Elise Deunf

Franck Dolhem

Dominique Guyomard

Jacques Simonet

Philippe Poizot
email: philippe.poizot@cnrs-imn.fr

Anodic oxidation of p-phenylenediamines in battery grade electrolytes

Keywords: phenylenediamine, cyclic voltammetry, redox-active amine, organic batteries, PF 6 decomposition, lithium hexafluorophosphate

The use of anion-inserting organic electrode materials represent an interesting opportunity for developing 'metal-free' rechargeable batteries. Recently, crystallized conjugated diamines have emerged as new host materials able to accommodate anions upon oxidation at potentials higher than 3 V vs. Li + /Li 0 in carbonate-based battery electrolytes. To further investigate the electrochemical behavior of such promising systems, comparison with electroanalytical data of soluble forms of conjugated diamines measured in battery grade electrolytes appeared quite useful. However, the literature on the topic is generally poor since such electrolyte media are not common in molecular electrochemistry. This contribution aims at providing relevant data on the characterization by cyclic voltammetry of unsubstituted, diphenyl-substituted and tetramethyl-substituted p-phenylenediamines. Basically, these three molecules revealed two reversible one-electron reaction upon oxidation corresponding to the electrogenerated radical cation and dication, respectively, combined with the association of electrolyte anions (i.e.,

M A N U S C R I P T A C C E P T E D

Introduction

Global warming, fossil fuels depletion and rapid population growth are confronting our technology-oriented society with significant challenges notably in the field of power engineering. One of the priorities in this domain is to promote reliable, safe but also lowpolluting electrochemical storage devices for various practical applications from mWh to MWh range. Since the invention of the first rechargeable battery in 1859 by G. Planté (leadacid cell), the current manufacturing of batteries is still dominated by the use of redox-active inorganic species but the organic counterparts appear today as a promising alternative displaying several advantages such as low cost, environmental friendliness and the structural designability [START_REF] Poizot | Clean energy new deal for a sustainable world: from non-CO 2 generating energy sources to greener electrochemical storage devices[END_REF][START_REF] Liang | Organic electrode materials for rechargeable lithium batteries[END_REF][START_REF] Song | Towards sustainable and versatile energy storage devices: an overview of organic electrode materials[END_REF][START_REF] Haeupler | Carbonyls: Powerful organic materials for secondary batteries[END_REF][START_REF] Zhao | Rechargeable lithium batteries with electrodes of small organic carbonyl salts and advanced electrolytes[END_REF][START_REF] Schon | The rise of organic electrode materials for energy storage[END_REF][START_REF] Muench | Polymer-based organic batteries[END_REF][START_REF] Zhao | Advanced organic electrode materials for rechargeable sodiumion batteries[END_REF][START_REF] Winsberg | Redox-flow batteries: from metals to organic redox-active materials[END_REF]. For instance, the operating redox potential of organic electrodes can be widely tuned by the choice of (i) the electroactive functional group (both n-or p-type1 [START_REF] Song | Towards sustainable and versatile energy storage devices: an overview of organic electrode materials[END_REF][START_REF] Gottis | Voltage gain in lithiated enolate-based organic cathode materials by isomeric effect[END_REF]), (ii) the molecular skeleton, and (iii) the substituent groups. Organic structures based on conjugated carbonyl/enolate redox-active moiety represent probably the most studied family of n-type organic electrode materials especially for developing Li/Na-based rechargeable systems. Conversely, p-type organic electrodes which involve an ionic compensation with anions [START_REF] Song | Towards sustainable and versatile energy storage devices: an overview of organic electrode materials[END_REF][START_REF] Muench | Polymer-based organic batteries[END_REF][START_REF] Gottis | Voltage gain in lithiated enolate-based organic cathode materials by isomeric effect[END_REF] makes development of 'molecular' ion batteries possible [START_REF] Yao | Molecular ion battery: a rechargeable system without using any elemental ions as a charge carrier[END_REF] since numerous metal-free anions do exist. In this regard, our group has recently reported (for the first time) that crystallized conjugated diamines can accommodate anions at E > 3 V vs. Li + /Li 0 in the solid state with an overall reversible two-electron reaction making them interesting for positive electrode applications [START_REF] Deunf | Reversible anion intercalation in a layered aromatic amine: A High-voltage host structure for organic batteries[END_REF][START_REF] Deunf | Solvation, exchange and electrochemical intercalation properties of disodium 2,5-(dianilino)terephthalate[END_REF][START_REF] Deunf | A dual-ion battery using diamino-rubicene as anion-inserting positive electrode material[END_REF].

In light of the opportunity offer by this new family of insertion compounds and to go further in the understanding of electrochemical processes, we revisited the anodic oxidation of pphenylenediamine derivatives by cyclic voltammetry but measured in typical (aprotic) battery grade electrolytes for which less than 0.1 ppm of both O 2 and H 2 O are guaranteed. More specifically, we report herein the electrochemical feature of three selected phenylenediamines

Experimental

Chemicals

The different electrolyte formulations were prepared in an Ar-filled glovebox (MBRAUN) containing less than 0.1 ppm of both O 2 and H 2 O from lithium perchlorate (LiClO 4 ), lithium hexafluorophosphate (LiPF 6 ), propylene carbonate (PC), ethylene carbonate (EC) and dimethylcarbonate (DMC) purchased from BASF (battery grade) and used as received.

Lithium trifluoromethanesulfonate (LiOTf, 99.995%, Aldrich) was dried at 100° C under vacuum for 15 h prior use. The common "LP30" battery grade electrolyte (i.e., LiPF 6 1 M in EC:DMC 1:1 vol./vol.) was directly employed as received from Novolyte. Amines was purchased from Aldrich with the following purities: N,N'-p-phenylenediamine PD (99%), N,N,N',N'-tetramethyl-p-phenylenediamine TMPD (≥ 97%), N,N'-diphenyl-pphenylenediamine DPPD (98%), and triethylamine (Et 3 N, ≥99%).

Electrochemical procedures

Cyclic voltammetric (CV) experiments were recorded on a SP-150 potentiostat/galvanostat (Bio-Logic S.A., Claix, France). All electrochemical experiments were systematically conducted from freshly prepared electrolyte solutions (except the "LP30" battery grade electrolyte, Novolyte) in a conventional three-electrode setup (V = 10 mL) placed inside an Ar-filled glovebox (MBRAUN) containing less than 0.1 ppm of both O 2 and H 2 O. The working electrode was constituted of a commercial platinum disk microelectrode with a diameter of 1.6 mm (ALS Japan). Facing the working electrode, a large platinum wire was

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT

5 used as the counter electrode. An Ag + /Ag 0 reference electrode made of a fritted glass tube filled with an AgNO 3 10 mM solution in acetonitrile [START_REF] Bard | Electrochemical Methods: Fundamentals and Applications[END_REF] was systematically used. However, reported potentials were also given against the Li + /Li 0 reference electrode for a better appreciation of the battery community. This second reference electrode, made of lithium metallic attached on a Pt wire, was experimentally checked versus the Ag + /Ag 0 reference electrode in each studied electrolyte giving a correction of the measured potentials of +3.6 V.

Results and discussion

The typical electrochemical activity of the simple PD molecule is preliminary reported being a representative member of this family of redox-active compounds. In addition, PC/LiClO 4 1 M electrolyte was first employed in order to be aligned with our former battery cycling tests performed on crystallized p-phenylenediamines derivatives [START_REF] Deunf | Reversible anion intercalation in a layered aromatic amine: A High-voltage host structure for organic batteries[END_REF][START_REF] Deunf | Solvation, exchange and electrochemical intercalation properties of disodium 2,5-(dianilino)terephthalate[END_REF]. Basically, the oxidation of PD in such an electrolyte shows two anodic peaks (I, II) located at 3.58 and 4.07 V vs.

Li + /Li 0 , respectively (Figure 1b). When reversing the scan, two corresponding cathodic peaks are observed. The peak-to-peak separation values for both steps (I/I', II/II') are equal to 60 mV, which indicates the occurrence of two fully reversible one-electron processes. The anodic events are assigned to the electrogeneration of the radical cation PD • • • • + at peak I further followed by the dicationic form (PD 2+ ) at peak II. The ratio between the peak currents and the square root of the scan rate at both anodic and cathodic waves shows linearity (Figure 1c), which confirms the two electrochemical processes are under diffusion control as expected for reversible systems [START_REF] Bard | Electrochemical Methods: Fundamentals and Applications[END_REF][START_REF] Batchelor-Mcauley | Voltammetry of multi-electron electrode processes of organic species[END_REF].

At this point it should be recalled that the typical mechanisms for the electrochemical oxidation of phenylenediamines have already been established in common aprotic solvents for molecular electrochemistry [START_REF]The solvent effect on the electro-oxidation of 1,4-phenylenediamine. The influence of the solvent reorientation dynamics on the one-electron transfer rate[END_REF][START_REF] Bewick | Anodic oxidation of aromatic nitrogen compounds: Spectroelectrochemical studies of EE and EECr processes with a coupled redox reaction[END_REF][START_REF] Fernández | Determination of the kinetic and activation parameters for the electro-oxidation of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) in acetonitrile (ACN) by chronocoulometry and other electrochemical techniques[END_REF][START_REF] Santana | In situ UV-vis and Raman spectroscopic studies of the electrochemical behavior of N,N'-diphenyl-1,4phenylenediamine[END_REF][START_REF] Maleki | Mechanism diversity in anodic oxidation of N,N-dimethyl-pphenylenediamine by varying pH[END_REF] and very recently in the battery grade PC/LiBF 4 1 M electrolyte for non-aqueous redox flow batteries [START_REF] Kim | A comparative study on the solubility and stability of pphenylenediamine-based organic redox couples for nonaqueous flow batteries[END_REF]. Their oxidation processes through this

M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 6
two reversible one-electron transfer with the formation of successive stable radical cation and dication species. However, in the case of primary and secondary amines -meaning the existence of labile protons -this behavior can be impacted by the presence of acid-base species in the electrolyte [START_REF] Santana | In situ UV-vis and Raman spectroscopic studies of the electrochemical behavior of N,N'-diphenyl-1,4phenylenediamine[END_REF][START_REF] Maleki | Mechanism diversity in anodic oxidation of N,N-dimethyl-pphenylenediamine by varying pH[END_REF]. Indeed, these labile protons are easily involved in an acidbase reaction competing with the electrochemical process, sometimes even leading to the loss of the reversible character. Interestingly, in PC/LiClO 4 1 M electrolyte one can observe that

PD • • •
• + remains stable on the voltammetry time-scale and can undergo a second electrochemical oxidation at higher potentials for producing PD 2+ . Similarly, this dicationic form is also stable enough towards chemical side-reactions and is reduced back on the reverse scan. For comparison, two other common lithiated salts used in Li-batteries (LiPF 6 and LiOTf) were also evaluated as supporting electrolytes using again PC as the solvent. The resulting CV curves of PD shows quite similar electrochemical steps (Figure 2a) with fully reversible peaks obtained at anodic potentials of 3.6 and 4.1 V vs. Li + /Li 0 , respectively. This result attests that neither the thermodynamic nor the kinetic of the stepwise one-electron oxidation reactions are impacted by the counter anions of the supporting electrolyte although exhibiting very different donor number (DN) in PC and van der Waals volume [START_REF] Ue | Mobility and ionic association of lithium and quaternary ammonium salts in propylene carbonate and γ-butyrolactone[END_REF][START_REF] Ue | Ionic radius of (CF 3 SO 2 ) 3 C and applicability of stokes law to its propylene carbonate solution[END_REF][START_REF] Linert | Anions of low Lewis basicity for ionic solid state electrolytes[END_REF]. These results are in agreement with a dominance of the solvation process in high polarity aprotic solvents such as PC (ε r ~ 66) in which a low ion-pairing is expected [START_REF] Barrière | Use of weakly coordinating anions to develop an integrated approach to the tuning of ∆E 1/2 values by medium effects[END_REF]. However, the use of were also selected as representative secondary and tertiary p-phenylenediamine derivatives of interest for this comparative study. In fact, the possible π-delocalization by mesomeric effect (+M) occurring with the DPPD structure should induce both a positive potential shift and higher acidic character of the secondary amine functional group. On the contrary, methyl substituent groups which are electron-donating by inductive effect (+I) would decrease the oxidative strength of the p-phenylenediamine backbone (lower formal potential) whereas no acidic protons do exist. Figure 3 summarizes the most striking features observed in both PC/LiClO 4 1 M and EC-DMC/LiPF 6 1 M electrolytes. As expected with DPPD, the reversible stepwise one-electron oxidation steps occur at 140 and 50 mV higher than the corresponding events observed with PD while TMPD shows the lowest redox potentials of the series. It is worth noting that the presence of substituent groups on the p-phenylenediamine backbone does not impact the reversibility of the processes and further illustrates the stability of both the neutral and the electrogenerated species. Table 1 shows the diffusion coefficient values experimentally determined from the voltammetric curves recorded at different scan rates (50 mV.s -1 to 10 V.s -1 ). These values are comparable to those reported in the literature [START_REF] Kim | A comparative study on the solubility and stability of pphenylenediamine-based organic redox couples for nonaqueous flow batteries[END_REF][START_REF] Ue | Mobility and ionic association of lithium and quaternary ammonium salts in propylene carbonate and γ-butyrolactone[END_REF]. The peculiar electrochemical feature previously observed with PD in presence of PF 6 anion (Figure 2a) is again noticed in the case of DPPD with the appearance this time of an obvious reversible pre-peak (III) prior to the second main electrochemical step (Figure 3).

One possible explanation could be related to the peculiar chemistry of LiPF 6 in high polarity aprotic solvents (denoted :S). Indeed, it has long been known in the field of Li-ion batteries [START_REF] Linert | Anions of low Lewis basicity for ionic solid state electrolytes[END_REF][START_REF] Sloop | Chemical reactivity of PF 5 and LiPF 6 in ethylene carbonate/dimethyl carbonate solutions[END_REF][START_REF] Tasaki | Decomposition of LiPF 6 and stability of PF 5 in Li-ion battery electrolytes[END_REF][30][31]] that undissociated LiPF 6 do exist at relatively high electrolyte concentrations (in the range of 1 mol.L -1 ) in equilibrium with F -and the strong Lewis acid, PF 5 :

[Li + PF 6 - ] (ion pair) + :S ⇌ [S:PF 5 ] + (sol) + LiF (1) M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 8
The dilemma is that in high polarity aprotic solvents the dissociation of [Li + PF 6 -] ion pairs is facilitated but the stabilization of PF 5 too. In addition, it has been shown that the presence of H + ion in the medium that also form ion pairs with PF 6 -catalyzes its decomposition according to the following equilibria due to the strong H … F interactions [30]:

H + + PF 6 - (sol) ⇌ [H + PF 6 - ] (ion pair) ⇌ H … F-PF 5 ⇌ HF + PF 5 (2)
In the case of PD and DPPD, the resulting radical cations electrogenerated at peak I exhibit more polarized N-H bonds in comparison with the pristine state. In the presence of PF 6 -(and potentially F -in the vicinity of the electrode), the acidic proton can be neutralized according to Eq. 2 for producing the corresponding radical, which is a more readily oxidizable species.

This hypothesis is further supported by the fact that no pre-peak is observed with TMPD for which no labile protons do exist. However, supplementary experiments were also conducted by adding a base to LiPF 6 -free electrolyte media in order to verify the deprotonation assumption. In practice, triethylamine (Et 3 N) was used as a common base in organic chemistry. Figure 4 summarizes the as-obtained results by selecting both DPPD and TMPD as the two representative cases bearing no labile proton. As expected, in the presence of trimethylamine (0.5 mM), the pre-peak appeared with DPPD (III) while the electrochemical behavior of TMPD was not affected. Note that a pre-peak (IV) was also observed prior to the first oxidation step (I), which can be attributed to the deprotonation reaction of DPPD itself at this concentration of base. The proposed overall mechanism is finally depicted in Figure 5 Table 1. Diffusion coefficients for the oxidation of phenylenediamines in the different electrolytes calculated with the Randles-Sevcik equation from the slope of the experimental curves i p = f (υ 1/2 ) .

  e., N,N'-p-phenylenediamine, PD; N,N'-diphenyl-p-phenylenediamine, DPPD; N,N,N',N'tetramethyl-p-phenylenediamine, TMPD-Figure1a) solubilized at millimolar concentrations in different electrolyte formulations including LiPF 6 as the most popular supporting salt used in the Li-ion battery by manufacturers and researchers.

LiPF 6

 6 supporting salt shows some slight differences with the appearance of a new contribution between the two regular steps and a peak-to-peak separation of the second anodic wave shifted from reversibility (∆E 1/2 = 90 mV) suggesting a quasi-reversible process. When excluding LiPF 6 , the solvent change does not affect the reversibility of the two electrochemical steps involved with PD. Figure2bshows for instance a comparison of CVs recorded in PC, DMC and EC-DMC, respectively, using a concentration of 1 mol.L -(DPPD) and tetramethyl-substituted p-phenylenediamines (TMPD)

in the presence of PF 6 - 6 --

 66 or by adding Et 3 N in PF free electrolyte. Interestingly, this particular electrochemical investigation focused on both substituted and unsubstituted pphenylenediamines supports well the few other reports pointed out the decomposition issues of LiPF 6 in aprotic media when labile protons are present.This study aimed at emphasizing the potentiality of p-phenylenediamines which can offer high potential and multi-electronic behavior as p-type materials for battery applications. A specific cyclic voltammetry study was then conducted to evaluate the electrochemical behavior of three selected p-phenylenediamines derivatives (PD, DPPD and TMPD) dissolved in several battery grade (carbonate) electrolyte media. Among the various electrolytes tested, it appeared a chemical instability of the electrogenerated radical cation in presence of LiPF 6 when labile protons do exist on nitrogen atoms due to the propensity of PF 6-to be decomposed in high-polarity solvents such as PC-or EC-based battery electrolytes;this phenomenon being catalyzed by labile protons. This electrochemical study provides also to the Li battery community a supplementary proof concerning the high reactivity of the most popular supporting salt versus any labile proton potentially present in a battery electrolyte.

14 Figure 1 .Figure 2 .

 1412 Figure 1. (a) Structural formula of studied p-phenylenediamines denoted PD, DPPD and

Figure 3 .

 3 Figure 3. Comparison of typical CV curves recorded on Pt disk microelectrode at a scan rate of 200 mV.s -1 using a concentration of 1 mM PC/LiClO 4 1 M or EC-DMC/LiPF 6 1 M

Figure 4 . 18 Figure 5 .

 4185 Figure 4. Typical CV curves recorded on Pt disk microelectrode at a scan rate of 200 mV.s -1

Note that n-type structures involve upon oxidation an ionic compensation with cation release whereas p-type structures imply an anion.

Acknowledgments

This work was partially funded by a public grant overseen by the French National Research Agency as part of the program "Investissements d'Avenir" [grant number ANR-13-PRGE-0012] also labeled by the Pôle de Compétitivité S2E2.


Thomas Constant
email: thomas.constant@cnam.fr

Axel Buendia
email: axel.buendia@cnam.fr

Catherine Rolland
email: catherine.rolland@ktm-advance.com

Stéphane Natkin
email: stephane.natkin@cnam.fr

A switching-role mechanic for reflective decision-making game

Keywords: serious game, game design, decision-making, overconfidence

This paper introduces issues about a methodology for the design of serious games that help players/learners understand their decisionmaking process. First, we discuss the development of a video game system based on a switching-role mechanic where the player becomes the game leader of the experience. Then, we introduce game mechanics designed to induce a specific behavior, overconfidence, that helps to understand the players' decision-making processes. Finally, we describe tools for measuring the players' self-reflection regarding their judgment process.

Introduction

Serious games for decision-making play an important role in management training [START_REF] Barth | Former les futurs managers à des compétences qui n'existent pas: les jeux de simulation de gestion comme vecteur d'apprentissage[END_REF]. But their use is too often limited to the training of a specific behavior, or to learn good habits. Video games offer the possibility of teaching a more reflexive experience [START_REF] Constant | Enjeux et problématiques de conception d'un jeu sérieux pour la prise de décision[END_REF]. They can be designed as decision-driven systems [START_REF] Schell | The Art of Game Design A Book of Lenses[END_REF], tools created to help learners reflect on how they play [START_REF] Gee | Surmise the Possibilities: Portal to a Game-Based Theory of Learning for the 21st Century[END_REF], how they interact with the system [START_REF] Papert | Mindstorms: Children, Computers, And Powerful Ideas[END_REF] ; thus, how they make a decision [START_REF] Shaffer | Epistemic Games[END_REF]. This paper presents issues about a game design methodology for serious games whose goal is to help learners gain a better understanding of their decision-making process, and to encourage players' reflexivity towards their own decision-making. The design is based on an asymmetrical gameplay: after the player has made a judgment task, and has taken a decision, s/he can become the "game leader"able to influence the other player. By switching roles, s/he may gain a better understanding of his/her own decision process. Our proposal to validate the mechanic's efficiency is to build a video game designed to develop and maintain an excessive confident behavior in the players' judgment, in order to promote the emergence of a reflexive stance of the player towards their decision processes. The first section of this paper introduces our model and its working conditions. The second section explains game mechanics useful for inducing overconfident behavior. These mechanics are, in effect, a translation of cognitive science principles regarding overconfidence into game variables. The third section proposes measurement tools for evaluating the game's efficiency.

2 Main issue: enlighten the player's decision-making 2.1 Switching-role mechanic and operating conditions Our main hypothesis is that a switching-role mechanic can help the players to develop a better understanding of their decision-making processes. However, we make the assumption that switching-role is not enough: the player can be good at playing but may not necessarily understand of how. To help players to be in a reflexive position about their abilities to make a decision, we introduce three conditions to support the switching-role mechanic:

-A main condition: when switching-role, the player must become the game leader of the game. In this role, s/he can use variables to impact the game experience. The game leader is the one who plays with the mechanics in order to alter the other player's judgment. S/he can achieve an optimal point of view of how the game works, and how it can alter the player's behavior. -A pre-condition: before becoming the game leader, it is necessary that the player has been in the position of taking a decision for which s/he is confident about. The confidence must be assumed even if the decision was made in an uncertain situation, and may be biased by the context of the game. Players' judgment about their decision must be unequivocal if we want to help them to understand how it can be affected. -A post-condition: after playing the game leader, it is necessary that the player is able to play his/her first role again, in order to measure the impact of the switching-role mechanic on his/her behavior. For a serious purpose, we need to help the player to achieve this state of selfreflection. His/her way to make a decision has to be easier to understand and, as a consequence, the decision mechanisms have to be underlined by the system. Our proposal is to use cognitive fallacies in order to highlight judgment processes and explain why the player decision is biased.

Heuristic judgment and decision making processes

Heuristics and biases research allows to understand more precisely human judgment under uncertainty. Confronted with a complex question, decision-makers sometimes unwittingly substitute the question with an easier one. This process, called "attribute substitution", is an example of heuristic operating [START_REF] Kahneman | A model of heuristic judgment[END_REF]. A heuristic represents a shortcut in the judgment process as compared with a rational approach to decision-making. Heuristics are "rules of thumbs" -simpler and faster ways to solve a problem, based on knowledge, former experiences, skills, and cognitive abilities (similar to memory or computational ability) [START_REF] Kahneman | Judgment under Uncertainty: Heuristics and Biases[END_REF][START_REF] Gigerenzer | Heuristic decision making[END_REF]. If heuristic strategies are efficient most of the time, they can, however, occasionally lead to failure comparatively to a rational resolution of the full problem. These errors are called biases: markers of the use of a judgment heuristic. Identifying these markers allows researchers to better understand decision-making processes and reveal heuristic at work. Based on this approach, our methodology entails focusing on a single behavior in order to underline the player's decision-making processchosen specifically because it frequently manifests itself in the comportment of game players: overconfidence.

Serious game concept and context of use

Before introducing specific game mechanics, we present the key elements of a gameplay chosen to illustrate the use of our methodology. The game is played by two players on two different computers. Players cannot see each other and cannot communicate directly, but they are aware of each other presence and role in the game. They play a narrative adventure game which apparent goal is to solve a sequence of criminal cases. Each player has a specific role. One of the players adopts the role of an investigator, gathering information to build a hypothesis for a given problem. S/he is confronted with various forms of influence, which are going to have an impact on his/her judgment. The other player personify the game leader, played by the other player, who is going to control the investigator access to information. S/he has access to multiple game variables useful to induce overconfidence in the other player's judgment (see below). After playing a sufficient number of levels in the same role (to be sure that the evaluation of the player's behavior is correct), the players exchange their roles: the game leader becomes the investigator, and reciprocally. By experimenting with these two gameplays, the player puts its own actions into perspective in order to understand how s/he made a decision.

3 Pre-condition: guiding the player's judgment

Variables to orient the player's confidence

The overconfidence effect has been studied in economic and financial fields as a critical behavior of decision-makers [START_REF] Bessière | Excès de confiance des dirigeants et décisions financières: une synthèse[END_REF]. It impacts our judgment of both our own knowledge and skills and those of others [START_REF] Johnson | The evolution of overconfidence[END_REF][START_REF] Russo | Managing overconfidence[END_REF]. Overconfidence can be explained as a consequence of a person's use of heuristics such as availability and anchoring (defined in Section 3) [START_REF] Griffin | The weighing of evidence and the determinants of confidence[END_REF][START_REF] Russo | Managing overconfidence[END_REF]. Overconfidence is also commonly observed in player behaviors. In a card game, for example, beginners as well as experts can be overconfident with regard both to performance and play outcomes [START_REF] Keren | Facing uncertainty in the game of bridge: A calibration study[END_REF]. If we want to induce this behavior, the player's judgment has to be driven in a given direction. As a consequence, game mechanics must be related to expressions or sources of overconfidence in human behavior [START_REF] Moore | The Trouble with Overconfidence[END_REF]. Then, based on game design methods for directing the behavior of the player [START_REF] Schell | The Art of Game Design A Book of Lenses[END_REF][START_REF] Adams | Fundamentals of Game Design[END_REF], we derived game mechanics that can be used to produce the overconfidence effect. Figure 1 presents some mechanics examples according to three major expressions of the overconfidence effect.

Core gameplay

At the beginning of the level, the game leader introduces a case to the other player, the investigator. The investigator's mission is to find the culprit: s/he is driven through the level to a sequence of places where his/her is able to get new clues about the case, mainly by questioning non-playable characters. But the investigator is allowed to perform a limited number of actions during a level, losing one each time s/he gets a new clue. Thus, the investigator is pushed to solve the case as fast as possible. The game leader is presented as the assistant of the investigator, but his/her real role is ambiguous: maybe s/he is trying to help the investigator, or maybe s/he has to push the investigator on the wrong track. This doubt is required to avoid biasing the investigator's judgment about the nature of the influence which target him/her. The investigator should not easily guess what the game leader is really doing, and should stay in a context of judgment in uncertainty. If this is not the case, the measure of the confidence of the player may be distorted. After several levels (several cases), the investigator

Difficulty Anchoring Confirmation

Definition A decision-maker can be overconfident if s/he thinks that the task is too easy or too difficult [START_REF] Griffin | The weighing of evidence and the determinants of confidence[END_REF][START_REF] Lichtenstein | Do those who know more also know more about how much they know?[END_REF].

Estimations are based on an anchor, a specific value they will easily memorize. The adjustments will be too far narrowed down towards this value to give an appropriate estimation. Anchor bias can induce overconfidence when evaluating an item or an hypothesis [START_REF] Kahneman | Intuitive prediction: Biases and corrective procedures[END_REF][START_REF] Russo | Managing overconfidence[END_REF].

Confirmation bias reveals the fact that decision-makers often seek evidences that confirm their hypothesis, denying other evidences that may refute them [START_REF] Koriat | Reasons for confidence[END_REF][START_REF] Russo | Managing overconfidence[END_REF].

Mechanic example 1

Setting up sensitive difficulty by restricting the player's exploration in time and space.

The game designer chooses a specific piece of information to use as an anchor. In order for it to be clear to the player that s/he has to use it the information must be important to the case.

The game designer classifies each piece of evidence according to how they support the investigation's solution and each of the red herrings.

Mechanic example 2

Setting up logical difficulty using puzzle game design, the intrinsic formal complexity of which can be controlled via given patterns and parameters.

In order to compare its impact on player judgment the game leader sets the anchor at different points and times in the game.

During the game, when giving evidence to the player, the game leader must give priority to evidence that favors a specific red herring. becomes the new game leader, and vice versa. To win, the investigator must find the probable solution of a case depending on the clues s/he might have seen, associated with a realistic measure of his/her confidence. At the opposite, the game leader wins if s/he has induced overconfidence in the investigator's judgment, and if the latter didn't discover the game leader role.

4 Post-condition: measuring the player's behavior

Evaluation of the player's confidence

Two kinds of evaluations are used to assess the effectiveness of a serious game based on our switching-role model. The first ones focuses on the player's judgment through the evaluation of his/her confidence. Measurements of the investigator's overconfidence are based on credence calculation, which is used in overconfidence measurement studies [START_REF] Lichtenstein | Do those who know more also know more about how much they know?[END_REF]. This score assesses the players' ability to evaluate the quality of their decision rather than assessing the value of the decision itself. Variations of this score from one game session to an other can show the evolution of the players' confidence regarding their decision-making process. After playing, players must fill out a questionnaire survey in order to give a more precise evaluation of their progression and confidence [START_REF] Stankov | Confidence and cognitive test performance[END_REF].

Evaluation of the player's reflexivity

The second evaluations highlight the players' ability to assess their self-efficacy in terms of problem solving. Judgment calibration may engage the decision-maker in a reflexive posture on his/her ability to judge the quality of his/her decision that the overconfidence effect may bias [START_REF] Stone | Overconfidence in Initial Self-Efficacy Judgments: Effects on Decision Processes and Performance[END_REF]. But it is not enough for a long-lasting understanding of the behavior [START_REF] Stankov | Realism of confidence judgments[END_REF]. Therefore, in order to extend its effects, we design a re-playable game which can be experienced repeatedly within one or various training sessions. The switching-role mechanic allows the player to engage in a self-monitoring activity, by observing the behavior of other players and by experimenting on them. After several levels from this perspective, the player discerns how the investigator develops overconfidence, or tries to reduce it. Then the player resumes his/her first role and starts by giving new self-evaluations. This time, the player should give a more realistic assessment of his/her ability to solve the case. The variation of the players calibration score can give us a precise measure of the evolution of their behavior, and by extension, of their understanding on how they make a decision in the game. Figure 2 presents the range of possible player behaviors that we can expect.

Not confident Very confident

The solution of the case given by the player is improbable

The player is aware of the weakness of his/her reasoning. Well calibrated Score multiplied

The player was too quick in his/her reasoning (and s/he has failed to seen the limits). S/he made a mistake in his/her reasoning. Uncalibrated Player loses his points The solution of the case given by the player is probable

The player was too quick in his/her reasoning (and s/he realizes this). S/he is correct, but has no confidence in his/her reasoning.

Uncalibrated Player loses his points

The player is correct as well as confident in his/her reasoning.

Well calibrated Score multiplied Fig. 2. Player behavior matrix

Conclusion and future works

This paper proposed a game design methodology for building serious games and the way of use to let the players gain a better appreciation of how they make a decision. This methodology is based on the heuristic approach to the analysis of human judgment as well as game design research that relates to decision-making and reflexivity. We then proposed rules and game mechanics designed to induce and control the overconfidence effect and to encourage the players' reflexivity regarding their decision-making. Finally, we introduced the idea of tools for measuring both the players' reflexivity and the effectiveness of the game itself. This methodology is currently being used to develop a prototype of the serious game, which will be evaluated in training courses at the Management & Society School of the National Conservatory of Arts and Crafts1 . The prototype will be able to verify the proper functioning of the switching-role mechanic, its impact and its durability on the player's behavior.

Fig. 1 .

 1 Fig. 1. Variables and game mechanics to orient the player's behavior

For more informations about the School and the Conservatory: http://the.cnam.eu


B G M Van Dijk

E Potier

Maarten Van Dijk

Marloes Langelaan

Nicole Papen-Botterhuis

Keita Ito

Reduced tonicity stimulates an inflammatory response in nucleus pulposus tissue that can be limited by a COX-2-specific inhibitor

Keywords: explant culture, disc herniation, inflammation, regenerative therapy, sustained release

In intervertebral disc herniation with nucleus pulposus (NP) extrusion, the elicited inflammatory response is considered a key pain mechanism. However, inflammatory cytokines are reported in extruded herniated tissue, even before monocyte infiltration, suggesting that the tissue itself initiates the inflammation. Since herniated tissue swells, we investigated whether this simple mechanobiological stimulus alone could provoke an inflammatory response that could cause pain. Furthermore, we investigated whether sustained-release cyclooxygenase-2 (COX2) inhibitor would be beneficial in such conditions. Healthy bovine NP explants were allowed to swell freely or confined. The swelling explants were treated with Celecoxib, applied either as a bolus or in sustained-release. Swelling explants produced elevated levels of interleukin-6 (IL-6) and prostaglandin E2 (PGE2) for 28 days, while confined explants did not. Both a high concentration bolus and 10 times lower concentration in sustained release completely inhibited PGE2 production, but did not affect IL-6 production. Swelling of NP tissue, without the inflammatory system response, can trigger cytokine production and Celecoxib, even in bolus form, may be useful for pain control in extruded disc herniation.

Introduction

Low back-related leg pain, or radicular pain, is a common variation of low back pain. Its main cause is extruded lumbar disc herniation [START_REF] Gibson | Surgical interventions for lumbar disc prolapse: updated Cochrane Review[END_REF], during which the central core of the intervertebral disc, the nucleus pulposus (NP), pushes through the outer ring, the annulus fibrosus (AF). This extruded NP tissue can cause inflammation of nerve roots, which has been recognized as a key factor in painful extruded herniated discs [START_REF] Takada | Intervertebral disc and macrophage interaction induces mechanical hyperalgesia and cytokine production in a herniated disc model in rats[END_REF].

Presence of inflammatory factors, interleukin-1 (3,4) and 6 (5,6) (IL-1 and IL-6), tumor necrosis factor alpha [START_REF] Takahashi | Inflammatory cytokines in the herniated disc of the lumbar spine[END_REF][START_REF] Yoshida | Intervertebral disc cells produce tumor necrosis factor alpha, interleukin-1beta, and monocyte chemoattractant protein-1 immediately after herniation: an experimental study using a new hernia model[END_REF][START_REF] Andrade | Tumor necrosis factor-alpha levels correlate with postoperative pain severity in lumbar disc hernia patients: opposite clinical effects between tumor necrosis factor receptor 1 and 2[END_REF] (TNF), and prostaglandin-E 2 (5,6) (PGE 2 ), is reported in herniated tissue. This is mainly attributed to the body's immune response: monocyte infiltration [START_REF] Doita | Immunohistologic study of the ruptured intervertebral disc of the lumbar spine[END_REF], macrophage maturation, and resorption of extruded tissue [START_REF] Komori | The natural history of herniated nucleus pulposus with radiculopathy[END_REF]. But, production of several cytokines have been measured, albeit in lower quantities, in protruded NP tissue, which is not exposed to the body's immune response [START_REF] Kang | Herniated lumbar intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2[END_REF]. Furthermore, Yoshida et al. [START_REF] Yoshida | Intervertebral disc cells produce tumor necrosis factor alpha, interleukin-1beta, and monocyte chemoattractant protein-1 immediately after herniation: an experimental study using a new hernia model[END_REF] have observed IL-1 and TNF positive cells, before infiltration of monocytes. Thus, NP tissue itself may initiate the inflammatory response through an unknown mechanism. We hypothesize that when NP tissue is extruded, it is exposed to a lower osmolarity and this osmotic shock in turn stimulates the native NP cells to produce inflammatory factors.

Radicular pain caused by herniation is generally treated conservatively, for example, oral analgesics, and 50% of patients recover spontaneously [START_REF] Frymoyer | Back pain and sciatica[END_REF]. However, in many patients, analgesia is insufficient [START_REF] Croft | Outcome of low back pain in general practice: a prospective study[END_REF], and they are treated with epidural steroid injections or surgery. Long-term results of surgery are not different from conservative treatment for radicular pain [START_REF] Jacobs | Surgery versus conservative management of sciatica due to a lumbar herniated disc: a systematic review[END_REF], and although epidural injections can be effective in 80% of cases, patients often need four to five injections within a year [START_REF] Manchikanti | Evaluation of the effectiveness of lumbar interlaminar epidural injections in managing chronic pain of lumbar disc herniation or radiculitis: a randomized, double-blind, controlled trial[END_REF]. Moreover, steroids might slow down the natural resorption of extruded NP tissue as shown in a rabbit tissue model [START_REF] Minamide | Effects of steroid and lipopolysaccharide on spontaneous resorption of herniated intervertebral discs. An experimental study in the rabbit[END_REF]. Reducing pain, while not inhibiting resorption, would be a promising approach to treat herniation.

One of the inflammatory factors in herniation is PGE 2 which can sensitize nerves and induce pain [START_REF] Samad | Interleukin-1betamediated induction of Cox-2 in the CNS contributes to inflammatory pain hypersensitivity[END_REF]. Two enzymes are involved in PGE 2 production, cyclooxygenase 1 and 2 (COX1 and 2). Contrary to COX1, COX2 is inducible and, therefore, COX2 inhibitors are used in pain management, and have been shown to reduce pain in rat models of disc herniation [START_REF] Kawakami | Epidural injection of cyclooxygenase-2 inhibitor attenuates pain-related behavior following application of nucleus pulposus to the nerve root in the rat[END_REF]. Celecoxib (Cxb) is a COX2-specific inhibitor and a candidate for treating herniated discs whose cells can produce PGE 2 , when stimulated by macrophages [START_REF] Takada | Intervertebral disc and macrophage interaction induces mechanical hyperalgesia and cytokine production in a herniated disc model in rats[END_REF]. However, because the half-life of Cxb is only 7.8 h (17), a biodegradable Cxb sustained release option is likely to be more successful than a single Cxb injection.

We have previously cultured bovine NP tissue explants in an artificial annulus, to prevent swelling and provide a near in vivo environment for the NP cells up to 6 weeks [START_REF] Van Dijk | Long-term culture of bovine nucleus pulposus explants in a native environment[END_REF]. In this study, we allow the tissue to swell, as in extruded herniation, and investigate if this simple mechanobiological stimulus can stimulate the tissue to produce cytokines, in the absence of the inflammatory system response. Two injectable sustained release biomaterials loaded with Cxb were then tested in this model and compared to a single Cxb injection.

Materials and Methods

Tissue

Tissue NP explants (150-350 mg) were isolated with an 8 mm biopsy punch (Kruuse, Sherburn, UK) from the center of fresh caudal discs of 24-month-old cows, obtained from a local abattoir in accordance with local regulations. After weighing, free swelling samples were immediately placed in 6 ml of culture medium (DMEM; Gibco Invitrogen, Carlsbad, CA), with 4 mM Lglutamine (Lonza, Basel, Switzerland), 1% penicillin/streptomycin (Lonza), 50 mg/l ascorbic acid (Sigma), and 5% fetal bovine serum (Gibco)). Control samples (non-swelling) were cultured in an artificial annulus system (18) in 6 ml of medium (Fig. 1). In this system, a jacket knitted from UHMWPE fibers (Dyneema, DSM, Heerlen, the Netherlands) lined with a 100 kDa molecular weight cut off (MWCO) semi-permeable membrane (Spectrum Laboratories, Breda, the Netherlands) prevents swelling.

Injectable gel

A hybrid thermo-reversible biodegradable hydrogel (Fig. 2) was used as one controlled release platform (TNO, Eindhoven, the Netherlands) [START_REF] Craenmehr | Liquid composition comprising polymer chains and particles of an inorganic material in a liquid[END_REF]. The hydrogel consists of a network of biodegradable nanoparticles linked to Lower Critical Solution Temperature (LCST) polymers. At room temperature, they are dispersed in water, injectable through a 32-gauge needle. However, at 37°C, they gel by crosslinks arising from hydrophobic interactions of the LCST polymers. The hydrogel was mixed with Cxb (LC Laboratories, Woburn, MA) in three different concentrations [START_REF] Jacobs | Surgery versus conservative management of sciatica due to a lumbar herniated disc: a systematic review[END_REF]120, and 1200 mg/ ml gel). Previous in vitro experiments, showed that 0.5 ml gel, loaded with these concentrations, resulted in average releases of 0.1, 1, and 10 mM, respectively, when added to 6 ml of medium.

Microspheres

Biodegradable microspheres (DSM, Fig. 2) were prepared with an average particle diameter of 40 mm [START_REF] Yang | Applicability of a newly developed bioassay for determining bioactivity of anti-inflammatory compounds in release studies-celecoxib and triamcinolone acetonide released from novel PLGA based microspheres[END_REF]. During production, microspheres were loaded with 8.5%w/w Cxb. Previous in vitro experiments showed that 2.7mg of microspheres (loaded with 22.9 mg Cxb) in 6 ml of medium resulted in an average release of 10 mM. Therefore 2.7, 0.27, and 0.027 mg microspheres were added to a well to release 10, 1, and 0.1 mM Cxb.

Treatment conditions

Six NP tissue samples from independent donors were cultured in every experimental group (n = 6/group). Control artificial annulus samples remained untreated. Samples in free swelling condition were (1) not treated; (2) treated with a bolus (10 mM Cxb in medium for 3 days); (3) treated with a sustained release control (1 mM Cxb in medium for 28 days); and (4-11) treated with the microspheres or gel, loaded for an aimed release of: 0, 0.1, 1, or 10 mM Cxb (Table 1). Explants were cultured in 12-well deep-well insert plates (Greiner) with cell culture inserts (0.4mm pore size, Greiner) to hold the sustained release biomaterials. Samples were cultured for 28 days at 37°C, 5% O 2 , and 5% CO 2 and medium was changed twice a week. During medium changes, temperature was kept at 37°C to prevent hydrogel liquefaction.

Biochemical content

Samples were weighed at the beginning and end of culture, and the percent increase in sample wet weight (ww) was calculated. Subsequently, they were stored frozen at -30°C, lyophilized overnight (Freezone 2.5; Labconco) and the dry weight (dw) was measured. The water content was calculated from ww-dw/ww. The samples were then processed as described earlier and used to determine their content of sulfated glycosaminoglycans (sGAG), hydroxyproline (HYP), and DNA, as well as the fixed charge density (FCD) [START_REF] Van Dijk | Long-term culture of bovine nucleus pulposus explants in a native environment[END_REF]. The amounts of sGAG, HYP, and DNA were expressed as percentage of the sample dw.

Cytokine release into the media

At every medium change, samples were collected and stored at -80°C. Release of cytokines into the medium was measured with ELISAs specific for bovine IL-1 and IL-6 (both Thermo Fisher Scientific, Waltham, MA), specific for bovine TNF (R&D Biosystems, Minneapolis, MN), and PGE 2 (Enzo Life Sciences, Farmingdale, NY) following manufacturer's instructions, and normalized to the original sample ww. All standard curves were dissolved in fresh culture medium, to account for any effects of the media on the analysis. Release was measured for all groups at days 3 and 28 as well as on days 14 and 21 for the best performing concentration of both loaded biomaterials and control groups.

Cxb concentration

At every medium change, samples were stored at -80°C, before Cxb concentration analysis by InGell Pharma (Groningen, the Netherlands). Samples were pre-treated as described before with slight modifications, that is concentration using liquid-liquid extraction with ethyl acetate, and mefenamic acid (Sigma) used as internal standard [START_REF] Zarghi | Simple and rapid highperformance liquid chromatographic method for determination of celecoxib in plasma using UV detection: application in pharmacokinetic studies[END_REF]. The samples were analyzed with UPLC (1290 Infinity, Agilent, Santa Clara, CA) where the detection-limit was 10 ng/ml Cxb. The medium concentration of Cxb was measured for bolus treatment at days 14 and 28 and the best performing concentration of both loaded biomaterials at days 3, 14, 21, and 28.

Statistics

Matlab (Mathworks, Inc., Natick, MA) was used for statistical analysis. For all biochemical data, one-way analysis of variance (ANOVA) was performed, followed by Dunnett's test for differences compared to artificial annulus. For the cytokine release data of the selected groups, Kruskal-Wallis analysis was performed at each time point, followed by Bonferroni corrected Mann-Whitney post hoc tests for differences compared to artificial annulus. To investigate the difference in Cxb release between the two biomaterials, two-way ANOVA was performed, followed by Bonferroni corrected post hoc t-tests. Statistical significance in all cases was assumed for p<0.05.

Results

The ww of the artificial annulus group decreased 10% during culture, while free swelling groups increased between 100 and 150% ww (Fig. 3A). In all free swelling groups, the water content increased significantly (Fig. 3B), and the sGAG content (Fig. 3C) and FCD (Fig. 3D) decreased significantly compared to the artificial annulus group. In addition, the DNA content increased five-fold compared to the artificial annulus group (Fig. 3E). There were no differences in hydroxyproline content (Fig. 3F).

With both biomaterials, 1 mM aimed release was the lowest dosage that completely inhibited PGE 2 at day 28 (Fig. S1). These were analyzed at the intermediate time points (days 14 and 21), together with the artificial annulus, free swelling, sustained control, and bolus groups (Fig. 4). In artificial annulus samples, high levels of PGE 2 were produced during the first 3 days, but were ameliorated from day 14 onwards (Fig. 4A). Free swelling samples produced PGE 2 throughout culture, with maximal levels from days 14 to 28. Due to the large variance in response, these levels were only significantly different from artificial annulus at day 28. Bolus, sustained control and both biomaterial samples were significantly different from the artificial annulus at day 3, with a complete inhibition of PGE 2 production. From day 14 onwards, these groups were not different from the artificial annulus group, indicating continuous inhibition of PGE 2 production. In artificial annulus samples, very low or undetectable levels of IL-6 were produced throughout culture (Fig. 4B). In free swelling explants, significantly higher levels of IL-6 were produced, from day 14 onwards, which were maximal at days 14 and 21. In all treated groups, high levels of IL-6 were produced throughout the culture, except for gel 1 mM where IL-6 production was partially reduced at days 14 and 21 and not significantly different from artificial annulus. Very low or undetectable levels of IL-1 and TNF were produced in all groups and at all time points, and there were no differences between groups (Fig. S2).

Both biomaterials, loaded for an aimed release of 1 mM, released Cxb for 28 days (Fig. 5). The average release, determined from these four time points, was 0.84 and 0.68 mM for the microspheres and gels, respectively, that is, close to the aimed concentration of 1 mM Cxb release in the gel was stable around 0.7 mM for 28 days. A burst release was observed in microspheres during the first 3 days, which was significantly higher compared to the gel. Thereafter, the release decreased in the microspheres and was significantly lower at each subsequent time point. Cxb release at days 21 and 28 was significantly lower in the microspheres compared to the gel. Cxb was still measured in the culture medium of the bolus group at day 14 and even at day 28, and was significantly larger than 0.

Discussion

The artificial annulus prevented swelling, kept NP tissue stable [START_REF] Van Dijk | Long-term culture of bovine nucleus pulposus explants in a native environment[END_REF][START_REF] Van Dijk | Culturing bovine nucleus pulposus explants by balancing medium osmolarity[END_REF] and showed no long-term inflammatory response. Free swelling of NP tissue induced a sustained inflammatory response, of the inflammatory cytokine IL-6 and the nociceptive stimulus PGE 2 . This supports our hypothesis that even without interaction with the immune system, the extruded NP may alone initiate and/or contribute to the inflammation seen in herniation. With its high tonicity, extruded NP tissue will absorb water and swell [START_REF] Van Dijk | Culturing bovine nucleus pulposus explants by balancing medium osmolarity[END_REF], causing a hypo-osmotic stress to the cells. Such a stimulus has been shown to also stimulate amnion derived cells in a COX2 dependent manner resulting in production of PGE 2 [START_REF] Lundgren | Hypotonic stress increases cyclooxygenase-2 expression and prostaglandin release from amnion-derived WISH cells[END_REF]. NP cells regulate daily changes in osmolarity through NFAT [START_REF] Burke | Human nucleus pulposis can respond to a pro-inflammatory stimulus[END_REF][START_REF] Tsai | TonEBP/ OREBP is a regulator of nucleus pulposus cell function and survival in the intervertebral disc[END_REF] which is produced more in hypertonic conditions. Although NP cells in monolayer stimulated with hypo-osmotic stress demonstrated MEK/ERK and AKT involvement [START_REF] Mavrogonatou | Effect of varying osmotic conditions on the response of bovine nucleus pulposus cells to growth factors and the activation of the ERK and Akt pathways[END_REF], further research is needed, to clearly understand the involved biological pathway(s), which may be exploited for alternative treatment strategies. Nevertheless, this simple mechanobiological stimulus, similar to the initial phase of extruded herniation, did consistently elicit an inflammatory response although the variance was large between samples. This could have been due to the differences in osmotic pressure between samples, but this will be even more variable in patients with their different states of disc degeneration. PGE 2 is involved in painful herniated discs [START_REF] Samad | Interleukin-1betamediated induction of Cox-2 in the CNS contributes to inflammatory pain hypersensitivity[END_REF]; thus, the observation that NP tissue itself is able to produce PGE 2 shows the promise of COX2-specific treatment for radicular pain. The role of IL-6 in extruded herniation is not clear. It may be beneficial to alleviate the negative effects of herniation by contributing to resorption of herniated tissue via upregulation of matrix metalloproteases [START_REF] Studer | Human nucleus pulposus cells react to IL-6: independent actions and amplification of response to IL-1 and TNF-alpha[END_REF], inhibiting proteoglycan production [START_REF] Studer | Human nucleus pulposus cells react to IL-6: independent actions and amplification of response to IL-1 and TNF-alpha[END_REF], and stimulating macrophage maturation [START_REF] Mitani | Activity of interleukin 6 in the differentiation of monocytes to macrophages and dendritic cells[END_REF]. On the other hand, as it can induce hyperalgesia in rats [START_REF] Deleo | Interleukin-6mediated hyperalgesia/allodynia and increased spinal IL-6 expression in a rat mononeuropathy model[END_REF], it may be detrimental as well. However, IL-6 can induce PGE 2 (29) so, it is possible that hyperalgesic effects of IL-6 were not direct but due to increased PGE 2 production. This ambiguous role of IL-6 in herniated disc disease should be further investigated, but if it is mostly beneficial, treating painful herniated discs with Cxb could be a promising alternative to currently used steroids that have been reported to slow down the natural resorption of extruded NP tissue in a rabbit tissue model [START_REF] Minamide | Effects of steroid and lipopolysaccharide on spontaneous resorption of herniated intervertebral discs. An experimental study in the rabbit[END_REF].

Interestingly, swelling healthy bovine NP tissue did not directly produce effective doses of TNF and IL-1, although both have been detected in herniated human tissue [START_REF] Takahashi | Inflammatory cytokines in the herniated disc of the lumbar spine[END_REF][START_REF] Andrade | Tumor necrosis factor-alpha levels correlate with postoperative pain severity in lumbar disc hernia patients: opposite clinical effects between tumor necrosis factor receptor 1 and 2[END_REF] and rabbits (4). However, similar to our results, culture of such specimens did not lead to presence of either cytokine in the medium, even after lipopolysaccharide stimulation [START_REF] Burke | Human nucleus pulposis can respond to a pro-inflammatory stimulus[END_REF]. This could be because TNF detected in homogenates of human herniated NP tissue was only membrane bound and no soluble TNF was found [START_REF] Andrade | Tumor necrosis factor-alpha levels correlate with postoperative pain severity in lumbar disc hernia patients: opposite clinical effects between tumor necrosis factor receptor 1 and 2[END_REF]. Furthermore, expression of these cytokines is associated with increasing degree of disc degeneration [START_REF] Maitre | The role of interleukin-1 in the pathogenesis of human intervertebral disc degeneration[END_REF], but can also be produced by activated macrophages (32) neither of which were present in our system. Low levels of TNF, though, can have already strong effects in NP-like tissues [START_REF] Seguin | Tumor necrosis factor-alpha modulates matrix production and catabolism in nucleus pulposus tissue[END_REF] and TNF inhibitors have the potential to stop radicular pain in patients [START_REF] Korhonen | Efficacy of in¯iximab for disc herniation-induced sciatica: one-year follow-up[END_REF]. Thus, the roles of TNF and IL-1 in painful herniation remain pertinent.

The DNA content in free swelling samples increased five-fold compared to artificial annulus. However, the PGE 2 and IL-6 production at day 28 increased 30-90-fold, respectively, thus, cannot be explained by the increased cell number alone. In a previous study [START_REF] Van Dijk | Culturing bovine nucleus pulposus explants by balancing medium osmolarity[END_REF], increase in DNA was attributed to cell cloning and formation of a fibrous layer at the sample periphery. In the present study, we did not investigate the contribution of this fibrous layer to the production of IL-6 and PGE 2 . As this fibrous layer most likely contains active dedifferentiated cells, its production of inflammatory factors might be considerable. However, the increased PGE 2 and IL-6 production in the first 3 days, during absence of the fibrous layer, indicate that NP cells within the tissue produce inflammatory cytokines as well. Furthermore, in vivo this layer will probably also be formed [START_REF] Specchia | Cytokines and growth factors in the protruded intervertebral disc of the lumbar spine[END_REF] and may initiate the formation of the granulation tissue seen in sequestered and extruded surgical samples [START_REF] Koike | Angiogenesis and inflammatory cell infiltration in lumbar disc herniation[END_REF]. Any contribution of this layer to the production of cytokines in this study will occur in vivo as well.

Artificial annulus samples also increased PGE 2 production but only for the first 3 days. This is most likely a result of the shrinkage step needed before placing samples in the system. In this step, samples received a strong hyperosmotic stimulus (50% PEG in PBS (w/v) for 100 minutes), which has been shown to induce COX2 in renal cells [START_REF] Moeckel | COX2 activity promotes organic osmolyte accumulation and adaptation of renal medullary interstitial cells to hypertonic stress[END_REF]. Besides this temporal production of PGE 2 , no other cytokines were produced, showing that healthy bovine NP explants do not produce inflammatory cytokines, unless they are triggered.

Cxb was able to completely inhibit elevated PGE 2 levels for 28 days, when delivered continuously at a concentration of 1 mM in the sustained control group. This result was expected, as this is in the order of the therapeutic plasma concentration of Cxb (17). Both biomaterials were able to release Cxb continuously for 28 days and, thus, inhibit PGE 2 . However, the release kinetics from the two biomaterials were significantly different, that is, microspheres showed a burst release, and a slowly declining release afterwards as observed earlier [START_REF] Yang | Applicability of a newly developed bioassay for determining bioactivity of anti-inflammatory compounds in release studies-celecoxib and triamcinolone acetonide released from novel PLGA based microspheres[END_REF], while the gels showed constant release for 28 days.

To our surprise, the 10 mM bolus was also able to inhibit PGE 2 production of swelling NP explants for 28 days. This dosage is 10 times higher than the biomaterials and 3 times higher than maximum serum levels (17), but as this is a local deposit it will not lead to high serum concentrations in patients. We did not test a lower bolus; thus, we do not know if the bolus' success is because of the higher dose. What was most surprising is that we still measured Cxb in the medium at days 14 and 28, although all initial culture medium was removed at day 3. If Cxb was not degraded and distributed evenly throughout the fluid in tissue and medium, only approximately 70% of Cxb was removed at each medium change. Therefore, the bolus is likely to provide an effective dose longer than 3 days, possibly even until day 14, but not until day 28. Cxb only decreased 50% between days 14 and 28, where a 99% decrease was expected, so it is probable that Cxb is binding to the tissue. In blood serum samples, 97% of Cxb is bound to serum albumin (17), which is also a component of our culture medium. Furthermore, albumin is detected in osteoarthritic cartilage tissue, bound to keratan sulfate, one of the main sGAGs in the disc, through disulfide bonds [START_REF] Mannik | Immunoglobulin-G and Serumalbumin isolated from the articular-cartilage of patients with rheumatoidarthritis or osteoarthritis contain covalent heteropolymers with proteoglycans[END_REF]. If this proposed mechanism of albumin binding to the tissue is correct, this will have clinical implications. In 80% of extruded herniation samples, blood vessels are observed (38) so, Cxb injected adjacent to extruded NP tissue can diffuse in and bind to pre-bound albumin, lengthening the therapeutic effect. Possibly, this might explain the relative success of epidural steroid injections, as the NP tissue can prolong the effect of a single drug injection. Nevertheless, more research needs to be done to determine the validity of this mechanism. It is also possible that Cxb is binding to other proteins in the NP. This interesting finding shows the value of using our tissue model for preclinical evaluation of therapies for disc herniation, as in cell culture this property of Cxb would be missed. However, a limitation of this model is the absence of the body immune response. Repeating this study in a suitable animal model will truly show if a single injection of Cxb will be as successful as a sustained release biomaterial. Nevertheless, treatment with a COX2 inhibitor like Cxb has clinical potential, as not only the swelling NP tissues in this study, but also infiltrated samples, produce PGE 2 .

Another interesting finding is that the gel alone was able to reduce IL-6 production to some extent, especially at day 14. This reduction was also observed at day 14 with the 10 mM loaded gels but also with the empty gels (Fig. S3), indicating that the biomaterial itself affected IL-6 production. As we used Transwell  systems (pore size 0.4 mm) in this experiment, there was no direct contact between tissue and biomaterials, but degradation products could have reached the tissue. When the gels degrade, magnesium ions (Mg 2 ) leach out, which can affect IL-6 production [START_REF] Nowacki | Highmagnesium concentration and cytokine production in human whole blood model[END_REF]. Furthermore, a relatively large amount of biomaterial was used in the gel groups (500 ml per sample in all concentrations). Nevertheless, any direct effect of the gel on IL-6 production is only partial and transient, and any benefits thereof remain to be investigated.

Conclusion

Exposing NP tissue explants to lower tonicity conditions, without the inflammatory system response, increased PGE 2 and IL-6. These cytokines are interesting candidates when treating acute herniation, as they are produced before infiltration of macrophages, and involved in pain and inflammation. Cxb could successfully stop PGE 2 , but not IL-6, production for 28 days when supplied by sustained release biomaterials. Interestingly, a bolus was able to achieve the same result, revealing that NP tissue itself can function as a carrier for sustained release of Cxb. Explants were cultured in a deep-well insert plate (bottom). In some groups, celecoxib (Cxb) was added to the medium directly. Both the injectable gel (top left), and the microspheres (top right) were added using a culture insert. LDH, layered double hydroxide; pNIPAAM, poly(N-isopropylacrylamide); RT, room temperature (drawn by Anthal Smits).

Figure S3. IL-6 release into the media.

Release at day 14, in pg/ml, during 3 days, normalized to original sample ww. Values are means ± standard deviation, n = 6.

Figure 1 .

 1 Figure 1. Tissue NP explant culture system.Image of nucleus pulposus (NP) explants in artificial annulus culture. Artificial annulus samples (right) were cultured in a deep-well Transwell plate with 6 ml of culture medium. As the artificial annulus samples might be buoyant, a stainless steel cylinder was added to the culture insert (left) to keep samples submerged (drawn by Anthal Smits).

Figure 2 .

 2 Figure 2. Schematic image of NP explants in free swelling culture.

Figure 3 .

 3 Figure 3. Biochemical content of NP explants after 28 days. Weight change (A), water content (B), sulfated glycosaminoglycan (sGAG) content expressed per dry weight (dw) (C), fixed charge density in mEq/g (D), DNA content expressed per dw (E) and hydroxyproline content expressed per dw (F). Values are means standard deviation, n = 6. * Different from artificial annulus; p < 0.05.

Figure 4 .

 4 Figure 4. Cytokine release into the medium over time.Release of prostaglandin E2 (PGE2, A) and interleukin-6 (IL-6, B), in pg/ ml, during 3 days, normalized to original sample wet weight (ww). Values are means +/-standard deviation, n = 6. * Different from artificial annulus at same time point, p < 0.05.

Figure 5 .

 5 Figure 5. Cxb concentration measured in the medium over time.Concentration in mM at days 3, 14, 21, and 28 for both biomaterials aimed for 1mM release, and at days 14 and 28 for bolus. Values are means + standard deviation, n = 6. * Different from gel at same time point, # different from previous time point of the same biomaterial, $ different from 0 mM, p < 0.05.

Figure S1 .

 S1 Figure S1. PGE 2 release into the media.Release at day 28, in pg/ml, during 3 days, normalized to original sample ww. Values are means ± standard deviation, n = 6.

Figure S2 .

 S2 Figure S2. Cytokine release into the medium over time. Release of interleukin-1β (IL-1β, a) and tumor necrosis factor α (TNFα, b), in pg/ml, during 3 days, normalized to original sample ww. Values are means ± standard deviation, n = 6.

Table 1 . Overview of the culture groups.

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Acknowledgments

This research forms part of the Project P2.01 IDiDAS of the research program of the BioMedical Materials institute, a Dutch public-private partnership. We would like to acknowledge Paul van Midwoud at InGell Pharma for celecoxib release measurements, Irene Arkesteijn at TU/e for the valuable contribution to the artificial annulus model, Renz van Ee and Klaas Timmer at TNO, and Detlef Schumann at DSM for supplying us with the biomaterials.

Disclosure Statement

The academic partners did not receive direct funding from the commercial partners and only non-commercial authors interpreted the data.