Shuobin Liang

Xinyuan Ma

Man Li

Yanliang Yi

Qianqian Gao

Yongmin Zhang

Lihe Zhang

Demin Zhou

Sulong Xiao
email: slxiao@bjmu.edu.cn

Novel β-cyclodextrin-based heptavalent glycyrrhetinic acid conjugates: Synthesis, characterization and anti-influenza activity

Keywords: Glycyrrhetinic acid, β-cyclodextrin, influenza virus, multivalency, hemagglutinin

In our continuing efforts toward the design of novel pentacyclic triterpene derivatives as potential anti-influenza virus entry inhibitors, a series of homogeneous heptavalent glycyrrhetinic acid derivatives based on β-cyclodextrin scaffold were designed and synthesized by click chemistry. The structure was unambiguous characterized by NMR, IR, and MALDI-TOF-MS measurements. Seven conjugates showed sufficient inhibitory activity against influenza virus infection based on cytopathic effect reduction assay with IC 50 values in the micromolar range. The interactions of conjugate 37, the most potent compound (IC 50 = 2.86 μM, CC 50 > 100 μM), with influenza virus were investigated using hemagglutination inhibition assay. Moreover, surface plasmon resonance assay further confirmed that compound 37 bound to influenza HA protein specifically with a dissociation constant of 5.15 × 10 -7 M. Our results suggest the promising role of β-cyclodextrin as scaffold for preparing a variety of multivalent compounds as influenza entry inhibitors.

INTRODUCTION

Influenza A virus is a highly contagious respiratory pathogen that can cause seasonal epidemics and irregular pandemics due to its rapid transmission and frequent genetic alteration [START_REF] Neumann | Emergence and pandemic potential of swine-origin H1N1 influenza virus[END_REF][START_REF] Lagacé-Wiens | Influenza epidemiology-past, present, and future[END_REF]. The structure of the viruses consists of a lipid envelope that is generated from the host cell, to which two dominant membrane proteins hemagglutinin (HA) (80%, ∼300 copies of trimer) and neuraminidase (NA) (17%, ∼50 copies of tetramer) are anchored [START_REF] Leser | Influenza virus assembly and budding in raft-derived microdomains: A quantitative analysis of the surface distribution of HA, NA and M2 proteins[END_REF]. Based on the antigenicity of HA and NA, they can be classified into different subtypes. To date, 18 major antigenic variants of HA and 11 antigenic variants of NA have been recognized, which are found in numerous combinations. In addition, newly mutated forms of the influenza virus appear every year. The current available options to combat influenza A viral infections include the prophylactic, yearly reformulated vaccines and two classes of anti-influenza drugs (the M2 ion-channel inhibitors and the NA inhibitors) [START_REF] Das | Antivirals targeting influenza A virus[END_REF]. Since the vaccines that are available now offer a very limited breadth of protection [START_REF] Van Dongen | A small-molecule fusion inhibitor of influenza virus is orally active in mice[END_REF], the antiviral drugs are still the first line of protection to treat acute influenza infection. Unfortunately, all circulating influenza A viral strains appear to be resistant to the M2 ion-channel inhibitors. Recently, the continually emerging of resistant virus against NA inhibitors has been also reported [START_REF] Hurt | Resistance to anti influenza drugs: Adamantanes and neuraminidase inhibitors[END_REF][START_REF] Van Der Vries | Emergence of a multidrug-resistant pandemic influenza A (H1N1) virus[END_REF][START_REF] Hurt | The epidemiology and spread of drug resistant human influenza viruses[END_REF]. For these reasons, novel anti-influenza viral agents with new targets and mechanisms of action are urgently required to overcome the rapid emergence of drug resistance [START_REF] Heo | Baloxavir: First global approval[END_REF][START_REF] Van Dongen | A small-molecule fusion inhibitor of influenza virus is orally active in mice[END_REF].

In the first step of infection, the virion adheres to the host cell surface through the binding of HA to sialic acid-terminated carbohydrates present on the cell membranes [START_REF] Mammen | Polyvalent interactions in biological systems: Implications for design and use of multivalent ligands and inhibitors[END_REF][START_REF] Skehel | Receptor binding and membrane fusion in virus entry: The influenza hemagglutinin[END_REF]. Therefore, HA plays a pivotal role in virus entry, making it a potential target for antiviral intervention. HA is a homotrimeric integral membrane glycoprotein, which has ca. 300-400 copies on the viral surface [START_REF] Mammen | Polyvalent interactions in biological systems: Implications for design and use of multivalent ligands and inhibitors[END_REF]. The monovalent interaction between HA and sialic acid is mediated essentially by hydrogen bonds, and the dissociation constant of them shows a relative weak binding (K D ~ 2 mM) [START_REF] Sauter | Hemagglutinins from 2 influenza-virus variants bind to sialic-acid derivatives with millimolar dissociation-constants: A 500-MHz proton nuclear magnetic-resonance study[END_REF]. Therefore, a multitude of HA is involved in order to increase the overall strength of the interaction between the virus and the host cell. The binding between multiple pairs of HA trimer and sialic acid ligand increases substantially as characterized by a multivalent affinity constant of estimated 10 13 M -1 [START_REF] Mammen | Polyvalent interactions in biological systems: Implications for design and use of multivalent ligands and inhibitors[END_REF]. This process provides important clues for the design of efficient multivalent ligands to mimic the binding of HA to sialic acid to block the viral protein-host receptor interactions [START_REF] Lu | Carbohydrate-protein interactions and multivalency: Implications for the inhibition of influenza A virus infections[END_REF]. Over the past few years, many efforts have been made for the design and synthesis of carbohydrate-based multivalent HA binders, including polymers, dendrimers, nanoparticles and so on, serving as potent influenza A virus inhibitors [START_REF] Chaudhary | Multivalent sialosides: A tool to explore the role of sialic acids in biological processes[END_REF][START_REF] Lu | Carbohydrate-protein interactions and multivalency: Implications for the inhibition of influenza A virus infections[END_REF].

β-Cyclodextrin (β-CD) is a torus shaped cyclic oligosaccharide consisting of seven (α-1,4)-linked D-glucopyranose units and it is well-known for their ability to form inclusion complexes with a broad variety of organic and inorganic compounds [START_REF] Crini | Review: A history of cyclodextrins[END_REF]. It is widely used in pharmaceutical formulations to enhance the solubility of poorly soluble drugs, increase drugs permeability through biological membranes and improve the bioavailability of drugs [START_REF] Jansook | Cyclodextrins: structure, physicochemical properties and pharmaceutical applications[END_REF]. The unique steric accessibility and acidity of the three types of hydroxyls in β-CD have been taken into account to conceive efficient position-selective and face-selective chemical functionalization methodologies [START_REF] Khan | Methods for selective modifications of cyclodextrins[END_REF]. It has been shown to serve as a multivalent scaffold in the design of glycoconjugates [START_REF] Martinez | Cyclodextrin-based multivalent glycodisplays: covalent and supramolecular conjugates to assess carbohydrate-protein interactions[END_REF], glycoclusters [START_REF] Gomez-Garcia | Probing secondary carbohydrate-protein interactions with highly dense cyclodextrin-centered heteroglycoclusters: The heterocluster effect[END_REF][START_REF] Gomez-Garcia | Probing carbohydrate-lectin recognition in heterogeneous environments with monodisperse cyclodextrin-based glycoclusters[END_REF]), glycodendrimers (Ortega-Caballero et al., 2001;[START_REF] Vargas-Berenguel | Dendritic galactosides based on a β-cyclodextrin core for the construction of site-specific molecular delivery systems: Synthesis and molecular recognition studies[END_REF] and star polymers [START_REF] Zhang | Cyclodextrin-centred star polymers synthesized via a combination of thiol-ene click and ring opening polymerization[END_REF][START_REF] Gonzalez-Gaitano | Drug carrier systems based on cyclodextrin supramolecular assemblies and polymers: Present and perspectives[END_REF]. In addition, target-specific multivalent drug delivery systems have shown significant improvement of anti-tumor drugs delivery and hence antitumor efficacy and prevention of clinical multi-drug resistance [START_REF] Kim | Polyglycerolated nanocarriers with increased ligand multivalency for enhanced in vivo therapeutic efficacy of paclitaxel[END_REF][START_REF] Zhou | In situ-induced multivalent anticancer drug clusters in cancer cells for enhancing drug efficacy[END_REF].

In spite of the progress, the design of multivalent ligands with optimized biological properties still remains a challenge considering the complexity of the multivalent ligand-receptor interactions [START_REF] Lepage | Toward a molecular lego approach for the diversity-oriented synthesis of cyclodextrin analogues designed as scaffolds for multivalent systems[END_REF]. Glycyrrhizic acid (also known as glycyrrhizin, 1) (Fig. 1), an oleanane-type pentacyclic triterpene saponin, is the primary bioactive constituent of the roots and rhizomes of Glycyrrhiza glabra (licorice) which is employed as an herbal medicine in both Western and Eastern countries [START_REF] Fiore | A history of the therapeutic use of liquorice in Europe[END_REF][START_REF] Asl | Review of pharmacological effects of Glycyrrhiza sp and its bioactive compounds[END_REF]. Compound 1 and its aglycone, glycyrrhetinic acid (GA, 2), have been reported to possess antitumor, anti-inflammatory and other pharmacological activities [START_REF] Yang | The pharmacological activities of licorice[END_REF][START_REF] Hussain | Therapeutic potential of glycyrrhetinic acids: A patent review (2010-2017)[END_REF] [START_REF] Lallemand | N-(2-{3-[3,5-Bis(trifluoromethyl)phenyl]ureido}ethyl)-glycyrrhetinamide (6b): A novel anticancer glycyrrhetinic acid derivative that targets the proteasome and displays anti-kinase activity[END_REF]. Recently, attention to GAs has been greatly increased due to their broad spectrum of antiviral activities, such as anti-hepatitis B virus (HBV) [START_REF] Wang | Synthesis, biological evaluation and structure-activity relationships of glycyrrhetinic acid derivatives as novel anti-hepatitis B virus agents[END_REF], anti-Epstein-Barr virus (EBV) [START_REF] Lin | Inhibitory effects of some derivatives of glycyrrhizic acid against Epstein-Barr virus infection: Structure-activity relationships[END_REF], anti-rotavirus [START_REF] Hardy | 18β-glycyrrhetinic acid inhibits rotavirus replication in culture[END_REF], anti-influenza virus [START_REF] Stanetty | Synthesis and antiviral activities of spacer-linked 1-thioglucuronide analogues of glycyrrhizin[END_REF], anti-herpes simplex virus (HSV) [START_REF] Zigolo | Chemoenzymatic synthesis of new derivatives of glycyrrhetinic acid with antiviral activity. Molecular docking study[END_REF], etc. 3-Thioglucuronide derivative 4 and several C-30 ester derivatives, such as 4-(trifluoromethyl) benzyl ester (5), 4-iodobenzyl ester (6) and 4-nitrobenzyl ester (7), show promising antiviral activity [START_REF] Stanetty | Synthesis and antiviral activities of spacer-linked 1-thioglucuronide analogues of glycyrrhizin[END_REF][START_REF] Wang | Synthesis, biological evaluation and structure-activity relationships of glycyrrhetinic acid derivatives as novel anti-hepatitis B virus agents[END_REF]. In our previous study, a series of monovalent β-CD-GA conjugates have been synthesized and several conjugates show weak antiviral activity at high concentration (~50 μM) [START_REF] Liang | Synthesis and structure-activity relationship studies of water-soluble β-cyclodextrin-glycyrrhetinic acid conjugates as potential anti-influenza virus agents[END_REF]. As a natural step in our efforts towards the design of new pentacyclic triterpene conjugates with antiviral activity [START_REF] Yu | Discovery of pentacyclic triterpenoids as potential entry inhibitors of influenza viruses[END_REF][START_REF] Xiao | Pentacyclic triterpenes grafted on CD cores to interfere with influenza virus entry: A dramatic multivalent effect[END_REF][START_REF] Si | Triterpenoids manipulate a broad range of virus-host fusion via wrapping the HR2 domain prevalent in viral envelopes[END_REF], we described herein the design and synthesis of a series of heptavalent GA functionalized β-CD conjugates and evaluated their in vitro anti-influenza activity. Figure 1. Chemical structures of glycyrrhizic acid (1), GA [START_REF] Wang | Synthesis, biological evaluation and structure-activity relationships of glycyrrhetinic acid derivatives as novel anti-hepatitis B virus agents[END_REF] and its derivatives (3-7). Thin-layer chromatography (TLC) was conducted on a pre-coated silica gel 60 F 254 plate (E.

MATERIALS AND METHODS

Materials

GA
Merck, Darmstadt, Germany) and eluted with a mixture of methanol/dichloromethane (15:1~10:1) for per-O-acetyl-β-CD-GA conjugates or methanol for the deacetylated β-CD-GA conjugates. For spot detection, the plates were immersed in a yellow solution of Ce(NH 4 ) 2 (NO 3 ) 6 (0.5 g)/(NH 4 ) 6 Mo 7 O 24 . 4H 2 O (24.0 g)/6% H 2 SO 4 (500 mL). The plate was heated by hot gun in order to visualize the spots. FT-IR spectra were obtained using a Nicolet Nexus 470 FTIR spectrometer (Thermo Electron Scientific Instruments LLC, USA). MALDI-TOF mass spectra were obtained on an AB Sciex TOF/TOF TM 72115 spectrometer using methanol or chloroform as solvents and α-cyano-4-hydroxy-cinnamic acid (HCCA) as matrix. NMR spectra were recorded on a Bruker DRX 400 or DRX 600 spectrometer at ambient temperature.

Madin-Darby canine kidney (MDCK) cells were donated by Crown Bioscience Inc. (USA).

Dulbecco's Modified Eagle Medium (DMEM) was purchased from Gibco BRL, Inc. (USA).

CellTiter-Glo ® reagent was purchased from Promega Corp Inc. (USA). Recombinant influenza HA was purchased from Sino Biological Inc. (China). Deionized double-distilled water was used throughout the biological study. Surface plasmon resonance (SPR) assay was analyzed using the Biacore 8k system (GE Healthcare, Uppsala, Sweden).

Synthesis of heptavalent β-CD-GA conjugates

Heptakis (2,3-di-O-acetyl-6-deoxy-6-azide-)-β-CD 11 was prepared in a three-step procedure as previously reported [START_REF] Gadelle | Selective halogenation at primary positions of cyclomaltooligosaccharides and a synthesis of per-3,6-anhydro cyclomaltooligosaccharides[END_REF]. The GA intermediate 12 was synthesized based on the method of Schwarz et al. with minor modifications [START_REF] Schwarz | Amino derivatives of glycyrrhetinic acid as potential inhibitors of cholinesterases[END_REF]. The terminal alkynyl substituted amines 13-16 were prepared as previously described [START_REF] Murelli | Chemical control over immune recognition: A class of antibody-recruiting small molecules that target prostate cancer[END_REF][START_REF] Zhang | A remote arene-binding site on prostate specific membrane antigen revealed by antibody-recruiting small molecules[END_REF][START_REF] Tran | A modular approach to triazole-containing chemical inducers of dimerisation for yeast three-hybrid screening[END_REF][START_REF] Sanhueza | Efficient liver targeting by polyvalent display of a compact ligand for the asialoglycoprotein receptor[END_REF]. Alkynyl-functionalized GA intermediates 17-20 and 30-32 were synthesized as previously described [START_REF] Liang | Synthesis and structure-activity relationship studies of water-soluble β-cyclodextrin-glycyrrhetinic acid conjugates as potential anti-influenza virus agents[END_REF].

Heptavalent GA functionalized β-CD conjugates were synthesized through a two-step process involving the copper-catalyzed alkyne-azide cycloaddition reaction (CuAAC) under microwaves (step 1) and de-O-acetylation reaction under Zemblén conditions (step 2).

General procedure A for the click reaction (step 1)

To a solution of heptakis (2,3-di-O-acetyl-6-deoxy-6-azide-)-β-CD 11 (189.8 mg, 0.10 mmol)

and alkynyl-functionalized GA derivatives (0.84 mmol) in 50% of THF and H 2 O (5 mL) was added CuSO 4 (15.7 mg, 0.10 mmol) and sodium ascorbate (30.7 mg, 0.15 mmol). The resulting solution was heated in a microwave reactor at 100 o C until the azide was completely consumed (typically about 1 h) as determined by TLC. Then the reaction mixture was extracted with CH 2 Cl 2 (10 mL × 3). The CH 2 Cl 2 fraction was dried over Na 2 SO 4 , filtered, evaporated, and purified by silica gel column chromatography using CH 2 Cl 2 /CH 3 OH (15:1~10:1 v/v) as eluent to give the acetylated intermediates as a white foam.

General procedure B for the deacetylation reaction (step 2)

The multivalent GA functionalized per-O-acetylated β-CD conjugates were dissolved in dry methanol (5 mL per 100 mg of compound), and a solution of sodium methoxide (30% in methanol, 0.1 eq per mol of acetate) was added. The solution was stirred (180 rpm) at room temperature for 4~6 h in a N 2 atmosphere. After completion (TLC), the reaction mixture was neutralized with Amberlite IR-120 (H + ) ion exchange resin, then filtered and concentrated. The crude product was purified by RP column chromatography using CH 3 OH as eluent to give the final products in 73-95% yields.

The 1 H and 13 C NMR and ESI-HRMS or MALDI-TOF MS data of the synthesized new compounds 21-28 and 33-38 are available in supporting information (Supplementary Data, S6-S14).

FT-IR analysis

FT-IR spectra of the heptavalent β-CD-GA conjugates as well as their parent compounds 11 and 17 were obtained using a Nicolet Nexus 470 FTIR spectrometer (Thermo Electron Scientific Instruments LLC, USA) over a scanning range 500-4,000 cm -1 . In addition, 1.0 mg of different samples was mixed with KBr pellets.

NMR study

1 H and 13 C NMR spectra of GA derivatives 17-20 and 29-32 were acquired on a Bruker DRX 400 MHz spectrometer. 1 H NMR, 13 C NMR, 1 H-1 H COSY NMR and 1 H-13 C HSQC NMR spectra of β-CD-GA conjugates 21-28 and 33-38 were acquired on a Bruker DRX 600 MHz spectrometer.

Sample solutions were prepared with CDCl 3 or CDCl 3 /CD 3 OD (2:1) as solvent. CDCl 3 or CD 3 OD were also employed as reference: proton (δ 7.26 ppm) and carbon (δ 77.00 ppm) for CDCl 3 , proton (δ 3.31 ppm) and carbon (δ 49.00 ppm) for CD 3 OD. Detection temperature was set at 25 °C.

Cell culture and viruses

MDCK cells were donated by Crown Bioscience Inc. (USA) and grown in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS) (PAA Laboratories, Linz Austria) at 37 °C under 5% CO 2 . The A/WSN/33 (H1N1) influenza virus used in this study was artificially generated from the 12-plasmid influenza virus rescue system.

Cytotoxicity assay

Cell viability assays were performed using CellTiter-Glo ® reagent following the manufacturer's instructions. Briefly, the cells (1×10 4 per well) were seeded into 96-well tissue culture plates and incubated for 16 h at 37 °C with 5 % CO 2 to allow the cells to adhere to the surface of the wells. The culture medium was then replaced with fresh medium containing compound or paclitaxel (PTX) at 10 and 25 μM in triplicate, and the negative control wells contained the equivalent volume of medium with 1% DMSO, after which they were incubated for 48 h at 37 °C with 5% CO 2 . CellTiter-Glo ® reagent was added, and the plates were read using a

Tecan Infinite M2000 PRO TM plate reader.

Cytopathic effect (CPE) reduction assay

MDCK cells (1 × 10 4 per well) were seeded into 96-well plates, incubated at 37 °C with 5 % CO 2 for 24 h and infected with influenza virus (MOI = 0.1). Cells were suspended in DMEM supplemented with 1% FBS, containing different concentrations test compound or Oseltamivir (OSV) and 2 μg/mL TPCK-treated trypsin, and a final DMSO concentration of 1% was added in each well. After 48 h of incubation at 37 °C with 5 % CO 2 , CellTiter-Glo ® reagent (Promega Corp., Madison, WI, USA) was added, and the cells were assessed with the CellTiter-Glo ® assay according to the manufacturer's instructions.

Hemagglutination inhibition (HI) assay

HI assay was performed as described previously [START_REF] Yu | Discovery of pentacyclic triterpenoids as potential entry inhibitors of influenza viruses[END_REF]. Briefly, compound from a 2-fold serial dilution in saline was mixed with an equal volume of influenza virus (2 HA units) in the V-bottomed 96-well microplates. Subsequently, 50 μL of freshly prepared chicken red blood cells (cRBC) (1% v/v in saline) was added to each well. The mixture was incubated at room temperature for 30 min before observing cRBC aggregation on the plate.

SPR assay

Interactions between influenza HA and the compounds were analyzed using the Biacore 8k system (GE Healthcare, Uppsala, Sweden) at 25 °C. Recombinant influenza HA (Sino Biological Inc., Beijing, China) was immobilized on a sensor chip (CM5) using EDC and NHS for activation.

Final HA immobilized levels were typically ~9,000 RU. Subsequently, compounds were injected as analytes at various concentrations, and PBS-T (10 mM phosphate buffer with 5% DMSO, 0.05% Tween, pH 7.4) was used as the running buffer. For binding studies, analytes were applied at corresponding concentrations in running buffer at a flow rate of 30 μL/min with a contact time of 60 s and a dissociation time of 180 s. Chip platforms were washed with running buffer and 50%

DMSO. Data were analyzed with the Biacore insight evaluation software (version 1.05) by curve fitting using a binding model of 1:1.

RESULTS AND DISCUSSION

Synthesis of heptavalent β-CD-GA conjugates

As shown in Scheme 1, the synthetic strategy was based on coupling of alkynyl-functionalized

Gas with heptaazide substituted β-CD via click chemistry. At first, oligo (ethylene glycols) (OEGs)

were chosen as linkers to enhance and optimize the properties of conjugates. Various heterobifunctional OEGs with amine and alkynyl terminals 13-16 were synthesized as described previously [START_REF] Murelli | Chemical control over immune recognition: A class of antibody-recruiting small molecules that target prostate cancer[END_REF][START_REF] Zhang | A remote arene-binding site on prostate specific membrane antigen revealed by antibody-recruiting small molecules[END_REF][START_REF] Tran | A modular approach to triazole-containing chemical inducers of dimerisation for yeast three-hybrid screening[END_REF][START_REF] Sanhueza | Efficient liver targeting by polyvalent display of a compact ligand for the asialoglycoprotein receptor[END_REF]. The coupling of OEGs 13-16 with compound 12, which obtained from commercially available GA 2 by activation of the carboxylic acid at C-30, was performed to provide the alkynyl-functionalized

GAs 17-20. Then 17-20 underwent a "click chemistry" reaction with heptakis

(2,3-di-O-acetyl-6-deoxy-6-azide)-β-CD 11, which was prepared from β-CD 8 in three steps using the conventional method as previously described [START_REF] Gadelle | Selective halogenation at primary positions of cyclomaltooligosaccharides and a synthesis of per-3,6-anhydro cyclomaltooligosaccharides[END_REF][START_REF] Ashton | Amino acid derivatives of β-cyclodextrin[END_REF][START_REF] Karginov | β-Cyclodextrin derivatives that inhibit anthrax lethal toxin[END_REF]

Structure characterization of heptavalent β-CD-GA conjugates

The structures and symmetrical substitution of the heptavalent β-CD-GA conjugates 21-28 as well as their precursors 33-38 were characterized by IR, NMR and MALDI-TOFMS spectroscopy.

FT-IR analysis

The FTIR spectra of the all synthesized heptavalent β-CD-GA conjugates were recorded over the range of 400-4,000 cm -1 in a Nicolet Nexus 470 FT-IR thermo-scientific spectrophotometer.

The spectrum of β-CD intermediate 11 contained the characteristic absorption band for the spectrum with OH stretching at 3,386 cm -1 , CH stretching around 2,924 cm -1 and CO stretching around 1,030 cm -1 (Supplementary data Fig. S1). For GA [START_REF] Wang | Synthesis, biological evaluation and structure-activity relationships of glycyrrhetinic acid derivatives as novel anti-hepatitis B virus agents[END_REF], several vibration characteristics of the triterpenic moiety were present. The characteristic band attributed to the stretching vibration of the OH group at C3 was found at 3,443 cm -1 . And the bands at 2,930 and 2,870 cm -1 were assigned to the stretching vibration of the aliphatic CH (Supplementary data Fig. S2). The spectra of these heptavalent β-CD-GA conjugates were very similar. In a typical example, Fig. 2 shows the IR spectra of the two intermediates 11, 17 and their conjugate 21. It was noteworthy that the strong absorption band located at 2,107cm -1 in Fig. 2A and the weak absorption band located at 2,120 cm -1 in Fig. 2B, which should be assigned to the stretching vibration of azide and terminal alkyne, respectively, were diminished in the spectrum of 21 (Fig. 2C). In addition, a few representative absorption bands ascribable to 1,2,3-triazole units were shown around 1,529, 1,455 and 1,047 cm -1 [START_REF] Rao | Contribution to infrared spectra of five-membered N-and N,S-heterocyclic compounds[END_REF]. Spectra were acquired between 500 and 4,000 cm -1 .

NMR and MALDI-TOF MS analysis

Except for those signals of the linkers, the heptavalent conjugates were similar in their NMR spectra, and Fig. 3 represents the HSQC spectrum of conjugate 23 as an example. The low-field regions of the conjugate were relatively well resolved and fully assigned. In the aromatic region, the signal at δ 7.76 ppm was assigned to triazole-CH according to the 1 H-13 C correlation spectra, indicating that it was indeed connected by triazole linker. The assignment of NH proton at δ 6.31 ppm was confirmed as there was no correlation for NH by HSQC spectral analysis.

In vitro cytotoxicity of heptavalent β-CD-GA conjugates

It has been reported that GA 2 and some of its derivatives are cytotoxic in cancer cells [START_REF] Satomi | Glycyrrhetinic acid and related compounds induce G1 arrest and apoptosis in human hepatocellular carcinoma HepG2[END_REF][START_REF] Lallemand | N-(2-{3-[3,5-Bis(trifluoromethyl)phenyl]ureido}ethyl)-glycyrrhetinamide (6b): A novel anticancer glycyrrhetinic acid derivative that targets the proteasome and displays anti-kinase activity[END_REF]. In this study, the cytotoxicity of heptavalent β-CD-GA conjugates 21-28 and 33-38 was first determined in MDCK cells using the CellTiter-Glo ® luminescent cell viability assay. Decrease in viability of MDCK cells was observed in a dose-dependent manner upon treatment of PTX (Supplementary data Fig. S3). Treatment of cells with 2 for 48 h resulted in decreased viability to 82.8% for the 25 μM concentration and no viability for the 10 μM concentration, indicating that 2 had weak cytotoxicity to cells and agreed with previously reported results that 2 shows weak cytotoxicity to eight cancer cell lines with IC 50 values between 70.48-136.40 μM [START_REF] Gao | Synthesis of novel heterocyclic ring-fused 18β-glycyrrhetinic acid derivatives with antitumor and antimetastatic activity[END_REF]. For all the tested conjugates, except that 25

and 28 showed weak cytotoxicity to uninfected MDCK cells with the reduced viability to 85.6% and 87.9%, respectively, at high concentration of 25 μM, no significant cytotoxicity for the other conjugates was observed at concentrations of 10 μM and 25 μM.

Structure-activity relationships (SAR) of heptavalent β-CD-GA conjugates

Concerning the fact that the use of multivalent sialopeptides as potential inhibitors of influenza infections has been proved to be a useful approach [START_REF] Mammen | Polyvalent interactions in biological systems: Implications for design and use of multivalent ligands and inhibitors[END_REF][START_REF] Marra | Synthesis of sialoclusters appended to calix[END_REF], our interest was aimed at evaluating the inhibitory effect of the heptavalent GA conjugates 21-28 and 33-38 by microscopic examination of the viral CPE at 2 days post infection [START_REF] Noah | A cell-based luminescence assay is effective for high-throughput screening of potential influenza antivirals[END_REF]. Oseltamivir (OSV) and DMSO were used as positive and negative controls, respectively.

The primarily screening results are shown in Fig. 4. A careful analysis and comparison of the data allowed us to reveal some interesting SAR trends. Regarding the OEG linkers between the triazole ring substituted GA and β-CD scaffold, we found that the anti-influenza activity of conjugates 21-24 was decreased in the order as follows: 21>22>>23>24. Similar results were also observed for conjugates 25-28, indicating that a length of four atoms was found to be optimal for the inhibitory activity (21 and 25). Especially for conjugate 28, the inserting of two more ethylene glycols spacer in 26 resulted in the loss of activity under 25 μM, suggesting the importance of the length of the linker between GA and β-CD for their anti-influenza activity. Concerning the nature of the linker's heteroatom, the replacement of the oxygen of ethylene glycol in conjugates 21 and 25 by methylene (33 and 34) showed slightly weaker or similar antiviral activity. Interesting, the increasing of the rigidity of the linker by further replacement of the four methylene with aromatic benzene at positions at 1,4 (35 and 36) still retained reasonable anti-influenza activity.

Surprisingly, the inserting of a piperazine space in the alkyl liner caused a substantial increase in activity of conjugates 37 and 38 (relative to 33 and 34, respectively), which were proved to be the most two potent multivalent conjugates. 2. In addition, no significant toxicity was observed for the 10 conjugates at concentrations up to 100 μM. Based on these data, we investigated in detail the potential of a representative heptavalent conjugate 37. The CPE produced by influenza A/WSN/33 virus in MDCK cells and its abrogation by treatment with 37 were further confirmed by direct observation under a microscope. No cytotoxicity was observed for 37 when incubated with the MDCK cells for 48 h (Supplementary data Fig. S4A vs. Fig. S4B), while 37 significantly reduced the CPE induced by influenza A/WSN/33 virus infection (Supplementary Fig. S4C vs. S4D).

HI assay

With the confirmation of the antiviral activity of the heptavalent conjugates, we were interested in identifying the molecular target of them. It is well known that the viral envelop glycoprotein HA plays a critical role in the initial step of influenza virus entry into host cells.

Therefore, HI assay was designed to detect whether the heptavalent conjugates could interfere with the interaction between HA and its sialic acid receptors. Two fold dilutions of the heptavalent conjugate 37 (from 10 μM) or anti-HA antibody (from 0.50 ng/mL) treated virus were incubated with cRBC, and agglutination was observed (Fig. 5). In addition, 37 completely inhibited influenza A/WSN/33 (H1N1) virus induced agglutination of cRBC in a dose-dependent manner at concentrations of 1.3 μM or more. Similar results were observed for anti-HA antibody at concentrations of 0.063 ng/mL or more. These results suggested that the heptavalent conjugates 37 shared the same target of anti-HA antibody, thus interfering with virus-receptor interaction.

Determination of the K D for the interaction between the heptavalent conjugate 37 with HA protein

To determine the binding affinity of conjugate 37 for the HA protein, SPR experiments were performed on a Biacore 8K instrument. The SPR response was reported in the resonance unites (RU). Sensor grams were analyzed with a 1:1 (Langmuir) binding mode. As shown in Fig. 6, the binding of recombinant influenza HA to GA (2) exhibited typical weak affinity binding with a calculated equilibrium dissociation constant (K D ) value of 2.45 × 10 -4 M, while β-CD (8) did not bind to HA protein at the same concentration. However, the multivalent conjugate 37 showed strong binding affinity to HA protein within the concentration range 0.16-5.00 μM. The calculated K D value for conjugate 37 was 5.15 × 10 -7 M, which seemed to be too large by about 470-fold of magnitude to that of 2 (Fig. 6A vs. Fig. 6C). In addition, kinetic analysis revealed that changes in multivalent conjugate and HA protein not only affected the association rate constant (K a ), but also affected the dissociation rate constant (K d ) (Supplementary data Table S1). Compared with our previous study in which multiple pentacyclic triterpenes attached directly on CD scaffold via 1,2,3-triazolyl group [START_REF] Xiao | Pentacyclic triterpenes grafted on CD cores to interfere with influenza virus entry: A dramatic multivalent effect[END_REF], the insertion of a suitable linker between them will helpful for the binding of multiple pentacyclic triterpenes with their target. To test the feasibility of the strategy, various types of linkers including flexible linear alkyl/ether chains combinations between 4 and 19 atoms, rigid aromatic space chain as well as semi-rigid charged piperzine chain have been successfully introduced in monovalent β-CD-GA conjugates in our recently study [START_REF] Liang | Synthesis and structure-activity relationship studies of water-soluble β-cyclodextrin-glycyrrhetinic acid conjugates as potential anti-influenza virus agents[END_REF], which allowed us to further design and synthesis of the second-generation multivalent pentacyclic triterpene-β-CD conjugates. This well-established method will provide an alternative approach to the construction of novel multivalent derivatives bearing different linkers based on natural or chemically modified CDs scaffolds. Combined with our previous studies, we found that the pentacyclic triterpenes pharmacophore, the CD scaffold and the linkers between the two parts were all affected their anti-influenza activity.

CONCLUSIONS

In this work, a series of multiple GA functionalized β-CD conjugates with different linkers were synthesized and characterized. Except that two conjugates (25 and 28) showed weak cytotoxicity to MDCK cells at concentration of 25 μM, all other conjugates showed no significant cytotoxicity based on an alamarBlue assay. When presented multivalently, seven β-CD-GA conjugates (21, 22, 25, 26, 34, 37 and 38) showed strong anti-influenza properties with IC 50 values at a micromolar level (IC 50 : 2.86-9.03 μM). By using of HI and SPR assays, conjugate 37 showed specific binding to HA protein with K D value at 515 nM, which was about 470-fold potent to its parent compound GA [START_REF] Wang | Synthesis, biological evaluation and structure-activity relationships of glycyrrhetinic acid derivatives as novel anti-hepatitis B virus agents[END_REF]. Taken together, the results reported here for multivalent β-CD-GA conjugates demonstrated that multivalent displays of anti-influenza entry agent afford macromolecules with a remarkable activity and a strong binding to HA protein relative to their parent compound alone and multivalency based on β-CD scaffold is a promising alternative to the current available treatments for the management of viral infection by blocking virus entry into host cells.

Figure 2 .

 2 Figure 2. FTIR spectra of compounds 11 (A, black), 17 (B, red) and their conjugate 21 (C, blue).

Figure 3 .

 3 Figure 3. The 600 MHz HSQC spectrum of 23 recorded in CDCl 3 at 298 K, with the 1D 1 H and 13 C NMR spectra along the top and the side, respectively.

Figure 4 .

 4 Figure 4. The CPE-based primarily screening of GA (2), β-CD (8) and their heptavalent β-CD-GA conjugates 21-28 and 33-38. MDCK cells were utilized as the host cells to test A/WSN/33 virus infection; OSV and DMSO acted as positive and negative control, respectively. Error bars indicate standard deviations of triplicate experiments. After the preliminary screening at two concentrations of 10 μM and 25 μM, 11 conjugates (21-22, 25-27 and 33-38) with the inhibition rate over 50% at concentration of 25 μM were selected to perform in dose response assays, and the concentrations required to inhibit viral replication by 50% (IC 50 ) are summarized in Table2. In addition, no significant toxicity was

Figure 5 .

 5 Figure 5. Effect of the heptavalent compound 37 (A) and anti-HA antibody (B) on HA by HI assay. The chicken red blood cells (cRBC) (1% v/v in saline) were incubated with influenza A/WSN/33 at 2 × HA with various concentrations of 37 (two-fold serial dilution from 10 μM) or anti-HA antibody (two-fold serial dilution from 0.50 ng/mL).

Figure 6 .

 6 Figure 6. SPR inhibition assay sensor grams showing response to influenza HA protein in the presence of varying concentration of GA (2), β-CD (8) and their heptavalent conjugates 37. The binding kinetics was analyzed by BIA evaluation 1.0.5 software with 1:1 (Langmuir) binding mode.

  . In a notable example,

	Lallemand	et	al.	have	reported	that	compound	3,	with	a
	2-(3-(3,5-bis(trifluoromethyl)phenyl)ureido)ethyl group at C-30 of 2, shows strong antitumor
	activity with IC 50 in signal-digit micromolarity in a panel of eight cancer cell lines

  The low-field peak at δ 5.64 ppm, referring to 1H, was assigned to H 12 , and one carbon appearing at δ 128.35 ppm should be assigned to C 12 of GA. With a C 7 -symmetry in the molecule, compound 23 showed only one set of CD-H 1 signal appearing at 5.49 ppm and one CD-C 1 signal appearing at 96.29 ppm.Similar observation was also made for other proton and carbon signals of β-CD unit, and the chemical shift data are summarized in Table1. As C b was connected with nitrogen rather than

Table 1 .

 1 Chemical shifts of 1 H and 13 C NMR of β-CD unit in conjugate 23.

	1 H NMR (ppm)	13 C NMR (ppm)
	CD-H 1	5.64	CD-C 1	96.29
	CD-H 2	4.73	CD-C 2	69.81
	CD-H 3	5.34	CD-C 3	70.67
	CD-H 4	3.53	CD-C 4	76.51

Table 2 .

 2 In vitro anti-influenza A virus activity of heptavalent conjugates 21

-22, 25-27 and 33-38 a

  Using influenza A/WSN/33 (H1N1) strain. b The concentration required for a test compound to reduce the virus-induced CPE by 50% relative to the virus control was expressed as IC 50 .

	27	27.02±0.11	>100	>3.7
	Compd	IC 50 , μM b	CC 50 , μM	SI	Compd	IC 50 , μM	CC 50 , μM	SI
	2	NA c	85±6.8	-	33	12.1±0.31	>100	>8.3
	8	NA	ND d	-	34	9.03±0.50	>100	>11.1
	21	6.64±0.31	>100	>15.1	35	20.7±1.02	>100	>4.8
	22	7.70±0.74	>100	>13.0	36	11.0±0.87	>100	>9.1
	25	6.96±0.39	>100	>14.4	37	2.86±0.22	>100	>35.0
	26	8.14±0.11	>100	>12.3	38	4.89±0.15	>100	>20.4

a c No activity. d Not detectable.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Lijun Zhong from the Centre of Medical and Health Analysis (PUHSC) for MALDI-TOF experiments. The authors would also like to thank Dr. Fen Liu and Dr Jing Wang from State Key Laboratory of Natural and Biomimetic Drugs (PUHSC) for their help in conducting the NMR and SPR experiments, respectively.

FUNDING

This work was supported by the International Cooperation and Exchange Program (NSFC-RFBR, No. 82161148006), the National Natural Science Foundation of China (Nos. 21877007 and 81821004) and Shenzhen Bay Laboratory Start-up Fundation (21230071).

DATA AVAILABILITY STATEMENT

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS

SL and XM: design, synthesis and characterization of the multivalent conjugates; ML and YC: contribution in anti-influenza viral assay, HI assay,cytotoxicity assay; QG: SPR assay; YZ: discussion of all the experiments and assist in writing the manuscript; and LZ, DZ and SX: designed the synthesis and biochemical experiments, supervised the project, and wrote the manuscript.


Amandine Caudal

Daphné Guenoun

Guillaume Lefebvre

Jean-Franc ¸ Ois Nisolle

Gabriel Gorcos

Valérie Vuillemin

Bruno Vande Berg
email: bruno.vandeberg@uclouvain.be

Medial meniscal ossicles: Associated knee MRI findings in a multicenter case-control study

Keywords: Knee joint Magnetic resonance imaging (MRI) Menisci, tibial Meniscal ossicle Anterior cruciate ligament ACL, Anterior cruciate ligament, ETL, Echo-train Length, FOV, Field of View, LCL, Lateral Collateral ligament, MCL, Medical Collateral ligament, MRI, Magnetic Resonance Imaging, PCL, Posterior Cruciate ligament, SE, Spin-Echo, TR, Repetition Time, TE, Echo-Time

Purpose: The purpose of this study was to assess and compare the prevalence of meniscal, ligament and cartilage lesions on knee MRI in a series of age-and sex-matched patients with and without medial meniscal ossicle. Materials and methods: Forty-two knee MRI examinations obtained in 42 patients (36 men, 6 women; mean age, 42.5 ± 22.2 [SD] years; range: 19-65 years) on which a medial meniscal ossicle was present were compared to 42 knee MRI examinations obtained in 42 age-and sex-matched patients (36 men, 6 women; mean age, 41.8 ± 20.6 [SD] years; range: 19-65 years) on which no medial meniscal ossicles were present. Two radiologists (R1, R2) blinded to the presence of meniscal ossicle by reading only the fat-saturated intermediate-weighted MR images separately assessed the presence of meniscal, ligament and cartilage lesions on these 84 knee MRI examinations. Prevalence of meniscal and ligament lesions and degree of cartilage degradation at MRI were compared between knees with and those without medial meniscal ossicle. Results: In knees with medial meniscal ossicle, R1 and R2 detected 33 (79%) and 38 (90%) medial meniscal lesions, respectively that involved the posterior root (n = 25/32 for R1/R2), the posterior horn (n = 19/14 for R1/R2) or the body (n = 8/10 for R1/R2). The prevalence of posterior root tear (60% [25/42]/76% [32/42] for R1/R2) and that of anterior cruciate ligament (ACL) lesions (48% [20/42]/57% [24/42] for R1/R2) as well as the medial cartilage degradation score (3.35 ± 0.87 [SD] for R1 and 3.92 ± 0.78 [SD] for R2) were significantly greater in knees with than in knees without medial meniscal ossicle (root lesions: P < 0.01 for both readers; ACL lesions and medial cartilage score: P < 0.01 for both readers). Conclusion: On MRI examination, knees with a medial meniscal ossicle demonstrate a greater frequency of medial posterior root tear and of ACL lesions and a greater degree of medial femoro-tibial cartilage degradation by comparison with knees without medial ossicle.

Introduction

A meniscal ossicle is defined by the presence of an ossification within the substance of the meniscus. Initially described as a meniscal variant [START_REF] Harris | Calcification and ossification in the semilunar cartilages[END_REF][START_REF] Jones | Calcification, decalcification, and ossification[END_REF][START_REF] Burrows | Two cases of ossification in the internal semilunar cartilage[END_REF], case reports suggested that some meniscal ossicles were associated with posttraumatic meniscal lesions [START_REF] Yoo | Meniscal ossicle: a case report[END_REF] or from an avulsion of the posterior root of the medial meniscus from the tibial spine [START_REF] Berg | The meniscal ossicle: the consequence of a meniscal avulsion[END_REF]. Meniscal ossicle can be detected on radiography and on magnetic resonance imaging (MRI) [START_REF] Yu | Meniscal ossicle: MR imaging appearance in three patients[END_REF][START_REF] Yao | Magnetic resonance imaging of a symptomatic meniscal ossicle[END_REF][START_REF] Tuite | MR imaging of a meniscal ossicle[END_REF][START_REF] Schnarkowski | Meniscal ossicle: radiographic and MR imaging findings[END_REF][START_REF] Martinoli | Multimodality imaging assessment of meniscal ossicle[END_REF]. Two previous non-controlled studies including a large number of MRI examinations of knees with medial meniscal ossicle demonstrated that medial meniscal ossicle was associated with posterior horn and posterior root tears of the medial meniscus [START_REF] Mohankumar | Meniscal ossicle: posttraumatic origin and association with posterior meniscal root tears[END_REF][START_REF] Bernard | Understanding the implications of the meniscal ossicle: patient presentation, treatment and outcomes[END_REF] and with anterior cruciate ligament (ACL) tears [START_REF] Mohankumar | Meniscal ossicle: posttraumatic origin and association with posterior meniscal root tears[END_REF]. The importance in detecting meniscal ossicle is twofold. First, a meniscal ossicle should not be confused with an articular loose body as it is embedded in the meniscal substance and is less likely to produce articular impingement than a free loose body [START_REF] Gursoy | The distribution of loose bodies determined on knee magnetic resonance imaging: joint compartments, recesses and bursae including arthroscopic blind spots[END_REF]. Second, presence of a meniscal ossicle most likely indicates a chronic meniscal lesion that is less likely to benefit from surgical repair than an acute meniscal avulsion [START_REF] Palisch | Posterior root meniscal tears: preoperative, intraoperative, and postoperative imaging for transtibial pullout repair[END_REF].

In the two large non-controlled studies, knee MRI examinations were read in consensus by radiologists who were aware of the presence of a meniscal ossicle [START_REF] Mohankumar | Meniscal ossicle: posttraumatic origin and association with posterior meniscal root tears[END_REF][START_REF] Bernard | Understanding the implications of the meniscal ossicle: patient presentation, treatment and outcomes[END_REF]. Therefore, we performed a study in which two separate radiologists blinded to the presence of the meniscal ossicle were asked to analyze a series of knee MRI examinations obtained in age-and sex-matched patients with and without medial meniscal ossicle.

The purpose of this study was to assess and compare the prevalence of meniscal, ligament and cartilage lesions at knee MRI in a series of age-and sex-matched patients with and without medial meniscal ossicle.

Materials and methods

Study population, definitions and data selection

The institutional review board of the organizing center (UCL Saint-Luc) approved this retrospective multicenter study and waived informed consent of the patients. During the 2017 Spring meeting of the Société d'Imagerie Musculo-Squelettique, 15 radiologists working in different imaging centers agreed to collect radiographic and MRI examinations of knees with a medial meniscal ossicle (study group) that had been obtained between January 2005 and December 2017 by using electronic research using key-words including "os meniscal" and "ossicule" (French words used for meniscal ossicle) (Fig. 1a). They also agreed upon collecting radiographs and MRI examinations of knees from age-and sex-matched patients without meniscal ossicle (control group) that had been obtained during the same year as their study group knees (Fig. 1a). All eligible study and control group patients had knee symptoms to justify MRI examinations.

A meniscal ossicle was defined either by the presence of a welldelineated intrameniscal high signal intensity area on T1-weighted spin-echo (SE) MR images [START_REF] Yu | Meniscal ossicle: MR imaging appearance in three patients[END_REF][START_REF] Tuite | MR imaging of a meniscal ossicle[END_REF][START_REF] Schnarkowski | Meniscal ossicle: radiographic and MR imaging findings[END_REF][START_REF] Martinoli | Multimodality imaging assessment of meniscal ossicle[END_REF][START_REF] Mohankumar | Meniscal ossicle: posttraumatic origin and association with posterior meniscal root tears[END_REF][START_REF] Bernard | Understanding the implications of the meniscal ossicle: patient presentation, treatment and outcomes[END_REF] or by the presence of a typical well-delineated ossification projecting in the medial femoro-tibial joint on radiographs [START_REF] Schnarkowski | Meniscal ossicle: radiographic and MR imaging findings[END_REF][START_REF] Martinoli | Multimodality imaging assessment of meniscal ossicle[END_REF]. Study group inclusion criteria were: (i), performance of MRI for internal derangement of the knee; (ii), age above 17 years; and (iii), presence of a meniscal ossicle at knee MRI and/or on available radiographs obtained within two months of the MRI examination. Exclusion criteria for the study group included: (i), lack of MRI examination; (ii), lack of non-fatsaturated T1-weighted SE sequence; and (iii), lack of fat-saturated intermediate-weighted MRI sequences. The lack of radiographs was not an exclusion criterion from the study group. Control group inclusion criteria were: (i), performance of knee radiographs and MRI examination for internal knee derangement within two-month interval; (ii), same age and gender as that of a patient of a study group; and (iii), lack of meniscal ossicle on knee radiographs and MRI examinations. The lack of radiographs was an exclusion criterion from the control group. Fourteen imaging centers provided 46 knees MRI examinations with possible medial meniscal ossicle with more than five MRI examinations in three imaging centers and 36 age-and sex-matched control knee MRI examinations. Radiographic and MRI examinations of the 10 missing control group MRI examinations were provided by a recruiting center that failed to find any study group knee.

Two radiologists with five (A.C.) and nine years (D.G.) of experience analyzed in consensus the radiographs and the T1-weighted MRI images of the study and the control group knees to confirm the presence or absence of a meniscal ossicle based on the abovementioned definitions (Fig. 1b). Four study group knees and the four matched patients were excluded. years; age range: 19-65 years). They noted the location of the ossicle and its signal intensity that was considered to be either high or low, equivalent to or lower than that of adjacent marrow fat, respectively. MRI examinations were obtained on 1.5T (57/84; 68%) or on 3.0T MR units (27/84; 32%) by using dedicated knee coils in all imaging centers. Different MRI protocols were used due to variability among centers and time span of the study. All MRI examinations included at least one T1-weighted SE sequence obtained in the coronal (27/84; 32%) or sagittal (57/87; 68%) plane with the following parameters: repetition time (TR), 422-700 msec; echo-time (TE), 9-19 msec; echo-train length (ETL), 1-4; and flip angle, 76-150 • . All MRI examinations included at least one fat-saturated intermediate-weighted sequence obtained in the coronal (n = 84), sagittal (n = 78) or transverse (n = 84) plane that were acquired in a two-dimensional (2D) acquisition mode or reformatted from a three-dimensional (3D) sequence. 2D MR images were obtained with the following parameters: TR, 1596-5440 msec; TE, 8-85 msec; ETL, 5-15; and flip angle, 90-130 • . 3D MR images were obtained with the following parameters: TR, 800-1600 msec; TE, 37-44 msec; ETL, 32-48; and flip angle was 90 • . All 2D images were obtained with a slice thickness of 2.5-4 mm and a field of view (FOV) between 14 and 20 cm. Reformatted images were obtained with a slice thickness of 2.0-4.0 mm and an FOV between 14 and 20 cm. Sixty (18 from study and 42 from control group) sets of antero-posterior and lateral knee radiographs were available. A data manager anonymized all fatsaturated intermediate-weighted sequences of the 84 knees and archived them on a picture and archiving communication system.

Image analysis

Two other radiologists with five (G.L., reader 1) and 27 (J.-F.N., reader 2) years of experience separately analyzed the available fat-saturated intermediate-weighted sequences of the 84 knees to detect meniscal, ligament and cartilage lesions by using standard diagnostic criteria on their standard 1 or 2 mega pixel workstations by connecting onto the central picture archiving and communication system. They were blinded to the clinical and radiographic data as well as to the T1-weighted sequences. They did not assess the presence or absence of a meniscal ossicle. A meniscal lesion was identified if there was increased signal intensity within the meniscus abutting an articular surface on two or more contiguous slices or associated change in morphology of the meniscus [START_REF] Nguyen | MR imaging-based diagnosis and classification of meniscal tears[END_REF]. The meniscal tear was classified as simple or complex if there was only one or more than one plane of exit of the abnormal signal at its articular surface. The predominant meniscal lesion was located in the posterior meniscal root, posterior horn, body and anterior horn according to standard anatomic features. The posterior root of the medial meniscus is the most lateral aspect of that meniscus that inserts along the downward slope of the intercondylar fossa, anterior to the posterior cruciate ligament [START_REF] Nguyen | MR imaging-based diagnosis and classification of meniscal tears[END_REF][START_REF] Lee | Radial tear of the medial meniscal root: reliability and accuracy of MRI for diagnosis[END_REF]. Peripheral meniscal extrusion is defined by medial translation of the meniscal body [START_REF] Nguyen | MR imaging-based diagnosis and classification of meniscal tears[END_REF][START_REF] Liu | Threshold for lateral meniscal body extrusion on MRI in middleaged and elderly patients with symptomatic knee osteoarthritis[END_REF] and can be assessed by measuring the distance between the capsular margin of the body of the medial meniscus and the adjacent tibial plateau margin on coronal images [START_REF] Nguyen | MR imaging-based diagnosis and classification of meniscal tears[END_REF][START_REF] Liu | Threshold for lateral meniscal body extrusion on MRI in middleaged and elderly patients with symptomatic knee osteoarthritis[END_REF]. Meniscal extrusion was defined by a measured distance equal or superior to 3 mm [START_REF] Nguyen | MR imaging-based diagnosis and classification of meniscal tears[END_REF][START_REF] Liu | Threshold for lateral meniscal body extrusion on MRI in middleaged and elderly patients with symptomatic knee osteoarthritis[END_REF]. Presence or absence of lesion of anterior cruciate ligament (ACL), medial collateral ligament (MCL), lateral collateral ligament (LCL) and posterior cruciate ligament (PCL) were assessed according to standard lesion definition without grading of the lesions.

Any change of the ligaments including partial or complete tear and mucoid degeneration were considered to be abnormal. Modified Outerbridge's criteria were used to grade the articular cartilage in the femoral condyles and tibial plateaus [START_REF] Curl | Cartilage injuries: a review of 31,516 knee arthroscopies[END_REF]. Cartilage of the four articular surfaces was scored as follows: 0: normal cartilage; 1: less than 50% thickness loss; 2: more than 50% and less than 100% thickness loss; 3: down-to-bone thickness loss. Cartilage score of the medial and lateral femoro-tibial compartments were calculated by adding the scores of the corresponding femoral and tibial surfaces. Medial and lateral cartilage scores ranged from zero to six depending on the value of each of the two articular surfaces.

Statistical analysis

Statistical analysis was performed using dedicated software (MedCalc, version, Mariakerke, Belgium). Descriptive statistics were performed for the assessed variables in the study and control groups and compared by using Student t-test. Comparison of proportion of associated meniscal lesions were performed by using Chi square test. P-values < 0.05 were considered to indicate significant difference. Frequency analysis was performed on the location of a meniscal tear, in particular the posterior horn and root tears of the meniscus, and its relationship to the meniscal ossicle. Interobserver agreement for the detection of meniscal and ligament lesions and for the scoring of cartilage by R1 and R2 in the series of 84 knee MRI examinations was determined by using Kappa statistics [START_REF] Benchoufi | Interobserver agreement issues in radiology[END_REF]. Strength of agreement was interpreted according to the Altman's scale as follows: kappa < 0.20 poor; 0.21 < kappa < 0.40, fair; 0.41 < kappa < 0.60; moderate; 0.61 < kappa < 0.80, good; kappa > 0.81 very good. Kappa values were calculated along with their 95% confidence intervals (CI).

Results

Medial meniscal ossicles and associated meniscal lesions

The 42 medial meniscal ossicles were located at the posterior root (n = 34), the posterior horn (n = 7) or anterior horn (n = 1) of the medial meniscus. On T1-weighted SE images, the signal intensity of the meniscal ossicle was equivalent to that of marrow fat in 38 ossicles (Figs. 2 and3) and lower than that of marrow fat in four ossicles (Fig. 4). Among the 42 knees with a medial meniscal ossicle, R1 and R2 detected 34/42 (81%) and 38/42 (90%) medial meniscal lesions respectively that involved the posterior root (n = 25 for R1/32 for R2), the posterior horn (n = 19 for R1/n = 14 for R2) or the body (n = 8 for R1/n = 10 for R2) (Tables 123) (Figs. 234). There were no differences in frequency of root, posterior horn and body tears between meniscus with either root or posterior horn ossicle for each reader. R1 and R2 detected medial meniscus extrusion in 12 (28%) and in 15 (36%) (80%) study group knees respectively and in 7 (17%) and in 8 (19%) control group knees, respectively (Table 1). MRI findings in the control group knees are given in Tables 1 and2. Interobserver agreement was very good for the detection of lesions of the body of the medial meniscus (kappa = 0.856; 95% CI: 0.734-0.977) and was good for the detection of any medial meniscal lesions (kappa = 0.731; 95% CI: 0.580-0.881), of lesions of the posterior root (kappa = 0.730; 95% CI: 0.583-0.877) and of lesions of the posterior horn (kappa = 0.673; 95% CI: 0.511-0.836) of the medial meniscus.

Other MRI and clinical findings in study and control group knees

R1 and R2 detected lateral meniscal tears in 7/42 (17%) and 14/42 (33%) study group knees and in 5/42 (12%) and 7/42 (17%) control group knees, respectively (Table 1). They detected ACL Qualitative variables are expressed as proportions; numbers in parentheses are percentages. Post: posterior. The number of meniscal tears is higher than that of the meniscal ossicles because several segment of the meniscus can be torn. There was no difference in frequency of root, posterior horn and body tears between meniscus with either root or posterior horn ossicle for each reader. The ossicle involving the anterior horn is not included in this table.

lesions in 20/42 (48%) and 24/42 (57%) knees with meniscal ossicle (Table 1). Frequencies of other ligament lesions and medial and lateral cartilage scores for both readers are given in Table 1. The frequency of partial medial meniscectomy in the study group (4/42; 10%) was not different from that in the control group (1/42; 2%) (P = 0.12). The frequency of ACL plasty in the study group (11/42; 26%) was not different from that in the control group (4/42; 14%) (P = 0.17).

Comparison of MRI findings between knees with or without medial meniscal ossicle

The frequency of tear of the posterior root but not of that of the posterior horn or the body of the medial meniscus was statistically significantly higher in the study than in the control group knees (P < 0.01 for R1 and R2) (Table 2). The frequency of medial meniscus extrusion was not different between the study group and control group (Table 1). The frequency of ACL lesions but not those of the other assessed ligaments was greater in the study than in the control group (P < 0.01 for R1 and R2) (Table 1). The cartilage score of the medial femoro-tibial joint was greater in the study group than in the control group for both readers (Table 1). The cartilage score of the lateral femoro-tibial joint was greater in the study group than in the control group for one reader (Table 1).

Discussion

Ossicle within the knee meniscus is a rare finding at imaging, that has been studied in multiple case reports and two large series (Table 4) [START_REF] Yoo | Meniscal ossicle: a case report[END_REF][START_REF] Berg | The meniscal ossicle: the consequence of a meniscal avulsion[END_REF][START_REF] Yu | Meniscal ossicle: MR imaging appearance in three patients[END_REF][START_REF] Yao | Magnetic resonance imaging of a symptomatic meniscal ossicle[END_REF][START_REF] Tuite | MR imaging of a meniscal ossicle[END_REF][START_REF] Schnarkowski | Meniscal ossicle: radiographic and MR imaging findings[END_REF][START_REF] Martinoli | Multimodality imaging assessment of meniscal ossicle[END_REF][START_REF] Mohankumar | Meniscal ossicle: posttraumatic origin and association with posterior meniscal root tears[END_REF][START_REF] Bernard | Understanding the implications of the meniscal ossicle: patient presentation, treatment and outcomes[END_REF]. The current study confirmed the greater prevalence of medial posterior root tears and of ACL lesions in knees with medial meniscal ossicle than in knees without medial meniscal ossicle. This result, in line with those of previous studies, would suggest that a traumatic origin either due to a single trauma or multiple micro-traumatic events is likely to participate in the ossicle development. In the current series, however, posterior root tear was less prevalent (60% for R1 and 76% for R2) than in the study of Bernard et al. (97.5%) suggesting that non-traumatic origin could still be possible (Table 4) [START_REF] Bernard | Understanding the implications of the meniscal ossicle: patient presentation, treatment and outcomes[END_REF]. The male predominance in our study population, which contrasts to the 1:1 sex ratio in the other two large studies (Table 4), is in favor of the traumatic hypothesis as knee injuries are more frequent in men [START_REF] Thein | The prevalence of cruciate ligament and meniscus knee injury in young adults and associations with gender, body mass index, and height: a large crosssectional study[END_REF] and knee osteoarthritis predominates in women [START_REF] Cross | The global burden of hip and knee osteoarthritis: estimates from the global burden of disease 2010 study[END_REF].

In our study, we found no differences between the study group and control group in prevalence of posterior horn tear and meniscal extrusion. The former observation is in line with the rarity of the ossicle in comparison with the high frequency of posterior horn lesion. Most likely, other mechanisms than simple meniscal degeneration are needed to favor ossicle development. The latter observation could be related to the fact that meniscal extrusion is a non-specific finding associated with several meniscal, ligament and cartilage changes [START_REF] Lee | Radial tear of the medial meniscal root: reliability and accuracy of MRI for diagnosis[END_REF][START_REF] Liu | Threshold for lateral meniscal body extrusion on MRI in middleaged and elderly patients with symptomatic knee osteoarthritis[END_REF][START_REF] Lerer | The role of meniscal root pathology and radial meniscal tear in medial meniscal extrusion[END_REF] that are frequently observed in an age-and sex-matched series without menical ossicle.

In our study, we observed more advanced medial cartilage loss in the study group than in the control group knees. Although it has been suggested that the ossicle itself could generate cartilage damage in the adjacent articular surface [START_REF] Yao | Magnetic resonance imaging of a symptomatic meniscal ossicle[END_REF][START_REF] Tuite | MR imaging of a meniscal ossicle[END_REF], the high prevalence in the study group of medial meniscal lesions and of ACL lesions prevents to draw any definitive conclusions regarding the direct causative role of the ossicle. Furthermore, medial posterior root tear is an important meniscal lesion that greatly affects knee biomechanics, predisposing the compartment to articular cartilage degeneration, subchondral insufficiency fractures and osteonecrosis [START_REF] Gursoy | The distribution of loose bodies determined on knee magnetic resonance imaging: joint compartments, recesses and bursae including arthroscopic blind spots[END_REF][START_REF] Nguyen | MR imaging-based diagnosis and classification of meniscal tears[END_REF][START_REF] Lee | Radial tear of the medial meniscal root: reliability and accuracy of MRI for diagnosis[END_REF].

Finally, the low signal intensity on fat-and fluid-sensitive sequences of four meniscal ossicles in our series has not been previously reported as meniscal ossicles consistently demonstrated fat-like signal intensity on MRI [START_REF] Yoo | Meniscal ossicle: a case report[END_REF][START_REF] Berg | The meniscal ossicle: the consequence of a meniscal avulsion[END_REF][START_REF] Yu | Meniscal ossicle: MR imaging appearance in three patients[END_REF][START_REF] Yao | Magnetic resonance imaging of a symptomatic meniscal ossicle[END_REF][START_REF] Tuite | MR imaging of a meniscal ossicle[END_REF][START_REF] Schnarkowski | Meniscal ossicle: radiographic and MR imaging findings[END_REF][START_REF] Martinoli | Multimodality imaging assessment of meniscal ossicle[END_REF][START_REF] Mohankumar | Meniscal ossicle: posttraumatic origin and association with posterior meniscal root tears[END_REF][START_REF] Bernard | Understanding the implications of the meniscal ossicle: patient presentation, treatment and outcomes[END_REF]. In the absence of histological specimen, it is not possible to determine whether this signal pattern reflects a high degree of mineralization or the presence of tissue associated with osseous metaplasia that does not contain fat until maturity has been reached [START_REF] Foley | Histopathology of periarticular nonhereditary heterotopic ossification[END_REF].

The current study has several limitations in addition to its retrospective design and to the identification of eligible patients by using keywords as in the two previous large studies [START_REF] Mohankumar | Meniscal ossicle: posttraumatic origin and association with posterior meniscal root tears[END_REF][START_REF] Bernard | Understanding the implications of the meniscal ossicle: patient presentation, treatment and outcomes[END_REF]. First, blinding of the readers to the presence or absence of meniscal ossicle was achieved by disabling them to analyze the radiographs, the T1-weighted and the non-fat-saturated fluid-sensitive sequences. We cannot exclude that they could have seen some intrameniscal signal changes suggestive of a meniscal ossicle although they were not asked to search for ossicle and intrameniscal signal intensity changes. The lack of conspicuity of meniscal ossicle on fat-saturated images should also be taken into consideration when using artificial intelligence for detecting meniscal lesions based on fat-saturated sequence only [START_REF] Couteaux | Automatic knee meniscus tear detection and orientation classification with Mask-RCNN[END_REF][START_REF] Roblot | Artificial intelligence to diagnose meniscus tears on MRI[END_REF]. A second limitation is the lack of reliable previous clinical history and of body mass index. This limitation is inherent to the large number of recruiting centers. Third, there was no arthroscopic demonstration of the meniscal ossicle and of the other knee lesions. Our reference standard for the presence or absence of a meniscal ossicle relied on the analysis of the radiographs and MR images of 46 eligible knees and the corresponding control group. Four eligible knees were excluded because the presumed ossicles were not in the meniscal substance and corresponded to intra-articular osteo-cartilaginous bodies. Presence of an ossicle was defined by the presence of high signal intensity within the meniscal substance and/or the presence of a bony nodule on the 18 sets of radiographs from the study group. Absence of a meniscal ossicle was defined by the lack of meniscal high signal intensity on T1-weighted images and the lack of osseous nodule on the 42 sets of radiographs from the control group. Finally, sequence acquisition parameters and image planes varied among MRI examinations because they were provided by several imaging centers over a 12-year inclusion period.

In conclusion, on MRI, knees with medial meniscal ossicle are associated with a greater prevalence of medial posterior root tear, ACL lesions and ipsilateral cartilage degradation but not with posterior horn lesions and meniscal extrusion than knees without meniscal ossicle in an age-and sex-matched population.

Fig. 1 .

 1 Fig. 1. (a) Flowchart illustrates the recruiting process performed in the 15 imaging centers to obtain imaging data of knees with meniscal ossicle (study group) and age-and sex-matched knees without meniscal ossicle. PACS indicates (b) flowchart illustrates the inclusion process performed by two radiologists who read the SE T1-weighted spin-echo MR images (n = 46) and available radiographs (n = 25) of eligible study group knees to exclude those which did not have a medial meniscal ossicle with a high degree of confidence. These two radiologists also determined the topography and the signal intensity of the meniscal ossicle at magnetic resonance imaging (MRI).

Fig. 2 .

 2 Fig. 2. Medial meniscal ossicle in a 19-year-old man: a: lateral radiograph of the right knee demonstrates a triangle-shaped osseous body (arrow) projecting on the posterior aspect of the femoro-tibial joint; b: coronal T1-weighted SE image demonstrates a triangle-shaped area of fat-like signal intensity in the lateral aspect of the posterior horn (arrow); c: on corresponding fat-saturated proton-density-weighted image, the ossicle has fat-like low signal intensity and is not visible. The posterior root of the medial meniscus (arrow) is torn. No meniscal extrusion was found.

Fig. 3 .

 3 Fig. 3. Medial meniscal ossicle in a 36-year-old man: a, b: two posterior coronal T1-weighted SE images demonstrate a triangle-shaped intrameniscal fat-like signal intensity area (arrows); c, d: on the corresponding fat-saturated proton-density-weighted images, the ossicle shows moderate increase in signal intensity. The posterior root of the medial meniscus is normal at magnetic resonance imaging (MRI).

Fig. 4 .

 4 Fig. 4. Medial meniscal ossicle in a 65-year-old woman: a, b: knee radiographs demonstrate a triangle-shaped osseous body (arrows) located in the posterior aspect of the medial femoro-tibial joint; c: a coronal T1-weighted SE image demonstrates no signal abnormality of the medial meniscus; d: on the corresponding fat-saturated protondensity-weighted image, there is a tear of the posterior root. An area of intermediate signal intensity can be seen at the meniscal free edge (arrow) that probably corresponds to the ossicle. No meniscal extrusion was found.

Table 1

 1 Clinical and MRI findings in patients with a medial meniscal ossicle (study group) and in age-and sex-matched patients without medial meniscal ossicle (control group).

		Reader 1			Reader 2			Kappa	[95% CI]
		Study group	Control group	P	Study group	Control group	P
	Medial meniscus tear	34/42 (81%)	18/42 (43%)	< 0.01	38/42 (90%)	24/42 (57%)	< 0.01	0.731 [0.580-0.881]
	Medial meniscus extrusion	12/42 (29%)	7/42 (17%)	0.19	15/42 (36%)	8/42 (19%)	0.08	0.873 [0.753-0.993]
	Lateral meniscus tear	7/42 (17%)	5/42 (12%)	0.51	14/42 (33%)	7/42 (17%)	0.09	0.666 [0.472-0.860]
	ACL lesion	20/42 (48%)	11/42 (26%)	0.03	24/42 (57%)	11/42 (26%)	< 0.01	0.850 [0.736-0.965]
	PCL lesion	2/42 (5%)	4/44 (10%)	0.38	2/42 (5%)	3/42 (7%)	0.70	0.708 [0.395-1.00]
	MCL lesion	2/42 (5%)	2/42 (5%)	> 0.99	7/42 (17%)	6/42 (14%)	0.70	0.429 [0.139-0.718]
	LCL lesion	3/42 (7%)	1/42 (2%)	0.27	1/42 (2%)	2/42 (5%)	0.45

0.255

[-0.193

to 0.703] Medial cartilage score 3.35 ± 0.87 [2-6] 2.85 ± 0.74 [2-6] < 0.01 3.92 ± 0.78 [2-6] 3.35 ± 0.71 [2-6] < 0.01 0.489 [0.346-0.632] Lateral cartilage score 2.85 ± 0.75 [2-6] 2.47 ± 0.61 [2-6] 0.01 3.06 ± 0.72 [2-6] 2.88 ± 0.75 [2-6] 0.26 0.487 [0.328-0.647] Qualitative variables are expressed as proportions; numbers in parentheses are percentages. Quantitative variables are expressed as means ± standard deviations; numbers in brackets are ranges. ACL: anterior cruciate ligament; PCL: posterior cruciate ligament; MCL: medial collateral ligament; LCL: lateral collateral ligament. Bold indicates significant P-value. CI: confidence interval.

Table 2

 2 Comparison

	Reader 1

of the frequency of MRI lesion patterns and topography of the medial meniscus between patients with (study group) or without (control group) meniscal ossicle according to two radiologists (reader 1 and reader 2) who were blinded to the presence or absence of a medial meniscal ossicle.

Table 3

 3 Associations between location of ossicle and that of tears of medial meniscus for reader 1 and reader 2.

		Reader 1		P	Reader 2		P
		Root ossicle(n = 34)	Post. horn ossicle(n = 7)		Root ossicle(n = 34)	Post. horn ossicle(n = 7)
	Root tear	20/34(59%)	5/7 (71%)	0.55	26/34 (76%)	6/7 (86%)	0.56
	Post. horn tear	17/34 (50%)	2/7 (29%)	0.31	12/34 (35%)	2/7 (29%)	0.76
	Body tear	6/34 (18%)	2/7 (29%)	0.51	7/34 (20%)	3/7 (43%)	0.19

Table 4

 4 Study design, patient characteristics and MRI findings in the current study and in those of Mohankumar et al.[START_REF] Mohankumar | Meniscal ossicle: posttraumatic origin and association with posterior meniscal root tears[END_REF] and Bernard et al.[START_REF] Bernard | Understanding the implications of the meniscal ossicle: patient presentation, treatment and outcomes[END_REF].

		Current study	Mohankumar et al.	Bernard et al.
	Study design
	Case-control study	Yes	No	No
	Recruiting centers	15	2	1
	Inclusion period	2005-2017	2007-2012	2000-2018
	Number of readers	2	3	2
	Reading method	Independent	Consensus	Consensus
	Blinding to meniscal ossicle	Yes	No	No
	Patients
	Number of patients	84	65	59
	Patients with medial ossicle	42/84 (50%)	58/65 (89%)	40 (68%)
	Sex
	Male (%)	36/42 (86%)	33/65 (51%)	N.A.
	Female (%)	6/42 (14%)	32/65 (49%)	N.A.
	Age
	All patients with ossicles	42.5 ± 22 [19-65]	51.4 ± N.A. [23-80]	51 ± 19 [N.A.]
	Men	42.9 ± 23 [19-62]	47.8 ± N.A. [23-80]	N.A.
	Women	41.8 ± 16 [32-65]	55.0 ± N.A. [30-77]	N.A.
	Prior meniscectomy	4/42 (10%)	4/58 (7%)	N.A.
	ACL plasty	11/42 (26%)	14/58 (24%)	11/40 (27%)
	MRI findings
	Posterior ossicle	41/42 (98%)	56/58 (97%)	39/40 (97%)
	Ossicle with high signal on T1W	38/42 (90%)	58/58 (100%)	40/40 (100%)
	Ossicle with low signal on T1W	4/42 (10%)	0/58 (0%)	0/40 (0%)
	Medial meniscal tear	34/42 (81%)38/42 (90%)	57/58 (98%)	40/40 (100%)
	Posterior root tear	25/42 (60%)32/42 (76%)	47/58 (81%)	39/40 (97.5%)
	Posterior horn tear	19/42 (45%)14/42 (33%)	13/58 (22%)	1/40 (2.5%)
	Body tear	8/42 (19%) 10/42 (24%)	0/58 (0%)	0/40 (0%)
	ACL lesion	20/42 (48%)24/42 (57%)	31/65 (48%)	N.A.

MRI: magnetic resonance imaging; N.A.: not available; ACL: anterior cruciate ligament; T1W: T1-weighted images. Qualitative variables are expressed as proportions; numbers in parentheses are percentages. Quantitative variables are expressed as means ± standard deviations; numbers in brackets are ranges.

Acknowledgment

We thank the following radiologists for participating in the current study by searching for and providing study and control cases: Loic Bouilleau (Tours), Joseph Yazbek (Bordeaux), Celine Brun (Lyon), Marine Brunet (Toulouse), Yannick Carillon (Lyon), Benjamin Dallaudière (Bordeaux), Frederic Defaux (Poitiers), Marie-Laurence Denis (Liège), Guillaume Etievent (Saint-Étienne), Marie Faruch-Bielfeld (Toulouse), Romain Gillet (Nancy), Patrick Omoumi (Lausanne), Jean-Noel Ravey (Grenoble), Nor-Eddine Regnard (Paris), Jerome Renoux (Paris), Flore Viry (Paris), Arnaud Monnier (Montreal), Quentin Monzani (Paris). We also thank Perrine Triqueneaux for technical and secretarial assistance.

Funding

This work did not receive any grant from funding agencies in the public, commercial, or not-for-profit sectors.

Human rights

The authors declare that the work described has been carried out in accordance with the Declaration of Helsinki of the World Medical Association revised in 2013 for experiments involving humans.

Informed consent and patient details

The authors declare that this report does not contain any personal information that could lead to the identification of the patients.

Author contributions

All authors attest that they meet the current International Committee of Medical Journal Editors (ICMJE) criteria for Authorship.

Disclosure of interest

The authors declare that they have no competing interest.