeed showed interactions between the hydrogel scaffold and collagen fibers (Figure 6a). In vivo images of the dPAA formed in situ (Figure 6b) and stained with Methylene Blue also displayed fibrous formation within the hydrogel matrix (Figure 6c). However, a deep investigation is currently ongoing to explain this behavior.

As already mentioned in ESD procedures the ability of mucosal lifting and its maintenance are essential to avoid perforation and safely complete the en bloc resection of the lesion.

We examined the formation and lasting of a SFC ex vivo comparing the dPAA with an isotonic saline solution. In particular, fresh resected porcine stomachs were used, and 2 ml of NS or DPAA solution were injected. Protrusions appeared at the injection site and the height changes in submucosal elevation were recorded after 10 seconds, 10 minutes and 1 hour, to cover the whole time of the ESD procedure, which is approximately 40-50 minutes (Figure 6d). Although both the examined solution and the hydrogel led to the mucosal elevation right after the injection, the dPAA nanocomposite displayed higher mucosal lifting: 8.3 mm vs 6.7 mm, for the dPAA and the NS respectively, with the same amount of solution injected. This showed that already part of the NS solution was absorbed by the surrounding tissues after 10 seconds. After the injection of the hydrogel solution, the formation of a solid SFC was detected, which showed only a small change in size over 1 hour, i.e. from 8.3 mm to 5.8 mm. No significant change in shape or consistency of mucosal lifting was observed. This behavior was due to the formation of the dPAA hydrogel under the submucosa.

In contrast, the elevation created with NS gradually collapsed, showing a reduction of 37% in size already after 10 minutes and of more than half after 1 hour (i.e. height from 6.7 mm to 2.9 mm).

In vivo ESD procedure

The higher performance of the dPAA nanocomposite in ex vivo was therefore evaluated in vivo in a living pig. We first set appropriate lesions sizes of approx. 3 cm in diameter in the porcine stomach and then 8 ml of the hydrogel solution were injected in the submucosa.

The ESD procedure was performed in triplicate in different areas of the same porcine stomach; NS solution was used as control.

In Figure 7a is reported the endoscopic view of the stomach at time 0 before the injection of the prehydrogel solution. It shows the set of an appropriate lesion of approx. 3 cm in diameter. We observed that in all the cases the injected dPAA solutions in the submucosa led to the gelation of the material in less than 3 minutes, which thus formed a clear and stable mucosal elevation (Figure 7b,c).

A comparison with the normal saline solution generally used showed initially no significant difference compared to the SFC formed by dPAA. However, the elevation of the SFC formed by NS had obviously reduced after 15 min, due to quick diffusion of the NS at the target site and absorption of the liquid by the tissue, thus it was necessary to repeat the injections to keep the submucosa lifted and be able to finish the surgery.

In contrast, the mucosal lifting obtained with dPAA allowed the surgeon to perform the entire ESD procedure (40 min) without requiring a second injection, therefore significantly simplifying the procedure and avoiding large injection of liquids.

The presence of the hydrogel allowed the use of the common electrocautery settings and the long-lasting conservation of the mucosal elevation created with the dPAA enabled the surgeon to smoothly accomplish the submucosal dissection (Figure 7d). We can conclude that no significant complications or perforation occurred during the procedure due to the reliable and long-lasting mucosal lifting achieved with the dPAA.

Then, the lesion was resected en bloc without any sign of mucosal or muscularis damage, confirming that the dPAA hydrogel formed in situ was able to "dissect" the submucosal layer (Figure 7e). Intact mucosal specimens were conveniently achieved via the performed ESD procedures. This is essential, as a definite resection provides accurate histological assessment and thus, can reduce the risk of neoplastic recurrence. To prove that fast gelation can occurs also in other tissues and that the procedure can be applied for other lesions the same procedure was also performed in the colon and in the esophagus with excellent results (Figure S5). Besides aiding the surgery, the protecting layer created with the gels can protect the layer underneath (Figure 7f). The possibility of releasing active components from the breakable capsules is highly beneficial at the end of such delicate procedure. Biomolecules, such as adrenaline, proton-pump inhibitors, corticoids or antibiotics could potentially be efficiently delivered to assist the cauterization of the resected tissue or the prevention of inflammations.

Conclusions

A degradable hybrid hydrogel was successfully developed by embedding breakable nanocapsules into a disulfide-containing polyamidoamines-based hydrogel.

We demonstrated that the network can be completely cleaved in 3 days upon incubation in a GSH solution mimicking the extra-cellular concentration (10 M). Most importantly, the obtained dPAA sustained the proliferation of HDFa and underwent complete degradation in response to cell-secreted molecules from HDFa seeded onto the scaffold, without any external stimulus.

The obtained dPAA can be delivered to the desired tissue by facile injection through a 23-gauge needle.

To prove its applicability in real models, the hydrogel solution was injected in the submucosa of a porcine stomach in vivo. It formed an elastic hydrogel in 3 minutes, most likely due to temperature increase and interaction with collagen fibers.

Such an important result, together with the low-cost starting materials employed for the synthesis, motivated us to employ the dPAA hydrogel as a novel submucosal injection agent in ESD. The hydrogel formed a reliable SFC in vivo, enabling a long-lasting mucosal elevation, which was superior to commonly used NS. This facilitated en bloc resection of the lesion, which was successfully accomplished with just a single injection. No perforation, major bleeding or tissue damage were observed during ESD. Moreover, part of the in situ formed hydrogel adhered tightly to the muscolaris, under the resected mucosa, allowing protection of the membrane during the procedure and after it.

The possibility to release active molecules and to biodegrade the hybrid hydrogel in few days opens interesting perspective for a faster regeneration of the tissue.

Experimental Section

Synthesis of dPAA: In a 5 ml round bottom flask methylenbisacrylamide (MBA; 200 mg) and N,Ndimethylethylenediamine (70 l) were added to a water solution of amino-functionalized BNCs freshly sonicated (1.5 ml; 1 mg/ml). Then, cystamine (65 mg) was added to the mixture that was stirred vigorously for 1 hour at room temperature, after which a homogeneous and clear pre-hydrogel solution was obtained. When the hydrogels were needed for in vitro experiments (i.e. GSH degradation and cellular viability and degradation), the obtained solution was transferred to glass vials (500 l per vial) and allowed to react in static conditions at r.t. Glass vials with inner diameter of 8 mm were used as molds. The hydrogels were obtained after 48 hours.

Once obtained, the disk-shaped hydrogels were freeze-dried and weighted. Dried hydrogels were used to study the swelling ratio at different pH and the degradation kinetics with different concentrations of GSH.

This step allowed us as well to sterilize the materials for in vitro experiments. Sterile and ultrafiltered water was used during hydrogel preparation for in vivo tests; the synthesis was carried out in closed sterile vials and protected from bacteria contamination, thus we assumed that the final product was free of bacterial contamination.

To assess the presence of unreacted MBA, the hydrogels were washed several times; the extracted washing solutions were then freeze-dried and analyzed by H-NMR spectroscopy, which confirmed that no unreacted monomers were present. We concluded that the final hydrogel composition was not significantly different from the feeding starting monomer mixture, as elsewhere observed. [START_REF] Fiorini | Nanocomposite Hydrogels as Platform for Cells Growth, Proliferation, and Chemotaxis[END_REF] Cell-mediated degradation of dPAA: Hydrogels nanocomposites were freeze-dried and weighed (Wi). Then 2.5x10 5 HDFa were seeded onto the samples (see paragraph 5.6.5). The cell-laden samples were collected at pre-determined time points and were freeze-dried to obtain their dry weight after degradation (Wf).

The cell-mediated degradation of the hydrogels, D, was calculated using the following equation:

𝐷 (%) = 𝑊 𝑖 -𝑊 𝑓 𝑊 𝑖 × 100
Acellular hydrogels were used as degradation control.

Creating submucosal cushion and performing ESD in a living pig: The pig was fasted for 1 day before operation. A standard endoscope (Karl Storz, Tuttlingen, Germany) was used in the pig under general anesthesia. Both the dPAA solution and the NS used as control contained a small amount of Methylene Blue as a color agent in order to facilitate visualization of the SFC.

After setting appropriate lesion sizes of approx. 3 cm in diameter in the porcine stomach, 8-10 ml of dPAA solution and NS were injected in the stomach submucosa through the endoscope accessory channel using a 23-gauge injection needle.

The mucosal elevation due to the injected dPAA at the target site was observed endoscopically before starting the ESD. It was compared under direct view with the elevation caused by NS during the procedure. After injection, the ESD was performed and a circumferential mucosal incision was accomplished using a TT knife (Olympus, Tokyo, Japan). Injection of dPAA and ESD were repeated three times. The animal was euthanized after completion of experiments; the whole procedure was followed and recorded using a Silver Scope tm Video Gastroscope (Karl Storz, Tuttlingem, Germany).

The main outcome measures were (1) the rapid gelation of dPAA when injected into the submucosa and

(2) the long-lasting SFC formed; (3) the feasibility of the dissection procedure during ESD; (3) the adhesion of dPAA to the muscolaris layer and thus the increase of protection during the procedure and after it.

ASSOCIATED CONTENT

The following files are available free of charge:

Supporting information (PDF), including details of the synthesis and functionalization of BNCs, degradation kinetic in presence of GSH, mechanical characterization of dPAA by shear rheology, invitro cell culturing, cell staining and viability studies, evaluation of the gelation and formation of SFC ex vivo.

Figure 1

 1 Figure 1 Scheme of the synthesis and functionalization of BNCs containing disulfide moiety in the framework and loaded with Cyt-C inside the silica capsule (a); SEM image of the monodispersed functionalized BNCs, in the insert SEM picture of a naked nanoparticle (b); degradation of the BNCs (c).

Figure 2 .

 2 Figure 2. Synthesis of dPAA, embedding BNCs (a); scheme of the network (b); FTIR trace of dPAA (c); SEM showing the porosity of the nanocomposite (d).

  10-wt% and 40-wt%, compared to the dPAA hydrogel which had 20 wt%. The degradation profiles of the three samples are reported in Figure S2. The degradation time was found to be proportional to the amount of disulfide crosslinker.

Figure 3 .

 3 Figure 3. Degradation of the dPAA hydrogel with 10 µM GSH (solid line) and with culture media (dashed line) (a). Breakability of the BNPs before (i) and after exposure to GSH (ii) (b).

  ,b), demonstrating a good agreement with the GSH degradation tests. It was observed that the degradation process resulted into a gradual movement of the cells from the nanocomposite to the bottom of the well containing the scaffold (Figure 4c). The viability of HDFa measured after the degradation of the scaffold showed that 87 % of the cells were viable, thus confirming that the degradation products were non-toxic. Previous studies on linear polyamidoamines systems have shown that the degradation products were completely non-cytotoxic, since the degradation produced oligomers. 37

Figure 4 .

 4 Figure 4. HDFa-mediated degradation as function of time of dPAA hydrogels, solid line, containing 2.5x105 cells and control (acellular dPAA), dashed line (a); macroscopic pictures showing the cellmediated degradation of the dPAA (b). Overlay images (i.e brightfield and DiD channel) of the HDFa growing onto the scaffold (24 h) and gradually transferring to the bottom well plastic as the dPAA gets degraded (48 and 96 h); scale bar is 100 µm (c).

Figure 5 .

 5 Figure 5. Shear storage modulus (G') and shear loss modulus (G'') of dPAA hydrogel as measured by oscillatory shear rheology (a). dPPA mechanical degradation upon GSH incubation. Lines are only eye-guides for better visualization (b).

d 6 .

 6 SEM image of the explanted tissue and hydrogel, showing collagen fibers within the hydrogel matrix (a), scale bar is 100 m; endoscopy images of the dPAA stained with Methylene Blue formed in vivo (b) and close up (c) showing with fibrous formation within the hydrogel matrix. Change in mucosal elevation as a function of time after the injection of NS or dPAA into a resected porcine stomach (d). Methylene blue was mixed as color agent. Height values (black bar) obtained for NS were 6.7 mm, 4.2 mm and 2.9 mm after 10 sec, 10 min and 1 h respectively; for dPAA were 8.3 mm, 6.4 mm, 5.8 mm after 10 sec, 10 min and 1 h respectively.

Figure 7 .

 7 Figure 7. Endoscopic views of the different steps of the ESD procedure performed using the dPAA stained with Methylene Blue. Setting of the lesion, approx. 3 cm in diameter (a); injection of the dPAA solution (b); formation of the SFC after gelation of the dPAA (c); circumferential cutting (d); complete resection with protective layer of dPAA that remained adhered to the muscolaris (e); wound left after ESD with layer of the dPAA (f).

ACKNOWLEDGMENT F.F. and L.D.C. acknowledge the European Research Council (ERC), grant no. 2009-247365 titled "MaGIC", for financial support. G.A. and L.D.C. acknowledge the European Union Horizon 2020 research and innovative programme under the Marie Sklodowska-Curie grant agreement No 642192. I. L-M acknowledge the ERC Starting Grant MITOCHON" (ERC-StG-2013-338133). I. L-M. and F.M. acknowledge Spanish Ministry of Economy MINECO (FIS2015-70339-C2-1-R) The authors gratefully acknowledge use of the services and facilities of the IRCAD and the Institute of Image Guided Surgery IHU-Strasbourg.

AUTHOR INFORMATION

Corresponding Author * Prof. L. De Cola, Institut de Science et d'Ingénierie Supramoléculaires, Université de Strasbourg, 8 rue Gaspard Monge, 67000 Strasbourg, France E-mail: decola@unistra.fr

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally to the work.


Cyril Bossard

Gilles Kermarrec
email: gilles.kermarrec@univ-brest.fr

Romain Bénard

Pierre De Loor

Tisseau Jacques
email: tisseau@enib.fr

Virtual Reality for Research and Training in Sport: an Illustration with CoPeFoot

Keywords: Simulation, Virtual Reality, Sport, Design

New possibilities for research and training in sport could be offered thanks to virtual reality simulations. The literature is examined, analysing the advantages and properties of current simulators. This paper aims to present an original experiment in virtual reality simulation design based on autonomy, interaction and evolving concepts. Our main suggestion is that believability of simulation for the user needs both regularity and surprises (in terms of virtual agents' behaviour). A naturalistic approach was used to study experts' decision-making processes in soccer settings. The decision-making model represented a guideline for choosing and implementing computational models. The advantages of virtual reality simulation are discussed in relation to training and research in sports science.

Introduction

The benefits of new technologies have led many researchers to develop research programmes relating to simulation design. Although simulations in the fields of driving, aviation and military or medical interventions is no longer questioned [START_REF] Sebrechts | Transfer of Training in Virtual Environments: Issues for Human Performance[END_REF], similar interest in the field of sport is much more recent [START_REF] Ripoll | The interest of simulation for research and training in sport: the exemple of football. Third International Sport Sciences Days, The analysis of elite performance in its contextual environment[END_REF]. There is therefore very little information available about the benefits of simulations for research and training in high-level sports. Simulation, that is to say pretending, imagining or performing an action outside of its regular context, can serve many purposes in the context of high-level sports:

preparing oneself for action, acquiring new technical skills, or studying particular techniques.

In order to interact with a virtual environment, the validity of the simulation relies on the design approach and on the models of the virtual agents involved [START_REF] Cobb | Development and evaluation of virtual environments in education[END_REF].

Virtual environments (VE) can be used to immerse an individual or a team within a believable and interactive computer-generated environment. Users must be able to interact with autonomous agents and/or other human users. In order for the interaction to be effective, the virtual agents must also be able to influence the user, and be altered by the user's actions.

The VE must be able to evolve due to these interactions. If the agents' behaviour evolves, it must be neither totally predictable, nor completely random. By making the virtual agents autonomous, we could expect more believable behaviours.

With a view to immersing high-level football players in a simulation, designing this believability depends on a balance between predictability and surprise in order to allow users to construct meaning in terms of their interaction with the VE. The aim of this paper is to present a multidisciplinary approach to designing simulations dedicated to sport. We will examine the methodological and scientific choices made in designing the CoPeFoot (Collective Perception in Football) virtual environment.

We will introduce our suggestion by a brief review of the simulators put forward up until now in the field of sports. We will show that these simulators respond poorly to the demands of autonomy, evolution and interaction. In our experiment, we will present four stages of a collaborative approach between sports science and computer science, with the aim of developing a believable simulation application for football. Finally, we will discuss the advantages of the simulation using virtual reality apparatus for sports research and training.

Sport simulation: a brief overview

According to [START_REF] Alessi | Five keys to successful simulations[END_REF], three main aims can be associated with simulation for sports research and training: improving cognitive aspects of performance, improving the physical aspects of performance, and improving the affective aspects of performance (emotion and attitudes). Two main approaches are linked to these objectives and can be used to facilitate them: virtual reality simulation and realistic physical simulators. Within the approaches that use virtual reality simulations, we can identify the uses of simulations for strategic analysis of sports situations, and those relating to user-player immersion. The aim of the training in question imposes, or at least suggests the use of one or other of these approaches.

Realistic physical simulators for studying technical movements

In physical simulators designed for studying technical movements, the aim is to recreate a more believable environment with regards to senses; mainly relating to sight, balance, touch and sound. The physical interaction between the user and the machine is an essential part of believability. In order to enhance the user's impression of immersion in these simulators, we use different mechanical systems to recreate the vibrations inherent to sports such as skiing or mountain biking, for example [START_REF] Mester | Biological response to vibration load[END_REF]. Immersion through physical interaction is also linked to the scale (1/1) of the simulator. These very characteristics can be observed in Peloton, an environment which recreates virtual cycle races [START_REF] Ensor | Accommodating performance limitations in distributed virtual reality systems[END_REF].

Similarly, [START_REF] Bideau | Real Handball Goalkeeper vs. Virtual Handball Thrower[END_REF] suggest immersing handball goalkeepers by confronting them with virtual players; [START_REF] Huffman | A motion based virtual reality training simulator for bobsled drivers[END_REF], and [START_REF] Kelly | Design and construction of a bobsled driver training simulator[END_REF] also presented a virtual training environment for piloting Bobsleighs. One of the issues raised by these simulators is the evaluation of their value for completing the exercise in real-life situations.

For example, [START_REF] Todorov | Augmented feedback presented in a virtual environment accelerates learning of a difficult motor task[END_REF] present a study based on the acquisition of technical moves in table tennis in a VE. They studied the transfer of these moves to real-life settings.

Studies examining the transfer of acquisitions from the virtual to reality [START_REF] Bossard | Transfer of learning in virtual environments: A new challenge?[END_REF] show the need to think both about the alternation between real and virtual training sessions, and about the content of the desired acquisitions and the information provided by the virtual environment in response to the users' actions.

Finally, simulations for technical movements aim to optimize believability by focusing primarily on the physical interaction. Although these systems are tightly controlled, lacking autonomy and evolution, this type of simulation can be used to improve the physical aspects of performance.

Virtual reality simulation for analysis and strategic assistance.

The advantage of virtual reality simulations is that it is possible to conduct risk-free experiments and to be able to analyse the consequences of choices made by the players in different situations. The aim is not to use simulations to put the player in the thick of the action, but, on the contrary, to enable the player to take a step back from the action. In much the same way as for analysing parts of a game from video footage, the idea is to offer the player a number of different viewpoints. From a technical point of view; in order to generate a believable environment, this tool uses image processing and synthesis. For example, [START_REF] Ziane | Contribution à la formation des entraineurs sportifs, caractérisation et représentation des actions de jeu : l'exemple du basket-ball[END_REF] suggests that basketball coaches could be trained to analyse their team's actions. A model recreating scenes from the game based on a film was used to identify specific movements of both the players and the ball. A kinematic study of the action enables the coaches to graphically represent and to accurately interpret the different ways the teams positioned themselves on the court, the surface area occupied by each team, the distance between the players, the ball and the hoop, and the speed of the players and the ball. Other models (Bayesian networks) can be used in order to identify the most relevant viewpoint amongst all the possible points of view in order to study the correlations between the players' movement, the teams, and the ball. This tool has the advantage of being able to alter the view of the action and to identify the phenomena which are difficult to perceive through spontaneous observation. [START_REF] Metoyer | Animating athletic motion planning by example[END_REF] also suggest a simulator with the aim of analysing the results of different combinations of movements in American football. These combinations are played by virtual agents. According to the authors, there is no point in modelling the virtual players to look realistic, as the illusion of reality is more likely to be lost if their behaviour is unnatural. Therefore, believability depends less on the appearance of the players than on their behaviour (their movements) in context. The aim is to test different movement strategies in order to evaluate the results before applying them to real-life settings. This type of simulation is therefore a useful tool in training high-level players.

Here, the believability of behaviours relies essentially on "visual" return from the images, or the conformity of the behaviours in the simulation compared to those from the knowledge base created by experts in the field. These approaches can be used to improve the strategic analysis of sports situations either outside of, or at a distance from the action. Furthermore, this type of simulator does not require the user to participate in the action but is rather concerned with the graphical quality of the environments or the conformity of the produced behaviours. The autonomy of the agents and the evolution of this kind of simulation are limited.

Virtual reality simulation for immersion in sports settings

When the aim is to immerse the human user within a believable virtual environment, simulators attempt to produce virtual agents with a certain degree of autonomy. Video games can therefore provide very effective simulations. The TeamVision system from Konami, for example, is an adaptive artificially intelligent system for Pro Evolution Soccer 2008. The creators of the game FIFA 2008 (Fédération Internationale de Football Association) refer to a system evaluating up to thirty action options at any given time as an opportunity map. In the current state of affairs, the achieved behaviours are realistic, as they are due to the relative autonomy of the virtual players. The choices are made by each agent independently of a metamodel. However, these simulations often lack evolution in terms of behaviours, and interactions can be limited by the options programmed into the system. [START_REF] Sanza | Adaptive behavior for cooperation: a virtual reality application[END_REF] present a football simulator called NeViS (Networked Virtual Soccer) enabling interaction between humans and virtual agents. The agents' behaviour is guided by a classifier system.

The virtual agents can practice their interactions together in order to learn (to select) new winning strategies. The classifier system implements reinforcement learning from an "actioncondition" set of rules. Each rule can be evaluated according to the quality of its attributes.

The system inspects the environment, selects one of the applicable rules, and carries out an action as a result of these rules. The chosen rule may be reinforced, depending on the result (compensation). The rules can be combined using genetic algorithms to create new laws. This computational modelling allows for a good deal of evolution in the agents' behaviours.

However, [START_REF] Sanza | Adaptive behavior for cooperation: a virtual reality application[END_REF] highlight the fact that, as the new rules are generated randomly, they create valuable behaviours, but sometimes, these behaviours which are absurd in terms of suitability in sports settings. Research into the believability of behaviours is essential if these simulations are to be considered significant by experts in the field. Indeed, this requirement is crucial for players to be able to transfer real skills to virtual situations [START_REF] Bossard | Transfer of learning in virtual environments: A new challenge?[END_REF]. [START_REF] Walls | Assessment of upwind dinghy sailing performance using a virtual reality dinghy sailing simulator[END_REF] present an experiment with a sailing simulator. The simulator reproduces the bridge of a boat, held up by two supports and dynamically controlled by the computer.

The visual elements of the manoeuvres such as steering, or adjusting the sails, are extremely realistic. Having familiarized themselves with the simulator, competent sailors complete a ranking of each subject on the simulator was then compared to their ranking in other competitions. The results confirmed the possibility of transferring decisional skills from reality to the virtual and, as a consequence, they reinforce the believability of the environment.

Simulations aiming to immerse users in sports settings thus require autonomous agents whose behaviour evolves, in order to produce believable virtual situations.

A User-centered Virtual Reality Simulation: the CoPeFoot project

We here present a user-centered design approach to virtual environments which is unique in that it is based on close collaboration between computer science researchers and researchers in sports science. This collaboration is founded on four stages, explained separately here, but which constitute an interactive process: 1) an analysis of the experts' activity is conducted in a naturalistic football setting (reality); 2) this analysis fuels the choice and implementation of computational models to improve the virtual agents' behavioural believability; 3) the result obtained, the VE, is used to engage in simulations (pairing subjects with the environment) and to improve the implemented computational model; 4) the believability of the virtual agents' behaviour and the simulator are evaluated using the simulation itself.

Modelling tactical decisions in naturalistic settings.

Unlike "anthropomorphic" simulations [START_REF] Burkhardt | Immersion, Representation and Collaboration in Virtual Reality: discussion and empirical perspectives in cognitive ergonomics[END_REF], the aim of our design approach to virtual environments is not to reproduce reality but to make the situation as believable as possible. In order for the simulation to be considered believable, agents need to be relatively autonomous, and the designer therefore needs a model for this autonomy. Each virtual entity must be able to make choices according to its past experience, its state, its perceptions and the possible actions it can execute, so that its behaviour appears coherent and unique. The role of the sports science researcher would be to participate in making these choices while this agent model is being defined. Although knowledge acquired from analyzing actions is starting to be utilized in the context of training settings [START_REF] Ria | Recherche et formation en « analyse des pratiques » : un exemple d'articulation. [Research and Training in "practical analysis": an example of articulation[END_REF], their use in simulation settings making the most of virtual reality techniques remains largely unexplored. In order to simulate a counter attack in football, we wanted to model the tactical decision of players from a naturalistic study.

Using the NDM (Naturalistic Decision Making) approach, which examines the relationship between user and context, we considered the tactical decision to be a process of recognition of spatiotemporal configuration [START_REF] Klein | Naturalistic Decision Making[END_REF]. This link can be described as a "cognitive package" (Ross et al., 2006, p. 406), a set of elements relevant to the user. These elements are recognized as relevant and represent significant relationships as they are perceived from a background structure deriving from experience. From our hypothesis, this functional structure for recognizing and reacting seems to refer back to the concept of schemata [START_REF] Piegorsch | Ergonomic decision-making: A conceptual framework for experienced practitioners from backgrounds in industrial engineering and physical therapy[END_REF].

In order to contribute to the empirical validation of this approach within the context of team sports, we tested a counter attack situation in football. Behavioural data was recorded from 12 national level football players (under 16s) supplemented by verbal data collected during self-confrontation interviews. Analyzing decision-making activity allows us to define activities and the most likely sequences (i.e. possible scenarios) of 16 schemata from experts in situations with tight time constraints. These schemata represent underlying structures which link perceptive and cognitive elements, and facilitate the recognition of situations during a counter attack.

Choosing and implementing computational models

The second stage of the design process consisted of working on narrowing the gap between the decision-making model for tactical recognition of relevant configurations [START_REF] Bossard | Transfer of learning in virtual environments: A new challenge?[END_REF]) and computational models. In fact, two computational models were required in order to obtain what we refer to as a simplified analogy: a problem-solving model (Case Based Reasoning; [START_REF] Aamodt | Case-Based Reasoning: Foundational Issues, Methodological Variations and System Approaches[END_REF] and a context representation model [START_REF] Kokinov | A Dynamic Approach to Context Modeling[END_REF].

The CBR (Case Based Reasoning) model is based on the hypothesis that a perceived problem (development) can be solved successfully by referring to past experience. Finding a solution depends on identifying the experience which best fits the current situation, within all the cases encountered in the past (recall). Each case thus represents an action potential which is defined according to its context (adaptation). This ability to adapt is one of CBR's most encouraging features as the solutions found in the base are not applied directly but are rather adapted to fit a specific context. The base set of cases acts as a guide and does not dictate the responses. This model stems from frames theory [START_REF] Minsky | A framework for representing knowledge[END_REF], which are similar to schemata concepts. The schemata can be adapted due to contextual representation.

Contextual representation originates from the model developed by [START_REF] Kokinov | A Dynamic Approach to Context Modeling[END_REF].

According to the dynamic theory of context, if the context of a decision is always general, we can distinguish its component dimensions through analysis and representation. He thus identifies internal and external context. External context refers to the physical properties of the situation, the elements that could be said to be objective from an exocentric point of view.

Internal context is defined as the active state of an agent' s cognitive system at a given moment t. These are the elements to which the agent will refer in order to make a decision: the context as perceived from an egocentric point perspective. From this initial distinction, the context of a CoPeFoot agent is then divided into a number of sub-contexts. The external, or environmental, context groups together all of the physical elements of the situation. Internal context is a representation of the agent's skills, or a representation of the agent's state, i.e. fatigue. Each sub-context is made up of a set of perceptions which, once assembled, form the context of the agent within the situation. This context-free representation has two roles. The first is to facilitate contextualised decision-making (figure 1); for each agent, the context is thus an active filter which generates the situation in progress. The second role is to authorise the graphical visualisation of each perception and context.

Figure 1. The computer-generated CBR and Context model [START_REF] Bénard | Raisonnement en contexte pour la simulation participative et l'étude des situations dynamiques collaboratives. Une application au sport collectif[END_REF] Using these two computational models, we can conduct a simplified analogy between a representation of the tactical decision, from a social-science perspective, and that of the autonomous virtual agents' decision-making, from a computer science perspective. Above all, we are trying to avoid contradiction between a tactical decision model in football and an autonomous virtual agent model.

Simulations and enhancing computational models.

In order to improve the behavioural believability of the virtual agents, the computational model can be assigned evolution possibilities. We considered the evolution of the agents' behaviour according to three categories which can be positioned on three levels: 1) First level: the empirical model stemming from the analysis of human activity is used (applied) in order to enhance the set of cases and to define the agents' perceptions in context. In this respect, the description of expert footballers' typical schemata constitutes an information base for computer science researchers. The computer program limits actions: there is explicit learning or learning through instructions.

2) Second level: the computational model is enhanced through use. Within CoPeFoot, an interaction is proposed: the users click on the zones of the context they find relevant. Thanks to the benefits offered by augmented virtuality, the user defines the action's significant elements. The information provided by the users, when they click on these elements, is used to revise the weighting associated with each contextual element according to an algorithm described in [START_REF] Bénard | Raisonnement en contexte pour la simulation participative et l'étude des situations dynamiques collaboratives. Une application au sport collectif[END_REF]. The computer program has only partial control of actions: learning is linked to the agents' imitation of the users' perceived behaviours.

3) Third level: enhancement is a result of the simulation. The computational model records the new cases which occur during interaction. The "case tree" expands in order to offer the virtual agents new possibilities in terms of actions. The computer program has very little control of actions: learning is implicit. This stage of the design process of an interactive simulation demonstrates the of the models' evolution, which in turn enables the behaviours arising from that simulation to evolve.

Experimentation and simulation validation

The believability of a simulation is not easy to evaluate. Some simulators are tested in terms of skills transfer (for a complete review of these tests, see [START_REF] Bossard | Transfer of learning in virtual environments: A new challenge?[END_REF]. The transfer of skills from the virtual to reality is considered as a measure of the VE's success as a training tool. Some experimental studies have shown effective skills transfer [START_REF] Rose | Training in virtual environments: transfer to real world tasks and equivalence to real task training[END_REF], while others have shown less significant results [START_REF] Kozak | Transfer of training from virtual reality[END_REF]. These evaluations, which are carried out after the study, do not participate in designing the virtual environment and therefore are not used to modify the computational model. In order to rectify this shortcoming, we implemented an exploratory in virtuo study which aimed to evaluate the behavioural believability of CoPeFoot during the design process. Inspired by the Turing test (1950), this experimentation tested the ability of a user to distinguish between players controlled by humans and autonomous virtual players. This "decoy effect" was studied by comparing two groups of subjects (experts and novices) during three consecutive measurements. Results show a more marked decoy effect for novices when compared to experts, and an improvement for both groups over time. These results show that our virtual agents seem to demonstrate acceptable believability. They also show a reduction of this believability over time, and highlight the importance of the evolutionary nature of computational models in ensuring these simulations remain believable.

Advantages of virtual reality simulation for training and research in sports sciences

Today, virtual reality provides a conceptual, methodological and experimental framework which is well adapted to imagining, modelling and experimenting complexity.

Virtual reality simulations therefore allow real interaction with the modelled system. These simulations let us observe phenomena as if we had a virtual microscope which could be moved and oriented as we wish, with a choice of focal points. User can test the resistance of the implemented model (reactivity and adaptability). They become spectator-actor-creators as they can focus on observing a specific type of behaviour, a subsystem's activity or the overall activity of the system. The user can also interrupt the phenomenon at any time, accurately focus on the bodies present and the interactions underway, and then restart the simulation where it was stopped. This new kind of experiment is called in virtuo experimentation.

Simulations in virtual reality allow the user to evolve with other users and/or virtual autonomous agents. Finally, users are "in situ" and "in action". Virtual reality fully involves the user in the simulation, which is closely akin to the participatory design approach preferring to consider users as human actors rather than human factors. Within this type of virtual environment, users must interact with autonomous agents and/or human team members. Various combinations of multiple human or virtual learners may be brought together for practice exercises.

Advances in virtual reality technologies can be used to enhance the acquisition of cognitive skills. As an example, to become progressively effective in a sport situation, the athlete must learn by acting. Putting the athlete into action can be expensive (in material terms) or risky (in human terms). This is the case when the aim is to learn how to act and react when faced with unpredictable events (like in team sports) or malfunctioning (material difficulties or psychological breakdown during competition). These problem-solving skills in dynamic situations are particularly difficult to deal with (uncertain, progressive and with strong time constraints) using the classical training approach of case studies, establishing general rules or instructions related to expected scenarios. On the contrary computer simulation makes it possible to immerse athletes in a VE where they can try things, choose, take initiatives, fail and try again. In virtual reality simulations, users can reconsider all previous research in order to study the operations induced by the sporting situation through the simulation. By confronting the athlete with more realistic environments than those traditionally used (i.e. pictures or videos), virtual reality allows, for example, to go from a mode of external or exocentric presentation to a mode of internal or egocentric presentation [START_REF] Petit | Scene perception and decision-making in sport simulation: A masked priming investigation[END_REF].

In conclusion, virtual reality simulations, as we developed them, allow the creation of game situations, which radically change the environments commonly used, in the perspective of both research and training. Of course, there is an obvious need for further investigation to acquire better and deeper understanding of the benefit of virtual reality simulations in sport. [START_REF] Loomis | Immersive virtual environment technology as a basic research tool in psychology[END_REF] have already highlighted the value of virtual reality technologies as a tool with high ecological validity that will greatly serve psychological research. We hope that, in view of this, sports science researchers will also be tempted by the new opportunities that virtual reality has to offer.

  Contextual information is reified by symbols indicating distance from the target, teammates and opposition players; symbols indicate whether or not teammates are marked or open.

Figure 2 :

 2 Figure 2: Representation of an agent's perceptions in context: exocentric perspective.


Kevin Spelman

Delphine Depoix

Megan Mccray

Elisabeth Mouray

Philippe Grellier

The traditional medicine Spilanthes acmella, and the alkylamides spilanthol and undeca-2E-ene-8,10-diynoic acid isobutylamide, demonstrate in vitro and in vivo anti-malarial activity

Keywords: Plasmodium falciparum, alkylamides, deca-2E,6Z,8E-trienoic acid isobutylamide, malaria, traditional medicine, phytotherapy

Spilanthes spp. are used as traditional herbal medicines in Africa and India to treat malaria. Yet, to date, there is no data on active constituents or most effective extraction methods for this indication. The isolated alkylamides, spilanthol and undeca-2E-ene-8,10-diynoic acid isobutylamide, found in S. acmella Murr., were shown to have IC50s of 16.5 μg/mL and 41.4 μg/ mL on Plasmodium falciparum strain PFB and IC50s of 5.8 μg/mL and 16.3 μg/mL for the chloroquine resistant P. falciparum K1 strain, respectively. Further investigations revealed that at relatively low concentrations, spilanthol and the water extract of S. acmella reduced the parasitemia 59% and 53% in mice infected with P. yoelii yoelii 17XNL at 5 mg/kg and 50 mg/kg, respectively. Unexpectedly, the 95% ethanol extract of S. acmella was less effective (36% reduction in parasitemia) at 50 mg/kg. These results provide the first evidence supporting S. acmella against malaria and demonstrating active constituents in S. acmella against P. falciparum.

INTRODUCTION

The majority of countries coping with malaria spend less than US $10 per capita annually on healthcare, resulting in a situation where drug costs of 50 cents or less are economic deterrents to treatment [START_REF] White | Antimalarial drug resistance[END_REF]. Perhaps due to such economics, and the lack of access to healthcare facilities, medicinal plant preparations remain a well utilized option for the treatment of malaria by the rural poor [START_REF] Spelman | Silver Bullet" Drugs Vs. Traditional Herbal Remedies: Perspectives on Malaria[END_REF]. Spilanthes acmella Murr. (Asteraceae; syn. Blainvillea acmella (L.) Philipson) is one such plant from the traditional pharmacopoeia that is reported to be useful in the treatment of malaria. A related species, S. oleracea L., is a component of a formula known as Malarial-5, produced and sanctioned by the National Institute of Public Health in Mali for the treatment of malaria, relying primarily on ethnobotanical indications as evidence for treatment [START_REF] Keita | Etude preliminaire sur la faisabilite d'un protocole d'essai clinique[END_REF].

Several bioactive compounds have thus far been isolated and characterized from S. acmella which includes alkylamides and flavonoids. The N-alkylamides are fatty acid derivatives and have been identified in several species of Spilanthes [START_REF] Greger | Comparitive phytochemistry of the alkylamides[END_REF]. Early work found spilanthol, also known as affinin or deca-2E,6Z,8E-trienoic acid isobutyl amide, a local anesthetic, as the main lipidic component [START_REF] Gerber | Ueber die chemischen Bestandteile der Parakresse (Spilanthes olearacea, Jacquin)[END_REF]. More recent work has found acetylenic alkylamides such as undeca-2E-en-8,10-diynoic acid isobutylamide (UDA) in lower quantities [START_REF] Bae | A validated liquid chromatography-electrospray ionization-mass spectrometry method for quantification of spilanthol in Spilanthes acmella (L.) Murr[END_REF]. However, these compounds and the extracts of S. acmella, have never been assessed for antiplasmodial activity.

MATERIALS AND METHODS

Plant Material and Extractions

Cultivation of S. acmella took place in Williams, OR at Horizon Herbs. Fresh, whole plants of S. acmella were harvested. Species was verified by Richard Cech (Horizon Herbs, Williams, OR) and voucher specimens were submitted to the University of North Carolina Herbarium in Chapel Hill, NC (accession numbers 583423 and 583424). The plants were one year old at time of harvest. A typical protocol for the manufacture of dry root ethanolic extracts was followed and has been described previously (Spelman et al., 2009b). After extraction, extracts were concentrated to 10 mg/mL by speedvac for in vitro and in vivo experiments.

Isolation of spilanthol

A CombiFlash system (Teledyne, Lincoln, Nebraska, USA) with a 130 g C18 column was used for the separation. A gradient of isocratic separation was conducted with mobile phase composition of 50% A and 50% B, where A = 1% acetic acid (Fisher Chemical, Pittsburgh, PA, USA) in nanopure water and B = HPLC grade acetonitrile (Pharmaco-AAPER, Shelbyville, KT, USA). A flow rate of 0.2 mL/min was used, the injection volume was 10 μL, detection wavelength 229 nm and the total analysis time was 10 min. An ion trap mass spectrometer with electrospray ionization source (LCQ Advantage, ThermoFisher, San Jose, CA) was employed for quantification of spilanthol content and for identification of other alkylamides and has been published previously. The purity of the spilanthol was estimated to be 84% spilanthol and 13% spilanthol isomers, and an alkylamide of (MH+) of 236 m/z at 2.2% previously identified as deca-2E,6Z,8E-trienenoic acid 2-methylbutylamide [START_REF] Bae | A validated liquid chromatography-electrospray ionization-mass spectrometry method for quantification of spilanthol in Spilanthes acmella (L.) Murr[END_REF]. UDA was purchased from Chromadex (Santa Ana, CA, USA).

Biological assays-in vitro and in vivo.

The IC50s of spilanthol and UDA were determined against the chloroquine-resistant K1 strain and the mildly chloroquine-resistant strain PFB of Plasmodium falciparum using a previously established method [START_REF] Desjardins | Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique[END_REF]. The growth inhibition was assessed in triplicate by comparison of the radioactivity incorporated into the treated culture with that in control culture from the same plate. The in vivo experiments were carried out with 5 week old male and female Swiss mice weighing 23.2 g (±0.67) (Elevage Janvier, La Genest Saint Isle, France) were housed in standard environmental conditions (24±1.8 C, 55% humidity). The experiments were conducted in accordance with the EEC Directive of 1986 (86/609/ EEC) on laboratory animals. Briefly, extracts of Spilanthes acmellla and spilanthol (25 mg/ kg and 2.5 mg/kg twice daily, respectively) in a PBS methylcellulose solution (1%) were injected intraperitonealy into each animal 2 hours after inoculation with 10 7 Plasmodium yoelii yoelii parasites. Injections were continued twice a day for 4 days and experimental groups consisted of 5 mice (1 male and 4 females) per group. Parasitaemia was monitored daily by microscopic examination of Giemsa stained-thin blood smears.

Statistical analysis

Data is expressed as the mean ± SEM and comparison of means was conducted using a two tailed t-test for paired data when differences were observed. The mean values were considered significantly different if p < or = 0.01.

RESULTS AND DISCUSSION

Figure 1 illustrates the IC50s for the tested alkylamides, spilanthol and UDA on P. falciparum in vitro. For the Brazilian mildy chloroquine resistant strain PFB (Figure 1A), the IC50s for spilanthol and UDA are 16.5 and 41.4 μg/mL, respectively. While for the Thailanese chloroquine resistant strain K1 (Figure 1B), the effect of the alkylamides is significantly greater, with IC50s of 5.8 and 16.3 μg/mL, respectively.

Because Spilanthes remedies are commonly prepared as tea, further studies into S. acmella were performed in vivo on P. yoellii yoelii-infected mice using a whole plant galenic extract (an extract of herb/vegetable origin) of 100% water (10 mg/mL). In addition, another form of traditional remedy, a fresh plant ethanolic extract (10 mg/mL, 70% ethanol final volume) was also utilized. Ethanol extracts have been shown to contain ten times the concentrations of spilanthol as compared to aqueous extracts [START_REF] Bae | A validated liquid chromatography-electrospray ionization-mass spectrometry method for quantification of spilanthol in Spilanthes acmella (L.) Murr[END_REF]. As seen in Figure 2, the control group had an average parasitemia of 17.7% (± 3.3) five days after infection. A significant reduction of parasitemia by treatment with spilanthol (5 mg/kg) and S. acmella water extract (50 mg/kg) was observed with parasitemia decreased to 7.3% ± 1.4 and 8.4% ± 1.7, respectively (p < 0.001). The average parasitemia of the Spilanthes acmella ethanol extract (50 mg/kg) group after 5 days was 11.3% ± 2.0 (p = 0.01). Thus, isolated spilanthol and water extract exhibited the highest activity, 59% and 53% reductions in parasitemia, while the ethanol extract showed a 36% reduction in parasitemia under these experimental conditions. This suggests that in addition to spilanthol, there may be water soluble constituents that are also active against Plasmodium. Common hydrophilic phytochemicals have previously been shown to potentiate known antimalarials [START_REF] Soh | In vitroand in vivo properties of ellagic acid in malaria treatment[END_REF]. Moreover, there is likely multiple modes of activity for the observed effect of Spilanthes extract. For example, the treatments could have also induced immunological activity contributing to a reduction in parasitemia. Recent studies on alkylamides structurally similar to spilanthol, and UDA specifically, demonstrate immunological activity, particularly cytokine modulation, at concentrations below 1.5 μM [START_REF] Matthias | Echinacea alkylamides modulate induced immune responses in T-cells[END_REF]Spelman et al., 2009a). Further investigations are necessary to determine the viability of this traditional medicine, and its lead compounds, for the treatment of malaria.

Figure 1 .

 1 Figure 1. Spilanthol and undeca-2E-ene-8,10-diynoic acid isobutylamide(UDA) in vitro inhibition of P.falciparum strains 1A Spilanthol and UDA show IC50son the P. falciparum strain PFB at 16.5 and 41.4 μg/ mL. 1B Spilanthol and UDA demonstrate IC50s of5.8 and 16.3 μg/mL on the chloroquine resistantstrain P. falciparum K1. 1C . Molecular structures of spilanthol and UDA. Growth inhibition was determined by comparison of the radioactivity incorporated into the treated culture with that in control culture from the same plate. Chloroquine served as a positive control (IC50s: PFB-28.4 nM; K1-100 nM). Values are mean± S.E.M.of experiments performed in triplicate.

Figure 2 .

 2 Figure 2. Spilanthol and extracts of whole plant S. acmella reduce parasitemia in Plasmodium y. yoelli-infected mice Spilanthol, water extract, or (70%) ethanol extract of Spilanthes acmella inhibit parasitemia as compared to the control group. Mice were inoculated with P. yoelli yoelli 17XNLand treatment started 2 hours later. Treatments (twice daily spilanthol 2.5mg/kg; water extract 25 mg/kg; ethanol extract 25mg/kg) were given two times aday for four days. Parasitaemia was determined 5 days after infection by microscopic examination of Giemsa stained-thin blood smears. Values are mean± S.E.M. (n = 5 for each group). †p = 0.01; *p< 0.001

Phytother Res. Author manuscript; available in PMC 2012 July 1.

Acknowledgments

This publication was made possible by grant number 1R15AT001466-01 from the National Center for Complementary and Alternative Medicine (NCCAM) at the National Institutes of Health and the Marie Curie Foundation which provided an early stage training fellowship for KS at the Muséum National d'Histoire Naturelle, Paris and the Laboratorie of Pharmacognosy, Université Paris 11. Additional support was provided by the National Science Foundation under grant number 0420292. Special thanks to Nadja Cech for providing the spilanthol and S. acmella extracts.

Abbreviations

K1

a strain of Plasmodium falciparum originating from Tailand PFB a strain of Plasmodium falciparum originating from Brazil UDA undeca-2E-en-8,10-diynoic acid isobutylamide