Sara Rojas

Ana Torres

Víctor Dato

Fabrice Salles

David Ávila

J García-González

Patricia Horcajada

Towards improving the antibiotic elimination capacity of UiO-66 from contaminated water

Antibiotics are found in natural waters, raising the concerns about their human and environmental toxicity and wide occurrence of antibiotic resistant bacteria. The antibiotic resistance crisis is attributed to the overuse and misuse of these medications. Particularly, sulfamethazine (SMT), an antibiotic commonly used in pigs and cattle for the treatment of bacterial diseases, has been severely detected in natural environments (soil and water). Among all the technologies developed to combat the deteriorating water quality and control antimicrobial resistance, the heterogeneous photocatalysis should be highlighted for the degradation of refractory organic compounds. Here, we described the SMT adsorption and photodegradation capacity of a highly porous and robust zirconium-based MOF UiO-66 under realistic conditions, and its potential recyclability. Futher, its SMT removal capacity was improved by nanosizing the MOF (100% of SMT degradation in only 4 h for nanoUiO-66), and funtionalizing the MOF porosity (28.5% in 24 h for nanoUiO-66-NH2), respectively. Finally, the safety of the formed byproduct during the SMT photodegradation was confirmed, reinforcing the potential of the applicaton of UiO-66 in water remediation.

Introduction

Antibiotics are essential for the maintenance of public health and life quality. Thousands of different active compounds are currently used in large quantities to treat human and livestock diseases. Concerning human maladies, only in 2018, 20.1 systemic doses of antibiotics were administered per 1000 inhabitants per day in the European Union, according to the annual report from the European Center for Disease Prevention and Control.

1 On the other hand, only in Spain, 1102 tons of veterinary antibiotics were commercialized in 2009 according to the Spanish Agency for Medicines and Health Products, 80% of them coming from four main groups of antimicrobials: 31% of tetracycline, 22% of sulfonamides and trimethoprim, 16% of β-lactams, and 8% lincosamides. [START_REF]Ministerio de Sanidad Política Social e Igualdad, Sales of veterinary antimicrobial agents in Spain in[END_REF] As a consequence of their extensive/intensive use, antibiotics are found in natural waters, raising the concerns about their human, animal, and environmental toxicity, and widening the occurrence of antibiotic resistant bacteria (ARB). [START_REF] Ding | [END_REF][4][5] In this sense, due to both their significance for human and veterinary medicine and their persistence, sulfonamides, quinolones, and trimethoprim are the most detected antibiotics in water. 6 Particularly, the sulfonamide sulfamethazine (SMT), frequently used in pigs and cattle, 7,8 has been detected in natural environments such as soil or water, ranging from a few nanograms to tens of milligram per litre or kg of soil (from 0.6 to 20 ppm). [9][10][11] Because of their continuous anthropogenic introduction into the environment, aquatic organisms are chronically exposed to SMT. 12 Thus, national programs to combat the deteriorating water quality and control antimicrobial resistance are trying to reduce the indiscriminate antibiotics use, and also to develop more efficient wastewater treatment technologies. Among these last, different advanced oxidation processes have emerged as a promising way to remove antibiotics from contaminated water in the last decades. 13 These processes enable the degradation of refractory organic compounds by the in situ generation of reactive oxygen species (ROS; mainly hydroxyl radicals), via Fenton processes, ozonation, electrochemical oxidation, heterogeneous photocatalysis, or their combination. 14 In particular, heterogeneous photocatalysis highlights for the treatment of hazardous organic pollutants since operates at mild conditions (ambient temperature and pressure) and is considered as a green technology, its mechanism relying on the light irradiation to convert toxic pollutants to non-toxic molecules by using O 2 as main reactive. 15 Basically, the photocatalyst generates a transient state by using light absorption (photon energy) and releasing the electron-hole pair to produce the chemicals (chemical energy). The increasing attention in the use of photocatalysts for water purification has resulted in the design and development of a wide variety of photocatalysts (i.e., metal oxides -TiO 2 , ZnO; metal sulphides -ZnS, CdS; ternary compounds -BaTiO 3 , Bi 2 WO 6 ; and multicomponent materials -BiVO 4 /Bi 2 OCO 3 ). 16 However, these materials show some limitations in water treatment, like toxicity (e.g., CdS medium oral lethal dose-LD 50 in rats = 7080 mg•Kg -1 ), high cost, low photochemical stability (e.g., metal sulphides), difficulty in recovery from water (e.g., ZnO, TiO 2 ), 17 and/or inefficient UV-vis light absorption (e.g., TiO 2 absorbs only 3% of the entire solar spectrum).

18

In this context, a new class of crystalline porous materials, known as Metal-Organic Frameworks (MOFs), has attracted an increasing attention as photocatalysts. MOFs are comprised of inorganic nodes and polydentate organic linkers that assemble into multidimensional periodic lattices through coordination bonds, providing a robust and well-defined network and also a high porosity. 19 Compared to the previously mentioned photocatalysts, MOFs present an exceptional versatility, with a broader range of possibilities regarding the interaction between their inorganic and organic parts. In this line, the metal clusters can be regarded as inorganic semiconductor quantum entities, which are in close contact with the organic ligands, serving these lasts as antennas to activate the semiconductor quantum dots. Recently, MOFs have been proposed as heterogeneous photocatalysts in organic pollutants elimination. 20 However, most of these studies address the removal of small molecules and organic dyes, remaining the elimination of macromolecular antibiotics from contaminated water poorly investigated. Further, in the elimination of antibiotics, pure MOFs have been mainly used as adsorbents, with only 3 examples of their use as photocatalyst. Coupled with persulfate (PS-persulfate oxidation process), MIL-88A(Fe) and MIL-101(Cr) were described as photocatalysts in the successful degradation of tetracycline hydrochloride (100% of removal in 90 min, 200 ppm, pH = 3.45) and sulfadimethoxine (97.8% of removal in 90 min at 60 ºC), respectively. 21,22 Very recently, a newly synthetized [Zn 6 (IDC) 4 (OH) 2 (HPRZ) 2 ] n composed by Zn(II) cations and piperazine (HPRZ) and imidazole-4,5-dicarboxylate (IDC) ligands was used as catalyst in the photodegradation of amoxicillin (37%, 180 min).

23

However, these reports do not consider MOF stability, and use purified water (Milli-Q or deionized) or other necessary species (i.e., PS) to perform the degradation process, which is far from complex river compositions or their functional application in WWTPs.

Here, we describe the SMT photodegradation capacity of a highly porous and robust benchmarked zirconium-based MOF, UiO-66 ([Zr 6 O 4 (OH) 4 (BDC) 12 ] H 2 BDC = 1,4-benzenedicarboxylic acid)UiO-66 is based on Zr(IV) oxoclusters and terephthalate anions, possessing octahedral and tetrahedral cavities (∼11 and ∼8 Å, respectively) accessible through triangular micropores (ca. 5 -7 Å). 24,25 Its very high important porosity, its essential structural stability in water, as well as its photocatalytic activity, makes UiO-66

an excellent candidate to be used in the photochemical degradation of SMT from water. First, we have evaluated the UiO-66 stability and SMT removal capacity (adsorption and photodegradation) in tap water. Remarkably, SMZ elimination studies were conducted under realistic conditions (i.e., tap drinking water, ambient temperature, neutral pH, in absence of other additives for the treatment, etc.) considering its potential recyclability, which highlights the interest of this work. Subsequently, in a way to further improve the SMT elimination capacity, the SMT elimination was rationalized by investigating the influence of crystal size and functionalization of UiO-66, by using the series of functionalized nanosized UiO-66s (nanoUiO-66-X; X = H, NH 2 , and OH). Last but not least, we have identified the formed by-products during SMT photodegradation, considering their potential toxicity, in order to assess the impact or the water treatment with UiO-66.

Results and discussion

A. Photodegradation of SMT using the robust UiO-66

To evaluate the ability of UiO-66 in water purification, the powdered material was first suspended in a tap water solution of SMT, using a concentration range within that one commonly found in wastewater (i.e., 10 ppm of SMT). [9][10][11] UiO-66 photocatalytic activity towards the degradation of SMT was investigated under UV-visible light irradiation, quantifying the contaminant elimination by high performance liquid chromatography (HPLC). In the same manner, the SMT adsorption capacity of UiO-66 was performed under dark. In addition, UiO-66 stability was monitored during the SMT photodegradation/adsorption processes by means of the quantification of the ligand leaching by HPLC. Remarkably, UiO-66 was able to photodegrade up to 77 ± 4 % of SMT within 24 h, while proving an excellent chemical stability (only 1.6 ± 0.9% of degradation, ca. 0.005 ppm; Figure 2a) and structural robustness (as confirmed by X-ray powder diffraction; XRPD, Supporting information, Figure S1a). These stability analyses are fundamental when MOFs are proposed for water remediation in order to avoid an extra contamination, originated from the matrix degradation.

26

Unlike its remarkable SMT photodegradation capacity, UiO-66 can eliminate by adsorption only 3.5% of SMT within 24 h. The excellent SMT removal (photodegradation) capacity and stability of UiO-66 prompt us to further investigate the potential recyclability of this MOF. Upon 4 cycles of the photodegradation process, UiO-66 efficiently removed SMT without a significant decrease in efficiency (Figure 2b). After 4 cycles, although the solid showed a significant broadening of the Bragg peaks, consistent with a loss of long range structural, chemical stability was fully kept (with no additional ligand leaching; see Supporting information and Figure S2).

Nanoscale

UiO-66

Functionalization NH 2 OH

Photodegradation Adsorption

Sulfametazine

In an attempt to further improve the SMT photodegradation capacity of UiO-66, we have taken advantage of MOFs' unique possibilities, and two strategies were considered: i) reduction of the particle size, and ii) tuneability of the light absorption by introduction of substituents on the aromatic linker. Regarding particle size, it is generally considered that smaller particles will favour the catalytic reactions. However, we know about the limitations of determining the particle size effects in catalyst, since MOFs' structure (e.g., density of defects and degree of crystallinity of MOF) may change during the synthesis as particle size decreases. 27 Tentatively, we have considered that if the reaction occurs in the inner surface, the transport of reactants and products will be faster in small particles. On the opposite, if the reaction occurs on the outer surface, the increased specific surface area will favour the reactivity. In relation to the second strategy, the modification of nanoUiO-66 by ligand functionalization could result in an enhancement of the photoconversion quantum yield and could allow the extension of the absorption band gap to visible light, enabling water remediation under sunlight. 28 Further, the introduction of functional groups into the MOF network will affect the reactivity with SMT (e.g., changing the hydrophobic/hydrophilic character of the framework, favouring the formation of hydrogen bonds, etc.). In this regard, we have modified the benzenedicarboyxlate of nanoUiO-66-X with an amine (NH 2 ) and hydroxyl group (OH). Cyclability tests of SMT photodegradation using UiO-66.

Thus, a series of nanoUiO-66-X (X = H, NH 2 , and OH) was synthetized at room temperature and ambient pressure, with some modification of the previously reported methodology. 29 The crystallinity and porosity of the nanoUiO-66-X series were confirmed using XRPD and N 2 sorption measurements at 77 K (Supporting information, Figure S3, Table S1). Further, the obtained series of porous polycrystalline nanoUiO-66-X were found to be smaller (81 ± 27, 38 ± 17, and 118 ± 68 nm; for X = H, NH 2 , and OH, respectively) than the initial UiO-66 material (672 ± 181 nm, Supporting information, Figure S4). The colloidal stability of the asprepared materials was studied under the working conditions (water at room temperature). As expected, the colloidal stability was strongly affected by the functionalization of the organic linker. DLS measurements in water of nanoUiO-66-X showed larger particles sizes, reaching 460 ± 30, 162 ± 4, and 673 ± 142 nm for X = H, NH 2 , and OH, respectively (see Supporting information, Table S2).

Considering its higher porosity (Brunauer-Emmett-Teller (BET) surface = 1130 m 2 •g -1

) and water colloidal stability, one could expect that the best material to be used in the SMT elimination is the nanoUiO-66-NH 2 derivative. To evaluate the potential application of the nanoUiO-66-X series in SMT removal from water, we studied their use in both processes (adsorption and photodegradation) following the same procedure as for UiO-66. When the matrixes were suspended in SMT tap aqueous solution in absence of irradiation, the crystalline structure of the nanoUiO-66 was kept intact, while there is an important crystallinity loss for nanoUiO-66-NH 2 and nanoUiO-66-OH derivatives (as confirmed by XRPD; Supporting information, Figure S6). SMT was successfully incorporated in the functionalized nanoUiO-66-NH 2 and nanoUiO-66-OH, with a 28 ± 1 and 6 ± 2 % removal capacity after 24 h, respectively (Figure 3; Supporting information, Figure S5). Although the pore size dimension of UiO-66 (ca. 5 -7 Å) are big enough to adsorb SMT (11 x 5 x 5 Å 3 , estimated by Vesta considering Van der Waal radii), no SMT elimination by adsorption was achieved when using the non-functionalized nanoUiO-66. These results agree with the results obtained previously for the microUiO-66, being able to absorb only 3.5% of SMT in 24 h. In addition, the solids showed an excellent stability since only ∼1% of the total H 2 BDC-X ligand was leached after 24 h contact (1.4 ± 0.0%, 0.0 ± 0.0%, and 0.0 ± 0.0% for X = H, NH 2 and OH, respectively). The adsorption of SMT on nanoUiO-66-X (X = NH 2 and OH) may be controlled by a sum of weak intermolecular forces, such as hydrophobic effect, electrostatic attraction, π-π electron donor-acceptor, and hydrogen bond interactions. 30 31 We will try to discuss the impact of each of them here. Hydrophobic/hydrophilic balance makes a critical difference during the adsorption process. We can assume that the hydrophobic/hydrophilic interactions are the only force in adsorption process at first. The adsorption can be judged by the n-octanol-water partition coefficient (K ow ) of SMT (log K ow = 0.14), indicating that SMT is an hydrophilic molecule. Therefore, SMT will preferentially be adsorbed in the more hydrophilic UiO-66-NH 2 and -OH derivatives. Aside from the hydrophobic/hydrophilic balance, the nature of the nanoparticles surface will determine the interaction with the different components of the contaminated water (water molecules, ions, SMT, etc.). In this respect, considering the pK a of SMT (pk a1 = 2.65aromatic amine; pK a2 = 7.65 -sulfonamide), SMT is predominantly neutral in tap water (pH = 6). Interestingly, the functionalization SMT@UiO-66

H 2 BDC_light H 2 BDC
of nanoUiO-66-X is a determining factor on surface charge (-17 ±1, -22 ± 1, and +23 ± 3 mV for nanoUiO-66, nanoUiO-66-OH, and nanoUiO-66-NH 2 , respectively). Therefore, considering that SMT is eliminated from water when using both negatively (nanoUiO-66-OH) and positively (nanoUiO-66-NH 2 ) charged MOFs, the intermolecular electrostatic attraction between SMT and nanoUiO-66-X should not be the driving force of the adsorption processes. Regarding the electrostatic interactions, SMT has the electron donor -NH 2 group (named as -NH 2_SMT ), which can interact with the benzene ring of nanoUiO-66 ligand in the π-π electron donor-acceptor interaction. Further, the π-π stacking should also be present between the benzene rings in SMT and organic ligand of nanoUiO-66-X. Again, considering that no SMT adsorption is achieved in the nanoUiO-66 material, we can claim that π-π interactions are not a predominant differential aspect in the SMT adsorption process. Finally, we have considered the hydrogen bonding, as it is regarded as an important mechanism to explain the adsorption of polar organics on functionalized MOFs. 32 The Fourier transform infrared (FTIR) spectra of the SMT-loaded nanoUiO-66-X (X = H, NH 2 , and OH) confirmed a bands shift associated to the carboxylate group found in the range of 1520-1650 cm -1 and 1280 to 1400 cm -1 for the γ asym and γ sym vibration bands in all the SMT loaded materials, supporting host-guest interaction by hydrogen bonding. In this regard, there is a shift from 1582 to 1578 cm -1 and 1395 to 1390 cm -1

for UiO-66-H, 1571 to 1563 cm -1 and 1384 to 1381 cm -1 for UiO-66-NH 2 , and 1582 to 1570 and 1415 to 1421 cm -1 for UiO-66-OH, respectively. Further, some of us have recently evidenced by Monte Carlo simulations that the NH 2 or -SO 2 groups from SMT can interact with the carboxylate groups of different MOFs.

On the other hand, in order to investigate the location and conformation of the SMT molecules in the pores of the nanoMOFs and try to explain the experimental results (to define the main interactions between the solid and the SMT molecules), Grand Canonical Monte Carlo (GCMC) simulations were performed by fixing the equilibrium saturation pressure of SMT molecules at a large value, which has already proved to be an efficient way to obtain the saturation description. 33,34 In this case, calculations failed to reproduce the saturation and results show no adsorption. Two explanations can be given to explain this failure: i) the force-fields considered to investigate the interactions between the solids and the SMT molecules are not consistent, ii) or the flexibility of the framework must be taken into account. To go further, both DFT calculations (which are not dependent of any force-field) and Monte Carlo simulations (where the loadings are fixed and impose the SMT molecules to be adsorbed, see Supporting information, Section 3) were performed. Snapshots obtained from these new calculations in Monte Carlo are given in Figure S8, while the extractions of the DFT calculations are disclosed in Figure S9. The comparison shows a general good agreement between the main interactions elucidated from these two techniques. The force field used in classical MC simulations was consistent, and therefore the flexibility of the framework should be considered for GCMC to reproduce adsorption of large molecules. Anyway, the comparison between the main interactions clearly evidenced a different behaviour between pristine UiO-66 and the functionalized UiO-66-X (X=NH 2 or OH), in agreement with the experimental results. Indeed, considering Figures S8a & S9a,b, weak interactions between the NH 2-SMT groups within the UiO-66 framework can be found (distances larger than 3.1 Å to 3.645 Å were found), and only stronger -stacking interactions were observed (distances ranging from 3.031 to 3.364 Å, and from 3.472 to 3.844 Å from MC and DFT calculations, respectively). Furthermore, no real difference was observed between the two types of pores regarding the interaction distances. In contrast, stronger interactions were found for UiO-66-X (X= NH 2 , OH), Figure 3. SMT photodegradation kinetics under dark (dashed line, squares) and UV-visible light irradiation (continuous line, triangles) in the presence of nanoUiO-66-X (X = H, NH2, and OH) (red, yellow, and green, respectively). For clarity, the data corresponding to MOFs degradation have been here omitted (although included in Supporting information, Section 3).

where NH 2_SMT interacts with the NH 2 of nanoUiO-66-NH 2 with distances ranging from 2.5 to 2.992 Å (from MC simulations, Figure S8b), or 2.316 to 2.982 Å (from DFT calculations, Figure S9c,d) or NH 2_SMT interacts with the OH of nanoUiO-66-OH (distances close to 2 Å from both simulations Figures S8c &S9e,f). In these structures, a strong -stacking was again observed with distances ranging from 3.1 to 3.6 Å in Figures S8 &S9). It can be noted that the larger pore seems to be the most preferential site due to the stronger interactions observed by DFT calculations. All these differences support the absence of SMT adsorption from experimental data for pristine UiO-66 (in which a weak affinity for SMT can be detected), while UiO-66-X (X=NH 2 , OH) can adsorb a larger SMT amount. Considering the experimental and computational results, the adsorption of SMT in UiO-66-X (X = NH 2 and OH) may be mainly governed by hydrophobic/hydrophilic and hydrogen bond interactions. However, the comparison between the interaction distances is not sufficient to explain the different adsorption behaviour between UiO-66-OH and UiO-66-NH 2 , considering the similar strong interactions between linker and SMT, or -stacking observed in both cases. Further, a different affinity for the solvent could also be invoked, and SMT and solvent adsorptions could therefore be in competition.

Remaining

SMT (%) Time (h)

SMT@nanoUiO-66 SMT@nanoUiO-66-NH 2 SMT@nanoUiO-66-OH SMT@nanoUiO-66_light SMT@nanoUiO-66-NH 2 _light SMT@nanoUiO-66-OH_light

Regarding the SMT photodegradation capacity, nanoUiO-66-X series (H = H, NH 2 , and OH) was remarkably able to degrade up to 100 ± 0, 90 ± 2, and 95 ± 5% of SMT in 4 h, while keeping its chemical integrity (Figure 3), 0.0 ± 0.0%, 0.1 ± 0.0%, and 0.0 ± 0.0% degradation for H = H, NH 2 , and OH, respectively, Supporting information, Figure S5). In view of these results, we can confirm that the modification of the band gap by linker functionalization does not improve the SMT photodegradation capacity of nanoUiO-66 (Eg = 3.89 eV for UiO-66 vs. 2.87 eV and 3.29 eV for nanoUiO-66-NH 2 and nanoUiO-66-OH, respectively; values obtained from the Tauc Plot, Supporting information, Figure S11). 35 Considering that the nanoUiO-66 was not able to adsorb SMT, one could conclude that the photodegradation process is not dependent on the MOF adsorption capacity, and may preferentially occur on the surface of the nanoparticle. The external surface was found to be similar for all the nanoparticles, ranging from the 387 and 386 m 2 •g -1 for the nanoUiO-66 and nanoUiO-66-NH 2 , respectively, to 208 m 2 •g -1 for the nanoUiO-66-OH. On the other side, the XRDP results

showed that only nanoUiO-66 keeps intact its crystalline structure, while nanoUiO-66-NH 2 and nanoUiO-66-OH suffer an important amorphization (Supporting information, Figure S6). In an attempt to rationalize these results, we could conclude that the lower activity found with NH 2 and OH functionalities should be attributed to both the electronic structure modulation that negatively affects the activity of the exposed Zr ions and the amorphization of the nanoUiO-66-NH 2 and nanoUiO-66-OH frameworks.

In order to compare the SMT degradation kinetics and to gain further understanding on the involved mechanism, the SMT degradation kinetics using all nanoUiO-66-X materials were fitted to a first order kinetics according to Eqn (1) (Supporting information, Figure S12). The best SMT degradation rate is obtained when using the nanoUiO-66. The fitting of the data gives rise to a k value of 1.7835 h -1

, ca. 20 times higher than the UiO-66, and 2.7 and 2 times higher than the nanoUiO-66-NH 2 and nanoUiO-66-OH, respectively (Supporting information, Table S3). Finally, to contextualize these results, nanoUiO-66 provided a fast SMT photodegradation than the micrometric UiO-66 (100 ± 0 vs. 35 ± 8% in 4 h, respectively), evidencing the relevance of the higher external surface area of nanoUiO-66 vs. UiO-66 (287 vs. 192 m 2 •g -1

, respectively). This removal efficiency can be compared with other proposed systems in SMT elimination (Supporting information, Table S3). Considering both, the adsorption and photocatalytic studies, these fittings also reveal that the rate-limiting step of the photodegradation is not the physisorption of the SMT on the MOF microporosity, but the photocatalytic reaction on the Zr-O active sites, after MOF photon absorption by the ligandto-cluster transfer mechanism. In view of these results, one could argue that the functionalization of the pore mainly impacts the adsorption process, while the particle size of the MOF affects the photocatalysis in SMT elimination from water.

Eqn (1)

The hazard of antibiotics in the environment does not only depend on their concentration but also their metabolites or degradation products, which can sometimes be more harmful than the parent compound. 37 Previous studies have described the photochemical behaviour of sulfonamides, determining that degradation occurs through cleavage at various positions. 38,39 The identification of the photodegradation products of SMT formed after 24 h-irradiation of the SMT + UiO-66 system in aqueous medium (pH = 6) was performed based on the results of ultra-high performance liquid chromatography coupled to mass spectroscopy (UHPLC/MS), supported with fragmentation patterns obtained from MS/MS experiments. Initially, the fragmentation pattern of SMT gave us the information needed to deduce several substituents and identify the unknown intermediates (Supporting information, Figure S13).

Although the determination of the SMT degradation pathway is out of the scope of this work, we have identified some intermediates deduced from their estimated molecular weight to assess the potential toxicity of the resulting products. The fragment ion m/z 215 (N-(4,6-dimethylpyrimidin-2-yl)benzene-1,4-diamine) was attributed to the product from SO 2 extrusion, a phenomenon frequently shown in sulfonamides. 40 Further, an attack of a hydroxyl radical at the carbon-nitrogen bond of the benzene ring might result in the fragment derived from the pyrimidinyl portion (m/z 124), leading to the formation of the 2-amino-4,6-dimethoxypyrimidine product. It should be pointed that the formation of the 2-amino-4,6-dimethoxypyrimidine as SMT photodegradation intermediate was also quantified by HPLC (3.6% after 24 h). Regarding the potential toxicity of the degradation products, although all molecules (N-(4,6-dimethylpyrimidin-2-yl)benzene-1,4-diamine; 2-amino-4,6-dimetoxypyrimidine and SMT) are classified as irritant (according to the Globally Harmonized System of Classification and Labelling of Chemicals-GHS), only SMT is described as health hazard, with a medium lethal dose (LD 50 , oral, mouse) of 50 g•kg -1

. 41 Notably, these results suggest that when using UiO-66 as photocatalyst, we are generating degradation products with lower toxicity than SMT, improving the quality of water.

C Conclusions

In this work, we present the successful utilization of the Zr-based MOF UiO-66 in the elimination of the emerging contaminant SMT. Its ability to remove (by photodegradation and adsorption) this challenging antibiotic can be improved by nanosizing the MOF (from 77 to 100% by photocatalysis) and functionalizing its porosity (from 0 to 28% by adsorption), respectively. The functionalization of the pore mainly impacts on the adsorption process controlled by hydrophobic/hydrophilic and hydrogen bond interactions, while the particle size and the stability of the MOF affects the photocatalysis in SMT elimination from water. Upon photodegradation of the antibiotic SMT, by using UiO-66 as photocatalyst we are generating products with lower potential toxicity than SMT, improving the quality water.

Materials and methods

All reactants were commercially obtained from Sigma-Aldrich (sulfamethazine (SMT, ≥99%), zirconium(IV) propoxide solution (70 wt.% in 1-propanol), zirconium (IV) chloride (ZrCl 4 , ≥99%), terephthalic acid (H 2 BDC, ≥98%), 2-hydroxyterephthalic acid (H 2 BDC-OH, ≥97%), 2-amino-4,6-dimethoxypyrimidine), Acros Organics (2-aminoterephtalic acid (H 2 BDC-NH 2 , 99%), Chemlab (N,Ndimethylformamide (DMF, 99.5%)), and Labkem (methanol (MeOH)), and used without further purification. Physicochemical characterization. Fourier transform infrared (FTIR) spectroscopic analyses were performed in a Nicolet 6700 (Thermo Scientific, USA) infrared spectrometer with the help of an attenuated total reflectance (ATR) diamond accessory. Nitrogen isotherms were obtained at 77 K using an AutosorbQ2 (Quantachrome Instruments, USA). Before the measurement, samples were evacuated at 200 °C overnight (on) under vacuum for UiO-66, and first at 80 ºC on and then at 150 ºC on under vacuum for all the nanoUiO-66 MOFs. Specific surface area was determined by applying Brunauer, Emmett & Teller equation (BET) in the relative pressure interval p/p 0 = 0.01-0.3 (being p 0 the saturation pressure). Pore volume and pore size distribution were calculated by the non-localized density functional theory (NLDFT) and the Horvath-Kawazoe (HK) methods, respectively. Routine X-ray powder diffraction (XRPD) patterns were collected using a conventional PANalytical Empyrean powder diffractometer (PANalytical Lelyweg, Netherlands, θ-2θ) using λCu K α1 , and K α2 radiation (λ = 1.54051 and 1.54433 Å). The X-ray powder diffraction (XRPD) patterns were carried out with a 2θ scan between 3-35° and 3-90° with a step size of 0.013° and a scanning speed of 0.1°•s -1 . Thermogravimetric analyses (TGA) were carried out in an SDT Q-600 thermobalance (TA Instruments, New Castle, DE, USA) with a general heating profile from 30 to 600 °C with a heating rate of 5 °C•min -1 under air using a flux of 100 mL min -1 . Scan and transmission electron microscopy (SEM and TEM) images were taken with a Hitachi TM1000, and a Technai 20 TEM microscope (Philips, Amsterdam, the Netherlands) with a 200 kV acceleration voltage, respectively. For sample preparation, 1 mg of sample was dispersed in 10 mL of ethanol and sonicated with an ultrasound tip (UP400S, Hilscher, Teltow, Germany) for 30 sec. For observation, 1 µL of the prepared solution was dropped over a copper TEM support with a carbon mesh (lacey carbon, 300 mesh, copper, approx. grid hole size: 63 µm, TED PELLA Redding, California, USA). Particle size was monitored via counting with ImageJ software. [START_REF] Abramoff | [END_REF] The mean particle size was statistically estimated (n > 200). The particle size and ζ-potential determinations were performed using a Malvern Nano-ZS, Zetasizer Nano Series. Ca. 1 mg of nanoUiO-X was dispersed in 10 mL of tap water using an ultrasound tip (Digital Sonifer 450, Branson; 10% of amplitude and for 1 min). UV-visible spectroscopic analysis of UiO-66-OH was run in a Perkin Elmer® Lambda 1050 UV/vis/NIR (Perkin Elmer, Waltham, MA, USA). The photoirradiated solutions were analyzed using a Shimadzu LCMS8030 accurate mass quadrupole coupled to ultra-high performance liquid chromatography (UHPLC) in the facilities of the Complutense University of Madrid using the same conditions as for the HPLC analysis (see below). Different organic molecules were analyzed by HPLC: the amount of degraded SMT, as well as the released corresponding linkers (H 2 BDC, H 2 BDC-NH 2 , or H 2 BDC-OH), and the degradation product 2-amino-4,6-dimethoxypyrimidine were determined using a reversed phase HPLC Jasco LC-4000 series system, equipped with a PDA detector MD-4015 and a multisampler AS-4150 controlled by ChromNav software (Jasco Inc, Japan). A Purple ODS reverse-phase column (5 µm, 4.6 x 150 mm, Análisis Vínicos, Spain) was employed. For the quantification of all chemical species, isocratic conditions were used. The flow rate was 1 mL•min -1

, and the column temperature was fixed at 298 K. In all cases, the injection volume was 30 µL. The mobile phase was based on a mixture of 50:50 MeOH:phosphate buffered solution (PBS; 0.04 M, pH = 2.5) for H 2 BDC, H 2 BDC-NH 2 , and H 2 BDC-OH ligands analysis, with a retention time (rt) and an absorption maximum of 3.6 min and 202 nm, 2.8 min and 225 nm, and 2.6 min and 209 nm, respectively. SMT was analyzed using a mixture of 35:65 acetonitrile:water, with a rt of 2.7 min and an absorption maximum of 263 nm. 2-amino-4,6-dimetoxipirimidine was analyzed using a mixture of 50: 50 MeOH:H 2 O with a rt of 4.3 min and an absorption maximum of 288 nm. Preparation of the phosphate buffered solution (0.04 M, pH = 2.5): 0.02 mol (2.4 g) of NaH 2 PO 4 and 0.02 mol (2.84 g) of Na 2 HPO 4 were dissolved in 1 L of Milli-Q water. The pH was then adjusted to 2.5 with H 3 PO 4 (≥85%) and to basic pH with NaOH 10 M.

Synthesis of UiO-66 and nanoUiO-66-X (X = H, NH 2 , and OH)

UiO-66 or [Zr 6 O 4 (OH)(C 8 H 4 O 4 ) 6 ] nH 2 O. 25 1 mmol of ZrCl 4 (233 mg) and 1 mmol of H 2 BDC (166 mg) were dispersed in 3 mL of DMF, placed in a teflon-lined autoclave and heated for 12 h at 200 °C. The resulting solid was recovered by filtration and washed with deionized water and acetone. 200 mg of the solid were suspended in 100 mL of DMF under stirring for 12 h. Then, the DMF-washed solid was suspended in 100 mL of MeOH under stirring for 12 h, recovering the activated solid by filtration. nanoUiO-66-X (X = H, NH 2 , and OH). 29 A mixture of 142 μL of a 70 wt.% zirconium propoxide [Zr(OnPr) 4 ] solution in 1-propanol (52 mg, 0.316 mmol), 14 mL of DMF, and 8 mL of acetic acid (140 mmol) was heated at 130 °C for 2 h under stirring. Then the solution was allowed to cool to room temperature (RT). Finally, the desired linker (149 mg, 0.90 mmol of H 2 BDC; 163 mg, 0.90 mmol of H 2 BDC-NH 2 ; or 42 mg, 0.23 mmol of H 2 BDC-OH) was added, and the obtained solution was briefly sonicated for 30 s and allowed to stir at 200 rpm for 18 h at RT. Then the nanoMOF was separated from the supernatant by centrifugation (5 min, 13000 rpm) and was washed 3 times with DMF (10 mL) and 1 time with acetone (10 mL). Samples were heated at 80 °C in a vacuum oven overnight before being analyzed.

Adsorption and/or photocatalytic degradation of SMT from water using UiO-66 or nanoUiO-66-X (X = H, NH 2 , and OH) series The adsorption/photocatalytic activity of UiO-66 and nanoUiO-66-X was evaluated in terms of elimination of the emerging organic contaminant SMT as challenging antibiotic in water. In a typical experiment, 4 mg of UiO-66 or nanoUiO-66-X (X = H, NH 2 , and OH) were suspended in 4 mL of a SMT tap-aqueous solution (10 ppm, according with the concentration of SMT found in the environment). 9 Adsorption/photodegradation reactions were performed under stirring. At certain intervals (0.25, 0.5, 1, 2, 4, 6, and 24 h), an aliquot of 100 µL was collected by centrifugation for HPLC analysis (SMT and the corresponding ligand). All experiments were performed at least in triplicate to ensure statistically reliable results. The crystallinity of all the remaining solids was analyzed by XRPD. First of all, the stability of an aqueous solution of SMT was studied under UV-vis light. It was verified that SMT was not degraded (after 24 h) under UV-vis light irradiation.

SMT photodegradation products: Particularly in the photodegradation studies, after 24 h of UV-vis irradiation, an aliquot was analyzed by mass quadrupole coupled to UHPLC to determine the final products of the photodegradation. A standard sample of SMT was measured to deduce through comparison the mass parents obtained.

The photodegradation experiments were performed in a photoreactor equipped with a 300 W Xe lamp (Oriel Instrument OPS-A500) under open air at RT, with the samples stirred and placed at a fixed distance of 21 cm from the irradiation source. It has to be pointed that prior to irradiation it is not necessary to stir the suspension until the adsorption-desorption equilibrium is reached. Although all the nanoUiO-66-X (X = H, NH 2 , and OH) materials show a different experimental band gap (3.89, 2.87 eV, and 3.29 eV for UiO-66, UiO-66-NH 2 , and UiO-66-OH, respectively; values obtained by the Tauc Plot), 35 we have irradiated all samples under all the UV-vis spectra.

Photodegradation kinetics: To shed some light on the SMT degradation kinetics and to gain further understanding on the involved mechanism, the first 2 h of SMT degradation were fitted to a first order kinetics according to Eqn (1), where [SMT] and [SMT] 0 is the remaining concentration of SMT (mg•L -1 ) at the time t (min) and the initial SMT concentration, respectively, and k is the first order kinetic constant (h -1

). Recyclability of UiO-66: The recyclability of UiO-66 was studied following the same procedure as previously described. 4 mg of UiO-66 were suspended in 4 mL of a SMT tap-aqueous solution (10 ppm) under UV-vis light irradiation. After 24h, the sample was centrifuged, an aliquot of 100 µL was collected for HPLC analysis (SMT and H 2 BDC quantification), and the solid was suspended again in a fresh SMT aqueous solution (10 ppm) for 24 h. This process was repeated for 6 cycles.

Monte Carlo simulations

Classical Grand Canonical Monte Carlo (GCMC) calculations using SORPTION (Materials Studio 2020) were performed to extract the distribution of SMT molecules inside the pores of the different UiO-66-X (X=H, NH 2 , OH) structures. [START_REF] Frenkel | Understanding Molecular Simulation[END_REF] For that purpose, a high pressure (10 4 kPa) and the temperature (T = 300 K) were fixed. Such a procedure has already been successfully used by some of us to determine the arrangement of drug and dye molecules. [START_REF] Rojas | [END_REF][45][46] Addition calculations, where the loading is fixed at 1, 2, 3 and 4 molecules per unit cell structure were also performed. Typically, 100 x 10 6 Monte Carlo steps for equilibration and 5 x 10 6 steps for production were used to reach the equilibrium. Regarding the calculations, the Ewald summation were computed to evaluate the electrostatic contribution, while atom-atom interactions at short range (cut-off fixed at 12.5 Å) were considered for the van der Waals part. In these calculations, no flexible force field was used for the solids and structures were therefore considered as rigid.

Multi-cell structures were considered in concordance with the use of the cut-off. Before Monte Carlo simulations, the partial charges used for the solids were calculated by the QEq methodology developed by Materials Studio. 47 For the SMT, DFT calculations were performed to calculate partial charges (see Supporting information, Figure S7) after geometry optimization of the molecule structure using the PW91 GGA density functional and the double numerical basis set containing polarization functions on hydrogen atoms (DNP) as implemented in DMol 3 code. Lennard Jones parameters were taken from UFF and mixed according the Lorentz-Berthelot rules.

48

DFT calculations

After Monte Carlo simulations, DFT calculations have been performed for UiO-66-X structures containing 2 and 3 SMT molecules (generally 1 molecule in the largest octahedral pore and 1 or 2 in the smaller tetrahedral pore). From these structures, a geometry optimization was performed to investigate the main interaction sites existing in the UiO-66-X structures and SMT molecules as a function of the pore nature. Again, DFT calculations were performed to obtain the final structure after geometry optimization using the PW91 GGA density functional and the double numerical basis set containing polarization functions on hydrogen atoms (DNP) as implemented in DMol 3 code.

Conflicts of interest

There are no conflicts to declare.

Figure 1 .

 1 Figure 1. Schematic view of the structure of UiO-66 (zirconium polyhedra, oxygen, and carbon are represented in green, red, and grey, respectively; hydrogen atoms are omitted for clarity). Structure of the antibiotic SMT is also given (sulphur is represented in yellow).

Figure 2 .

 2 Figure 2. a) SMT photodegradation and adsorption kinetics under UV-vis irradiation (black triangles, left y axis) and darkness (black squares, left y axis) in the presence of UiO-66, respectively. The MOF degradation, by means of H2BDC leaching, was also represented under UV-vis light (red triangles, right y axis) and dark (red squares, right y axis) conditions. b). Cyclability tests of SMT photodegradation using UiO-66.

Acknowledgements

The work has been supported by IMDEA Energy Foundation and MOFSEIDON project (PID2019-104228RB-100, MCI/AEI/FEDER, UE). SR and P.H. acknowledges the financial support of Madrid Community (Recruitment of Young doctors 2017, Talento Modality 2, 2017-T2-IND-5149). PH acknowledges the Spanish Ramón y Cajal Programme (Gran Agreements 2104-15039). S.R. acknowledges the Spanish Juan de la Cierva Incorporación Fellowship (grant agreement no. IJC2019-038894-I).

Notes and references


Keywords: bruxism, osteopathic manipulative treatment, movement analysis, range of motion, cervical spine

Introduction: This study explores the interest of using motion analysis to evaluate cervical and jaw ROM on students with or without bruxism when comparing Osteopathic manipulative treatment (OMT) to sham in school settings.

Methods: A feasibility study was run with 48 volunteer students from an osteopathic training institution. Random sequence for allocation was generated using a 1:1 ratio and block size of four to either OMT or sham treatment (control group). The chosen motion outcomes of interest were the lateral jaw range of motion, jaw opening, cervical rotation and side-bending.

ROM was measured averaging over three repeated movements at baseline, immediately after the 1st treatment, one week before the 2nd treatment, and immediately after second treatment using video-based analysis with 15 body landmarks.

Results: Repeated measures of joint motion at baseline showed high levels of reliability (ICC ranging from 0.953 to 0.985). Motion analysis detected clinically important differences between OMT and sham one-week post-treatment for jaw lateral ROM (3.3°; p=0.018) and cervical rotation ROM (12.0°; p=0.003) on participants with bruxism but not on those without. Magnitude of differences were increased for all parameters following the second treatment (lateral jaw movement; 4.8°, p=0.005; jaw opening; 5.5°, p=0.002; cervical sidebending; 9.2°, p=0.023; cervical rotation; 18.2°, p<0.001).

Conclusion:

Motion analysis is capable of detecting the effects of OMT on cervical and jaw ROM in students with bruxism but not without. Finally, the study showed the feasibility of introducing usual standards for clinical trials and sham treatment in school led studies with students.

INTRODUCTION

Background

Bruxism is a common behaviour that is associated with several other clinical musculoskeletal symptoms (neck pain, headache, tinnitus, sleep disorders, temporomandibular pain, etc.) [START_REF] Lobbezoo | Bruxism Defined and Graded: An International Consensus[END_REF][START_REF] Lobbezoo | International Consensus on the Assessment of Bruxism: Report of a Work in Progress[END_REF][START_REF] Martinot | Bruxism Relieved Under CPAP Treatment in a Patient With OSA Syndrome[END_REF][START_REF] Martynowicz | Evaluation of Relationship Between Sleep Bruxism and Headache Impact Test-6 (HIT-6) Scores: A Polysomnographic Study[END_REF][START_REF] Ohlmann | Correlations between Sleep Bruxism and Temporomandibular Disorders[END_REF][START_REF] Yap | Sleep Bruxism: Current Knowledge and Contemporary Management[END_REF].

Its prevalence is similar worldwide and concerns 20% of the population, decreases with age and appears to be very common among students [START_REF] Cavallo | Perceived Stress and Bruxism in University Students[END_REF][START_REF] Manfredini | Epidemiology of Bruxism in Adults: A Systematic Review of the Literature[END_REF][START_REF] Soares | Prevalence of Bruxism in Undergraduate Students[END_REF][START_REF] Wetselaar | The Prevalence of Awake Bruxism and Sleep Bruxism in the Dutch Adult Population[END_REF].

Preliminary studies have highlighted interrelations between cervical spine and jaw musculoskeletal functions [START_REF] Gouw | Coherence of Jaw and Neck Muscle Activity during Sleep Bruxism[END_REF]. Many associations supported by anatomical, physiological, biomechanical and neurophysiological studies support the idea that the musculoskeletal system of the head, the cervical spine and/or the craniocervical-mandibular system are linked [START_REF] Gouw | Coherence of Jaw and Neck Muscle Activity during Sleep Bruxism[END_REF][START_REF] Armijo Olivo | The Association between the Cervical Spine, the Stomatognathic System, and Craniofacial Pain: A Critical Review[END_REF][START_REF] Armijo-Olivo | Cervical Musculoskeletal Impairments and Temporomandibular Disorders[END_REF][START_REF] Olivo | The Association between Neck Disability and Jaw Disability[END_REF][START_REF] Piekartz | Temporomandibular Dysfunction and Cervical Impairments in Females -Results from an Observational Study[END_REF]. However, other causes that regulate motion, such as central neurological processes, could be at play. Therefore, alternative approaches that address other body regions or use different approaches (ex. mindfulness, stress management, lifestyle change, etc.) could also be beneficial.

Osteopaths make a substantial contribution to primary healthcare provision to a diverse population [START_REF] Thomson | Osteopaths' Professional Views, Identities and Conceptions -A Qualitative Grounded Theory Study[END_REF][START_REF] Vaucher | The Role of Osteopathy in the Swiss Primary Health Care System: A Practice Review[END_REF]. This also includes the management of patients with bruxism and neck pain [START_REF] Vaucher | The Role of Osteopathy in the Swiss Primary Health Care System: A Practice Review[END_REF][START_REF] Bill | Osteopathy in the French-Speaking Part of Switzerland: Practitioners' Profile and Scope of Back Pain Management[END_REF][START_REF] Morin | Primary Reasons for Osteopathic Consultation: A Prospective Survey in Quebec[END_REF][START_REF] Steel | Person-Centred Care and Traditional Philosophies in the Evolution of Osteopathic Models and Theoretical Frameworks: Response to Esteves et Al[END_REF]. Osteopathy is a person-centred, primary health care discipline that often relies on touch to help to optimize, to restore, and to maintain functional balance and well-being [START_REF] Oia | Osteopathy and Osteopathic Practice; A Global View of Practice, Patients, Education and the Contribution to Healthcare Delivery[END_REF][START_REF]Benchmarks for Training in Traditional /Complementary and Alternative Medicine: Benchmarks for Training in Osteopathy[END_REF].

A large debate persists in the literature about osteopathic ability to evaluate motion and dysfunction; however, most studies agree that osteopathic tests lack fidelity and accuracy [START_REF] Basile | Diagnostic Reliability of Osteopathic Tests: A Systematic Review[END_REF][START_REF] Fryer | Somatic Dysfunction: An Osteopathic Conundrum[END_REF][START_REF] Vaucher | Questioning the Rationality of Clinical Osteopathic Tests : Future Perspectives for Research[END_REF][START_REF] Johnston | A Call for Osteopathic Descriptive Research: Use of a Functional Model to Distinguish Segmental Motion Behavior[END_REF]. Specific and functional tests help osteopaths to analyse and evaluate the structural and functional integrity of the body in line with critical reasoning using osteopathic principles [START_REF] Vaucher | Questioning the Rationality of Clinical Osteopathic Tests : Future Perspectives for Research[END_REF][START_REF] Digiovanna | An Osteopathic Approach to Diagnosis and Treatment[END_REF]. Osteopathic motion tests remain very subjective and seem inappropriate to detect "somatic dysfunctions" as a simple dichotomous answer [START_REF] Fryer | Somatic Dysfunction: An Osteopathic Conundrum[END_REF]. Pattern recognition could be the J o u r n a l P r e -p r o o f way clinicians use motion tests thereby making it possible to detect subtler nuances in motion.

Tests might be interpreted in a broader manner than we think and also use motion detection to detect abnormal muscular response during active self-induced polyarticular motions.

Osteopathic motion tests could falsely target specific joint functions when in reality they provide information on a much more complex motion behaviour. Some behavioural pattern could reveal the intricate relationship of a whole chain of interlinked subcomponents that can only be correctly appreciated when considering motion in its entirety [START_REF] Chenaut | Biomechanical Analysis of the Lumbar-Pelvic-Femoral Complex during the One-Sided Tilt Test: A Pilot Study in Triathletes[END_REF][START_REF] Ménard | Analyse biomécanique du complexe lombo-pelvi-fémoral lors du test d'inclinaison unilatérale du bassin : étude pilote sur des triathlètes[END_REF][START_REF] Retailleau | New Insights into Lumbar Flexion Tests Based on Inverse and Direct Kinematic Musculoskeletal Modeling[END_REF]. This approach for clinical interpretation is close to Johnston's concept of "bony segments as mobile units within a mobile system" [START_REF] Johnston | Functional Methods[END_REF]. His pioneering work highlighted the importance of interpreting local motion of body parts by using gross motion tests within a broader functional system [START_REF] Osteopathy | The Johnston Academy Yearbook honors[END_REF]. Polyarticular motion could therefore help to understand dysfunction and guide treatment. This is of major importance for education and research in osteopathic healthcare [START_REF] Steel | Person-Centred Care and Traditional Philosophies in the Evolution of Osteopathic Models and Theoretical Frameworks: Response to Esteves et Al[END_REF][START_REF] Esteves | Models and Theoretical Frameworks for Osteopathic Care -A Critical View and Call for Updates and Research[END_REF][START_REF] Ménard | Finding a Way between Osteopathic Principles and Evidence-Based Practices: Response to Esteves et Al[END_REF]].

An interesting joint to test this principle is the temporomandibular joint. Some preliminary studies have reported that osteopathic care could decrease significantly cervical spine and/or temporomandibular joint pain [START_REF] Cuccia | Osteopathic Manual Therapy versus Conventional Conservative Therapy in the Treatment of Temporomandibular Disorders: A Randomized Controlled Trial[END_REF][START_REF] Gesslbauer | Effectiveness of Osteopathic Manipulative Treatment versus Osteopathy in the Cranial Field in Temporomandibular Disordersa Pilot Study[END_REF][37][START_REF] Monaco | Osteopathic Manipulative Treatment (OMT) Effects on Mandibular Kinetics: Kinesiographic Study[END_REF][START_REF] Burns | Gross Range of Motion in the Cervical Spine: The Effects of Osteopathic Muscle Energy Technique in Asymptomatic Subjects[END_REF] even if the rationale and mechanics by which osteopathic care may decrease bruxism remains unclear. Previous authors have suggested that osteopathic care could increase spine range of motion [START_REF] Branney | Does Inter-Vertebral Range of Motion Increase after Spinal Manipulation? A Prospective Cohort Study[END_REF][START_REF] Fryer | The Influence of Contraction Duration in Muscle Energy Technique Applied to the Atlanto-Axial Joint[END_REF][START_REF] Ménard | Modélisation du système musculo-squelettique : Implications cliniques, prévention des blessures et perspectives pour la recherche en ostéopathie[END_REF][START_REF] Millan | The Effect of Spinal Manipulative Therapy on Spinal Range of Motion: A Systematic Literature Review[END_REF] but few studies have focused on functional assessments on patients with bruxism. This is probably due to the difficulty in objectifying dynamic motion without impeding the movement itself [START_REF] Steel | Person-Centred Care and Traditional Philosophies in the Evolution of Osteopathic Models and Theoretical Frameworks: Response to Esteves et Al[END_REF][START_REF] Alvarez | A Methodological Review Revealed That Reporting of Trials in Manual Therapy Has Not Improved over Time[END_REF]. Detailed range of motion analysis may nevertheless help identify specific patterns of movement alterations that are not easy to identify with other means of measurement. This has now become possible with new methodologies developed in biomechanics and human movement analysis [START_REF] Ménard | Modélisation du système musculo-squelettique : Implications cliniques, prévention des blessures et perspectives pour la recherche en ostéopathie[END_REF][START_REF] Begon | Modélisation Anthropométrique Pour Une Analyse Mécanique Du Geste Sportif: Partie 1 : Modèles, Leurs Caractéristiques et Leur Validation[END_REF][START_REF] Begon | Modélisation Anthropométrique Pour Une Analyse Mécanique Du Geste Sportif.: Partie 2 : Estimation Des Centres Articulaires et Détermination de La Cinématique Du Squelette[END_REF]. Video capture systems make it possible to evaluate motion in a constraint-free environment [37,[START_REF] Ren | Biomechanical Effects of Cranio-Cervical Positions on Cervical Musculoskeletal Disorders[END_REF][START_REF] Salem | La colonne cervicale de la physiologie intersegmentaire tridimensionnelle à la manipulation ostéopathique par haute vitesse basse amplitude études in vivo[END_REF].

J o u r n a l P r e -p r o o f

However, experimental observations are lacking to support the use of such analysis in clinical settings. Motion analysis is expensive, complex, and time-consuming making it difficult to apply in pragmatic clinical trials. However, the applications drawn from motion analysis could bring a better understanding of assessment procedures used in usual care and improve functional global assessment [START_REF] Balsalobre-Fernández | The Concurrent Validity and Reliability of a Low-Cost, High-Speed Camera-Based Method for Measuring the Flight Time of Vertical Jumps[END_REF][START_REF] Bernardina | Are Action Sport Cameras Accurate Enough for 3D Motion Analysis? A Comparison With a Commercial Motion Capture System[END_REF][START_REF] Elwardany | Reliability of Kinovea Computer Program in Measuring Cervical Range of Motion in Sagittal Plane[END_REF][START_REF] Guzmán-Valdivia | Therapeutic Motion Analysis of Lower Limbs Using Kinovea[END_REF][START_REF] Puig-Diví | Validity and Reliability of the Kinovea Program in Obtaining Angles and Distances Using Coordinates in 4 Perspectives[END_REF].

This study aims to investigate whether motion analysis is capable of detecting subtle changes in jaw and cervical motion in a reliable way when comparing OMT to sham therapy and identifying which outcome is best as primary outcome.

METHODS

Trial design

This study consisted of a pilot monocentric 1:1 randomised parallel trial on symptomatic and asymptomatic students. It explores the usefulness of motion analysis for future phase I and II clinical trials. Approval for this study was obtained from the institution's ethical and scientific board of XX. The study was conducted in accordance with the amended Declaration of Helsinki.

Participants, randomization and allocation

Recruitment of participants was conducted through an online questionnaire sent to all 314 students from the Institute. Answers were collected from 124 osteopathic students.

Inclusion criteria were [START_REF] Lobbezoo | Bruxism Defined and Graded: An International Consensus[END_REF] being aged between 18 and 35 years, (2) being a student at the IO-RB, (3) having no musculoskeletal disorders reported in the cervical spine or the jaw, (4) and for the bruxism group, presenting chronic bruxism for more than a year. Exclusion criteria for both groups were: (1) having dental or orthodontic treatment in progress, (2) recent tooth J o u r n a l P r e -p r o o f extraction, (3) known disc-condyle disunion, (4) bite splints, (5) orthognathic devices or [START_REF] Yap | Sleep Bruxism: Current Knowledge and Contemporary Management[END_REF] recent direct trauma.

Self-reported bruxism was not evaluated other than with the questionnaire on symptoms.

Symptoms related to TMJ disorders were also ruled out.

Participants received written and oral instructions about the intervention, test protocol, and possible risks and benefits of the study. Students were told that different manual treatment approaches were to be compared without specifically mentioning one of them to be sham. This was necessary to avoid students discussing their treatment and guessing their allocation.

Once data collection was over, students were informed of the true nature of both groups.

Written informed consent was obtained from all participants before their inclusion in the study.

All participants agreed to avoid other forms of manual treatment during their follow-up and if required to notify this protocol deviation to investigators. Participants in both group bruxism or no-bruxism were randomly allocated in a 1:1 ratio to two sub-groups OMT (Treated Group) or sham treatment (Control Group) using a random sequence of blocks of size 4 generated by R. The study flowchart is presented in Figure 1. Allocation was then attributed following the order of responses to the first online questionnaire. A staff member then prepared the material for the treating osteopaths. Allocation was revealed to the osteopaths on the arrival of each patient when opening each folder.

Interventions

OMT consisted of two treatment sessions (45') separated by seven days that were personalized and included patient history taking, osteopathic diagnostic assessment and OMT.

Participants in the treatment group were randomly allocated to one of six osteopaths with over 10 years of practice experience. OMT was tailored for the patient by combining gentle J o u r n a l P r e -p r o o f techniques on areas of restriction consisting of articulatory techniques, myofascial release techniques, functional techniques and visceral and cranial techniques, etc. The entire body was included in the osteopathic evaluation and treatment with special attention to the head, jaw and cervico-thoracic areas. Despite some expected variation in their application by different practitioners, all the techniques used were familiar to most recently trained osteopathic practitioners and taught in all French institutions. Sham intervention (45') included patient history taking, osteopathic diagnostic assessment and two different protocols of three sham manual techniques pseudo-randomly assigned to each participant in order to reproduce, as similar as possible, the contextual variables of the sessions [START_REF] Rossettini | Clinical Relevance of Contextual Factors as Triggers of Placebo and Nocebo Effects in Musculoskeletal Pain[END_REF]. The sham protocol included palpation and tissue mobilisation that was predefined and targeted to specific regions without intent to change motion. All details about OMT and sham treatment interventions are reported in Table 1 using TIDieR-Placebo guidelines [START_REF] Howick | TIDieR-Placebo: A Guide and Checklist for Reporting Placebo and Sham Controls[END_REF].

Outcomes

All participants were assessed four times; first at baseline, then immediately after the first session, then seven days after the first session, and finally immediately after the second session. A research team member who was not involved with the interventions and was blinded to allocations was responsible for collecting all the baseline measures and follow-up outcomes.

The chosen motion outcomes of interest were the lateral jaw range of motion, jaw opening, cervical rotation and side-bending after the 1st and 2nd treatment, and one week after the first treatment. This was done through a video-based system developed for this study. Participants were equipped with 15 body landmarks indexed in Figure 2. Markers trajectories were J o u r n a l P r e -p r o o f obtained from three sport cameras (Gopro Hero7Black, Gopro Hero6, Gopro Hero5) placed around the participant. The experimental setup was illustrated in Figure 3.

To evaluate the cervical spinal range of motion, participants were instructed to perform three trials of active maximal side-bending and rotation on each side (right and left). To evaluate jaw range of motion, students were also asked to perform three trials of active open/close and right and left lateral movement of the jaw. A randomized sequence of functional active movement of cervical and jaw movement was proposed for all students. Kinovea® software (v0.8.15) was used to analyse the experimental data and to compute the range of motion of the cervical spine (side, rotation) and the temporomandibular joint (lateral and opening movement of the jaw).

Blinding

The researchers involved in the evaluation procedures had no access to the allocation of the participants. All participants were blinded to group assignment.

Statistical methods

Outliers were defined for each measure and identified. These were most often related to misreading of motion markers which had to be manually framed. Such errors were corrected.

Reliability of measures was evaluated using a one-way random-effect model with absolute agreement to calculate intraclass coefficient (ICC) for individual measures before taking their mean. After verifying that the range of motion was similar between the left and right side, a single outcome was summarized by adding ROM from the left to the right side. Baseline comparison between OMT intervention group and the sham group was compared using Student T-test. Linear regression was used to measure between-group differences at follow-up adjusting for baseline values. All analysis were intention-to-treat. Correlation between J o u r n a l P r e -p r o o f outcomes was using Pearson correlation coefficients. Differences between students with or without bruxism were evaluated adding an interaction term in the logistic regression. The interaction term identified those who were both in the intervention arm and had symptoms. In other terms, the logistic regression modelled the added effects of treatment in participants with bruxism. Significance level was set at p<0.05. No adjustments were made for multiple testing. Sensitivity analysis was done post-hoc to verify that results were consistent even after adjusting for baseline imbalance.

RESULTS

Participants and baseline values

A total of 48 participants were recruited from a XX osteopathic educational institution, the XX, between September and October 2019. Two participants did not attend their appointment (for medical reasons outside the context of the study) and were excluded from further analysis, resulting in a total sample of 23 participants in the bruxism group and 23 participants in the no-bruxism group. All participants received allocated treatments. No major protocol deviation was reported.

Most participants were female (34/46; 73.9%), their age ranged from 18 to 25 years, their BMI was of 22.4 on average (ranging from 18.3 to 32.4 kg/m 2 ). Details on baseline values for each group are provided in Table 2. Except for cervical rotation in the group with bruxism (p=0.012), all other measures were well balanced between groups (p>0.05).

Reliability

Repeated measures of joint motion at baseline showed high levels of reliability. Individual measures had an ICC of 0.953 (CI95% 0.925 to 0.972) for jaw lateral movement, an ICC of J o u r n a l P r e -p r o o f 0.975 (CI95% 0.959 to 0.985) for jaw opening, an ICC of 0.975 (CI95% 0.959 to 0.985) for cervical side-bending, and an ICC of 0.985 (CI95% 0.975 to 0.990) for cervical rotation.

Objectivation of OMT on jaw lateral movement

One week after treatment, compared to sham, OMT treatment had a significant effect on increasing lateral jaw ROM (+3.3°; CI95% 0.6 to 5.9°; p=0.018) on participants with bruxism (Table 3). This effect was absent in the group without bruxism (-0.4°; CI95% -2.4 to 1.7; p=0.720). Effects of treatment were restricted to those having symptoms. Having bruxism was determinant for OMT to have effects on jaw lateral movement in the studied population (interaction test shows superiority of 4.1°, CI95% 0.9 to 7.2, p=0.012). These results remained similar even after adjustment for baseline imbalance.

Effects on other ROMs

For students with bruxism, at one week, OMT had a significant effect over sham intervention on cervical rotation ROM (+12.0°; CI95% 4.7° to 19.3°, p=0.003), but not on jaw opening or cervical side-bending (Table 3). Similar results were observed for short term effects, immediately after the first treatment with lower magnitudes for cervical rotation (+7.8°, p<0.001) and significant effects for cervical side-bending (+5.2°, p=0.045). Following the second treatment, effects were significant for all parameters (Table 3).

For students without bruxism, compared to sham intervention, at one week there were no effects on jaw opening (+0.4°; p=0.821), cervical side-bending (+0.6°, p=0.851) or cervical rotation (+6.4°, p=0.09). This was also the case in the short term, immediately after treatment and immediately after the second treatment except for cervical rotation which was increased by 10.4° in the OMT group (CI95% 1.7 to 10.0; p=0.021).

J o u r n a l P r e -p r o o f

Correlations between parameters

Changes over time at one week were correlated between jaw and cervical ROM. Students that gained in lateral jaw movement also gained in cervical side-bending (=0.313, p=0.036) and cervical rotation (=0.378, p=0.010). These same correlations became stronger for students with bruxism (cervical side-bending: =0.595, p=0.003 ; cervical rotation: =0.440, p=0.036) and optimal for cervical side-bending in students with bruxism and OMT (=0.640, p=0.018).

In this same sub-population, there was also a correlation between gain in cervical rotation and reduced jaw opening (=-0.567, p=0.043) that was however absent in other students.

Efficacy of blinding

At the 15-day follow-up, participants were asked whether they believed they were in the active or sham group. Proportion of those believing they were in the active treatment group was similar in the OMT group (11/25; 44%) and in the sham group (7/20; 35%; p=0.760).

Adverse events

Adverse events were monitored and self-reported one week after each treatment. No adverse events were reported by the participants.

DISCUSSION

The main finding of this study was that a video-based approach can objectively identify effects of OMT on cervical and jaw range of motion during two sessions of osteopathic care.

Results showed that motion analysis made it possible to show that osteopathic manual care, as compared to sham, induces increased cervical and jaw ROM. Results suggest lateral jaw movement to be a good choice for primary outcome in studies evaluating effects of OMT on J o u r n a l P r e -p r o o f patients with bruxism. This pilot study also suggests blinding to be feasible using sham treatment on students. Finally, the gain in mobility was correlated between jaw lateral movement and cervical side-bending supporting our initial hypothesis of the interest of investigating movement over more than one joint.

The methodology developed in this study allowed to observe and analyse, through different time intervals measurements, the effect of the osteopathic manual care over time in an outstanding reliable way (ICC>0.95). The consistency of results over-time also suggests that treatment effects are conserved over at least seven days and could be emphasized by a second treatment session (Figure 4). Contrary to clinical motion tests, motion analysis can be an efficient and objective way to evaluate functional changes in movement following osteopathic manual care. However, our results should be interpreted with caution. Joint range of motion is by no means the only mechanism involved in bruxism and several etiological factors (psychosocial, anatomical, and/or neuromuscular factors) also need to be explored during patient management. Improved range of motion may be simply a surrogate of improved motor control. Functional motion disorders have been shown to be closely related to alteration of motor control, reduced discriminatory sensation, catastrophization, fear-avoidance, and loss of self-confidence [START_REF] O'sullivan | Diagnosis and Classification of Chronic Low Back Pain Disorders: Maladaptive Movement and Motor Control Impairments as Underlying Mechanism[END_REF][START_REF] Wernli | Does Movement Change When Low Back Pain Changes? A Systematic Review[END_REF][START_REF] Lin | Patient-Centred Care: The Cornerstone for High-Value Musculoskeletal Pain Management[END_REF].

However, even if our results support the hypothesis that OMT could alter motor control during movement, they do not make it possible to know whether these changes represent any meaningful change for patients (i.e. reduced jaw tension, reduced pain). Range of motion often investigates movements that are most often not done naturally. The underlying theoretical construct linking range of motion with daily symptoms infers that the underlying changes also affect movement before reaching physiological limits. This assumption was not tested. This would require collecting more descriptive and tangible evidences of individual J o u r n a l P r e -p r o o f motor behaviours, not only in a traditional structural model of articular dysfunction but during specific functional motion tests [START_REF] Johnston | A Call for Osteopathic Descriptive Research: Use of a Functional Model to Distinguish Segmental Motion Behavior[END_REF]. Also, additional determinants could be explored during functional motion-tests both with instrumental approaches (electromyographyjaw muscle activity) and non-instrumental approaches (self-reported -perceived pain, stress levels and impact on daily life).

Practical applications for education and research

One of the challenges of education is to find a way to facilitate the transition from academic learning to training for their future profession but also to research [START_REF] Launay | Impact of Different Types of Revision Materials on the Learning of Musculoskeletal Techniques[END_REF]. Biomechanics has been introduced as an official teaching unit in the education of osteopathic students since 2014 in France [START_REF]Relatif à La Formation En Ostéopathie[END_REF]. The challenge is to apply both anatomical/physiological bases and mechanical concepts (mathematics and physics) to observe/describe/measure human movement from a number of different applications that cut across the sub-areas of movement sciences (sport, ergonomics, injury, clinical rehabilitation, etc.) [START_REF] Hamill | Biomechanics Curriculum: Its Content and Relevance to Movement Sciences[END_REF][START_REF] Wallace | The Effect of Course Format on Student Learning in Introductory Biomechanics Courses That Utilise Low-Tech Active Learning Exercises[END_REF]. Additionally, new teaching innovative methodologies to evaluate human movement (Wii Balance Board platform, Kinect, smartphone applications, etc.) were proposed to facilitate the comprehension of biomechanics laws/applications and to make it more "touchable" and inform students about these tools and their limits [START_REF] Clark | Reliability and Validity of the Wii Balance Board for Assessment of Standing Balance: A Systematic Review[END_REF][START_REF] Keogh | Reliability and Validity of Clinically Accessible Smartphone Applications to Measure Joint Range of J o u r n a l P r e -p r o o f Motion: A Systematic Review[END_REF]. The challenge is also to educate future osteopaths in the use of objective tools to evaluate their treatments in order to promote research in osteopathy and to transfer knowledge to education and clinical practice [START_REF] Steel | Person-Centred Care and Traditional Philosophies in the Evolution of Osteopathic Models and Theoretical Frameworks: Response to Esteves et Al[END_REF][START_REF] Browning | Teaching Osteopathic Students Technique; Using Research to Identify Good Teaching Practice[END_REF][START_REF] Fryer | Teaching Critical Thinking in Osteopathy -Integrating Craft Knowledge and Evidence-Informed Approaches[END_REF]. In this sense, a large number of research themes must be proposed involving students and educators to continue building evidence by validating or disproving the knowledge or beliefs related to osteopathic care [START_REF] Martinot | Bruxism Relieved Under CPAP Treatment in a Patient With OSA Syndrome[END_REF][START_REF] Martynowicz | Evaluation of Relationship Between Sleep Bruxism and Headache Impact Test-6 (HIT-6) Scores: A Polysomnographic Study[END_REF][START_REF] Ohlmann | Correlations between Sleep Bruxism and Temporomandibular Disorders[END_REF][START_REF] Yap | Sleep Bruxism: Current Knowledge and Contemporary Management[END_REF][START_REF] Ménard | Finding a Way between Osteopathic Principles and Evidence-Based Practices: Response to Esteves et Al[END_REF]. Future challenges for the profession are to develop collaborations with other disciplines to create new knowledge and the assessment of the effectiveness of osteopathic manual care in the light of scientific methods but also to associate J o u r n a l P r e -p r o o f applied biomechanics and neurosciences to propose new findings in the impact of osteopathic healthcare [START_REF] Ménard | Finding a Way between Osteopathic Principles and Evidence-Based Practices: Response to Esteves et Al[END_REF][START_REF] Esteves | Models and Theoretical Frameworks for Osteopathic Care -A Critical View and Call for Updates and Research[END_REF].

Limitations

This study involved a modest cohort of 46 students (divided into 4 experimental groups), it would be interesting for future phase 2 and 3 clinical trials to use a similar methodology measuring ROM on a larger population with a follow-up by combining, it with other measurement tools (Patient Reported Outcome Measures, validated questionnaires, electromyography, etc.) to assess the impact of the global effect of osteopathic care on behaviour.

The study also might be limited in external validity given it targets a specific population particularly concerned by bruxism [START_REF] Cavallo | Perceived Stress and Bruxism in University Students[END_REF][START_REF] Soares | Prevalence of Bruxism in Undergraduate Students[END_REF][START_REF] Wetselaar | The Prevalence of Awake Bruxism and Sleep Bruxism in the Dutch Adult Population[END_REF]. Slight differences in treatment approaches between osteopaths cannot be ruled out. All six osteopaths however work in the same school clinic and are used to follow similar approaches. Nevertheless, future studies need to consider training osteopaths to assure they deliver similar treatments and include fidelity analysis to verify if treatment was delivered as planned. Even if blinding was assured, the students' response to treatment could be influenced by their familiarity with the discipline and communication between students.

We collected motion with three sport cameras but not really in 3D with an optoelectronic system for example. However, after comparing the data from this study with previous authors who have established an important database on cervical and mandibular kinematics from 22 experimental studies using recognized systems, it appears that the data obtained are close to the standards they established [START_REF] Koeppel | Analyse cinématique de l'appareil manducateur humain : constitution d'une base de données de sujets asymptomatiques et comparaison avec sujets à dysfonction[END_REF][START_REF] Boussion | Étude cinématique tridimensionnelle du rachis cervical. Comparaison entre sujets Asymptomatiques et pathologiques[END_REF]. In this way, we observed in this pilot study, the effect of OMT only in maximal ROM which focuses on assessing the patient's maximum capacity to perform selected active movements. In order to assess dynamic functional behaviour, it could J o u r n a l P r e -p r o o f be useful to track/estimate all the 3D kinematic of each degree of freedom to explore the evolution of the pattern of motion during the functional assessment before and after OMT. This appears to be a better way to explore motion behaviour which is used for the quantified analysis of walking, running or rowing for example [START_REF] Chehab | Age, Sex, and Body Mass Index Provide a Rigorous Basis for Comparing the Kinematic and Kinetic Profiles of the Lower Extremity during Walking[END_REF][START_REF] Ménard | A Musculoskeletal Modelling Approach of the Assessment of the Risk of Hamstring Injuries in Professional Soccer Players: A Pilot Study[END_REF][START_REF] Retailleau | Kinematics of the Lumbar Muscles in Rowing: A Preliminary Study[END_REF][START_REF] Riley | A Kinematic and Kinetic Comparison of Overground and Treadmill Walking in Healthy Subjects[END_REF]. A potential limitation to future use of motion analysis in clinical trials is the availability and access to sophisticated material and skilled personnel making it possible to acquire meaningful data. Therefore, future studies with optoelectronic, medical system imagery or inertial sensors are needed to go further with the data collection/analysis but also to confirm the benefits of the treatment and the assessment method [START_REF] Cazzola | Cervical Spine Injuries: A Whole-Body Musculoskeletal Model for the Analysis of Spinal Loading[END_REF][START_REF] Plocharski | Motion Analysis of the Cervical Spine during Extension and Flexion: Reliability of the Vertebral Marking Procedure[END_REF][START_REF] Raya | An Inexpensive and Easy to Use Cervical Range of Motion Measurement Solution Using Inertial Sensors[END_REF][START_REF] Salem | In Vivo 3D Kinematics of the Cervical Spine Segments during Pre-Manipulative Positioning at the C4/C5 Level[END_REF]. Finally, the subjectivity of active voluntary movements could have introduced social desirability bias to the observed improvements in ROM due to OMT.

New insights on interoceptive perception and the eventual role of osteopathic care in helping solve misrepresentation of one's body perception and movement are promising to the profession [START_REF] Esteves | Models and Theoretical Frameworks for Osteopathic Care -A Critical View and Call for Updates and Research[END_REF][START_REF] Esteves | Predictive Processing and Allostatic Regulation in Clinical Practice[END_REF].

CONCLUSION

This study highlighted that a video-based approach may allow analysing musculoskeletal function of the jaw and cervical spine before and after OMT. Motion analysis can reveal improvements of cervical and jaw ROM following OMT in osteopathic students with bruxism compared to sham treatment. A longitudinal investigation with a larger sample is needed to confirm the benefits of the treatment and the assessment method. These outcomes are promising for future studies investigating the role of osteopathic care in preventing musculoskeletal conditions in people with bruxism. This approach could be implemented as an additional tool to objectify the effect of osteopathic healthcare and also to the global advancement of the profession in thriving, growing osteopathic research culture necessary in the contemporary health care landscape.

J o u r n a l P r e -p r o o f

Tables Table 1: Account for non-specific, contextual and natural effects and isolate the OMT component of the treatment.

PROCEDURES

The session was personalized and included patient history taking (past experience, present state), physical examination, diagnostic osteopathic assessment, OMT, counselling, lifestyle change. Physical examination was done before and after treatment and included observation, global functional tests, pain provocation tests, orientation tests and local regional mobility assessment. Osteopaths were instructed to specifically evaluate jaw, cervical and upper thoracic motion.

The session was personalized and included patient history taking (past experience, present state), physical examination, diagnostic osteopathic assessment, and two sham procedures that mimicked OMT, counselling, lifestyle change. Physical examination was done before and after sham treatment and included observation, global functional tests, pain provocation tests, orientation tests and local regional mobility assessment. Osteopaths were instructed to specifically evaluate jaw, cervical and upper thoracic motion.

WHO PROVIDED

Participants were randomly allocated to receive treatment from one of six experienced osteopaths. All practitioners were educators with supervision experience within the school clinic. They had at least 10 years of experience in practice. All osteopaths received the same training for the procedures. There was no calibration on patient management.

HOW

OMT was personalized for the patient by combining a wide range of osteopathic techniques "as usual" without receiving any instructions on decision criteria. Patient preferences were accounted for. OMT included soft tissue, MET and articulatory techniques, visceral and cranial techniques. Level of patient participation was not standardised across treatments.

Osteopaths performed a predefined OMT technique on a specific region without any intent to induce changes in tissue mobility. Sham treatments were administered at two occasions in a random order. The sham treatment was the same for all participants and consisted of:

WHERE

School clinic

HOW MUCH

Two treatment sessions (45') separated by one week

TAILORING

The intervention was adapted and tailored to each participant including anamnesis, tests and treatment. Fidelity to protocol was assessed by analysing the reported interpretation and actions of osteopaths.

The sham procedure was of two types described above. Fidelity to protocol was assessed by analysing the reported interpretation and actions of osteopaths.

MEASURING THE SUCCESS OF BLINDING

Fifteen days' follow-up, participants were asked whether they believed they were in the active or sham group.

J o u r n a l P r e -p r o o f

Figure 1 .

 1 FIGURES legends

Figure 2 .

 2 Figure 2. Diagram and anatomical localization of markers used during the study; THD: Top

Figure 3 .

 3 Figure 3. Experimental set up proposed and developed in this school-based protocol.

Figure 4 .

 4 Figure 4. Jaw and cervical range of motion in the bruxism group before and after OMT or

  Description of the Osteopathic Manipulative Treatment (OMT) and sham interventions (TIDieR-Placebo checklist) OMT SHAM WHY Provide usual osteopathic care expected to improve jaw and cervical mobility.

( 1 )

 1 Palpation of cranial mobility / MET for an inferior glenohumeral / Tissue technique of the 4th Left rib. (2) Palpation of lower thorax orifice mobility / Soft tissue technique of zygomatic bones / MET on the right 5th rib posterolateral.

J o u r n a l P r e -p r o o f

We acknowledge thank all students for their enthusiastic participation. We gratefully acknowledge Franck Sutre (DO), Florence Delauriere (DO, MSc), Alexis Guery (DO), Ludovic Lancelot (DO) and François Launay (DO) for their contribution to this study. The author also acknowledges Marylène Bourgin (DO, MM) and Emmanuel Harel (MM) for their logistic support during the two sessions of experimentation.

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