Baptiste Pignon

MD Mohamed Lajnef

Ophelia Godin

PhD Marie-Maud Geoffray

PhD Paul Roux

PhD Laurent Marulaz

MD Sylvain Leignier

Myrtille Andre

Isabelle Chéreau

PhD Pierre-Michel Llorca

Msc Ophélia Godin

MD Romain Rey

MD Jasmina Mallet

MD Caroline Dubertret

PhD Christine Passerieux

MD Lore Brunel

Msc Julien Dubreucq

MD Delphine Capdevielle

PhD Myrtille André

MD Bruno Aouirzerate

PhD David Misdrahi

PhD Fabrice Berna

PhD Pierre Vidailhet

PhD Isabelle Chereau

PhD Guillaume Fond

PhD Christophe Lançon

PhD Marion Leboyer

PhD Franck Schürhoff

PhD The Fondamental

Andrianarisoa Md

PhD ; Blanc

Msc ; Brunel

Msc ; Bulzacka

Msc ; Capdevielle

PhD ; Chereau-Boudet

Danion Jm

MD D ' Amato

PhD ; Deloge

PhD ; Delorme

Msc ; Denizot

MD Dorey Jm

PhD ; Dubreucq

MD ; Faget

MD ; Fluttaz

MD ; Fonteneau

Msc ; Gabayet

; Giraud-Baro

MD Hardy-Bayle Mc

PhD ; Lacelle

Msc ; Lançon

PhD ; Le Gloahec

Msc ; Le Strat

PhD ; Llorca

MD Pm

PhD ; Mallet

PhD ; Metairie

Msc ; Misdrahi

MD ; Offerlin-Meyer

PhD ; Passerieux

MD, PhD ; Peri

Msc ; Pires

Msc ; Portalier

Msc ; Sebilleau

; Schandrin

PhD ; Tessier

Tronche Am

MD; Vaillant F j,l , Msc; Vehier A f,l , Msc; Vidailhet P b,l , MD, PhD ; Vilà

Msc ; Yazbek

PhD ; Zinetti-Bertschy

Relationship between childhood trauma and level of insight in schizophrenia: A path-analysis in the national FACE-SZ dataset

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ABSTRACT

Introduction

This study aimed was to investigate the relationship between different types of childhood trauma and the level of insight (i.e., awareness of having a psychiatric disorder) in subjects suffering from schizophrenia, as well as the putative role of clinical mediators.

Methods

294 community-dwelling subjects with stable schizophrenia were included into FACE-SZ, a multicentre cross-sectional study. All patients were assessed by specialized multidisciplinary teams. The level of insight was assessed by the Scale to assess Unawareness of Mental Disorder (SUMD), and childhood trauma by the Childhood Trauma Questionnaire (CTQ).

Path analyses from the five CTQ subscales (physical abuse and neglect, emotional abuse and neglect, and sexual abuse) and the SUMD, with current symptomatology (i.e., positive, negative, global psychopathology and depression) as mediator, was performed.

Results

Physical neglect (β = 0.14) and abuse (β = 0.13) were significantly associated with poor insight. Negative symptoms were a clinical mediator of the relationship between physical neglect and poor insight. Moreover, positive (β = 0.21) and negative (β = 0.30) symptoms were associated with poor insight, whereas depression (β = -0.14) was associated with higher levels of insight.

Discussion

For the first time, this study shows a significant relationship between childhood trauma, specifically physical neglect and abuse, and poor insight. The level of insight was linked to different clinical dimensions. Among subjects with schizophrenia, these results provide support for a role of childhood trauma in poorer management outcomes, and the need to provide treatment, including psycho-education that better targets the consequences of childhood trauma.

Key-words: Schizophrenia, Childhood trauma, insight, negative symptoms, path-analysis

INTRODUCTION

Poor insight is defined as the presence of an impaired awareness of having a psychiatric disorder, as well as of its symptoms and implications. Poor insight has been reported in 50 to 80 % of patients with schizophrenia [START_REF] Lincoln | Correlates and Long-Term Consequences of Poor Insight in Patients With Schizophrenia. A Systematic Review[END_REF]. A low level of insight is associated with negative attitudes towards taking antipsychotic medication, and to the decision to decline treatments [START_REF] Lysaker | Insight in schizophrenia spectrum disorders: relationship with behavior, mood and perceived quality of life, underlying causes and emerging treatments[END_REF]. Similarly, it is associated with higher rates of rehospitalisation and poor global functioning [START_REF] Lysaker | Insight in schizophrenia spectrum disorders: relationship with behavior, mood and perceived quality of life, underlying causes and emerging treatments[END_REF][START_REF] Pousa | A deeper view of insight in schizophrenia: Insight dimensions, unawareness and misattribution of particular symptoms and its relation with psychopathological factors[END_REF]. Moreover, several studies have shown that poor insight is associated with the overall severity of schizophrenia, and in particular with positive, negative and disorganisation symptoms [START_REF] Amador | Awareness of Illness in Schizophrenia and Schizoaffective and Mood Disorders[END_REF][START_REF] Brohan | Self-stigma, empowerment and perceived discrimination among people with schizophrenia in 14 European countries: the GAMIAN-Europe study[END_REF][START_REF] Mintz | Insight in schizophrenia: a meta-analysis[END_REF][START_REF] Zhou | Insight in inpatients with schizophrenia: relationship to symptoms and neuropsychological functioning[END_REF].

Conversely, high levels of insight are associated with depression, self-stigma, and suicidality [START_REF] Misdrahi | Depression in schizophrenia: The influence of the different dimensions of insight[END_REF][START_REF] Schrank | Insight, positive and negative symptoms, hope, depression and self-stigma: a comprehensive model of mutual influences in schizophrenia spectrum disorders[END_REF]. Finally, insight appears to be temporally stable [START_REF] Cuesta | Reappraising insight in psychosis: Multi-scale longitudinal study[END_REF][START_REF] Smith | Insight and recovery from psychosis in chronic schizophrenia and schizoaffective disorder patients[END_REF], with cognitive-behavioural and psycho-education therapies having a significant (but small to moderate) positive effect on insight levels [START_REF] Pijnenborg | Changes in insight during treatment for psychotic disorders: a meta-analysis[END_REF].

Childhood trauma (CT) is defined by a range of severe adverse experiences, including physical, sexual and emotional abuse, neglect, or bullying. These experiences may affect approximately one-third of the general population [START_REF] Kessler | Childhood adversities and adult psychopathology in the WHO World Mental Health Surveys[END_REF][START_REF] Scher | Prevalence and demographic correlates of childhood maltreatment in an adult community sample[END_REF].

There is an accumulating body of evidence suggesting an association between such CT experiences and a range of negative social and health outcomes, including higher gastrointestinal disorders, chronic pelvic pain, and cardiometabolic disorders, as well as several psychiatric disorders [START_REF] Mclaughlin | Childhood adversities and adult psychiatric disorders in the national comorbidity survey replication II: Associations with persistence of DSM-IV disorders[END_REF][START_REF] Paras | Sexual Abuse and Lifetime Diagnosis of Somatic Disorders: A Systematic Review and Meta-analysis[END_REF][START_REF] Tebeka | Parental divorce or death during childhood and adolescence and its associations with mental health[END_REF][START_REF] Wickrama | Stressful Life Experiences in Adolescence and Cardiometabolic Risk Factors in Young Adulthood[END_REF]. Moreover, CT is a well-known risk factor for psychotic disorders, in interaction with genetic risk, and in an addition of other environmental risk factors, such as urbanicity, or cannabis use [START_REF] Guloksuz | The slow death of the concept of schizophrenia and the painful birth of the psychosis spectrum[END_REF][START_REF] Kraan | Child Maltreatment and Clinical Outcome in Individuals at Ultra-High Risk for Psychosis in the EU-GEI High Risk Study[END_REF][START_REF] Pries | Evidence That Environmental and Familial Risks for Psychosis Additively Impact a Multidimensional Subthreshold Psychosis Syndrome[END_REF][START_REF] Varese | Childhood Adversities Increase the Risk of Psychosis: A Metaanalysis of Patient-Control, Prospective-and Cross-sectional Cohort Studies[END_REF]. Some studies have even suggested a dose-dependent relationship between CT and psychosis [START_REF] Schäfer | Childhood trauma and psychosis -what is the evidence?[END_REF]. More specifically, sexual and physical abuse [START_REF] Fisher | Gender differences in the association between childhood abuse and psychosis[END_REF][START_REF] Shevlin | Cumulative traumas and psychosis: an analysis of the national comorbidity survey and the British Psychiatric Morbidity Survey[END_REF], separation from the parents [START_REF] Baudin | Childhood trauma and psychosis: Beyond the association[END_REF][START_REF] Morgan | Parental separation, loss and psychosis in different ethnic groups: a case-control study[END_REF], and peer-victimisation or bullying [START_REF] Trotta | Prevalence of bullying victimisation amongst first-episode psychosis patients and unaffected controls[END_REF] are associated with future psychotic disorders. Moreover, among patients with psychotic disorders, CT are identified as modifier factors, i.e., factors that are associated with specific clinical features or specific course of the disease [START_REF] Misiak | Toward a unified theory of childhood trauma and psychosis: A comprehensive review of epidemiological, clinical, neuropsychological and biological findings[END_REF]. For instance, several studies showed associations between CT history and more severe positive symptoms [START_REF] Duhig | The prevalence and correlates of childhood trauma in patients with early psychosis[END_REF][START_REF] Mccabe | Relationship between childhood adversity and clinical and cognitive features in schizophrenia[END_REF][START_REF] Read | Childhood trauma, psychosis and schizophrenia: a literature review with theoretical and clinical implications[END_REF][START_REF] Schalinski | Impact of childhood adversities on the shortterm course of illness in psychotic spectrum disorders[END_REF], in particular auditory-verbal hallucinations [START_REF] Bailey | Childhood Trauma Is Associated With Severity of Hallucinations and Delusions in Psychotic Disorders: A Systematic Review and Meta-Analysis[END_REF][START_REF] Bentall | Do Specific Early-Life Adversities Lead to Specific Symptoms of Psychosis? A Study from the 2007 The Adult Psychiatric Morbidity Survey[END_REF]. Interestingly, among patients with bipolar disorders, childhood abuse was also associated with the presence of auditory-verbal hallucinations [START_REF] Smith | Characterizing the experience of auditory verbal hallucinations and accompanying delusions in individuals with a diagnosis of bipolar disorder: A systematic review[END_REF]. Consistent with these results, recent studies in the FACE-SZ dataset show CT to be associated with the number of hospitalizations, and worse overall outcome, as well as positive, and negative psychotic symptoms, and low global functioning [START_REF] Andrianarisoa | Childhood trauma, depression and negative symptoms are independently associated with impaired quality of life in schizophrenia. Results from the national FACE-SZ cohort[END_REF][START_REF] Baudin | Differential effects of childhood trauma and cannabis use disorders in patients suffering from schizophrenia[END_REF][START_REF] Roux | Mediation Analyses of Insight, Quality of Life, Depression, and Suicidality: Results From the FACE-SZ Cohort[END_REF].

CT may have a negative impact on insight levels, and clinical mediators may be involved. However, the specific impact of CT on the level of insight has never been studied.

Moreover, studying the associations between CT, clinical dimensions and insight could help to define subsets of patients for whom specific interventions, such as psychotherapy, may be of utility. This study uses path-analysis methods to investigate factors modulating insight levels. We hypothesise that CT could be negatively associated with insight in schizophrenia. This association may be both direct and indirect, and partly mediated by positive and negative symptoms dimensions. As such, this study aims was to analyse the relationship between CT and insight, in association with several clinical dimensions, in a sample of patients with schizophrenia. Anonymized data was entered into a shared national database for use in multi-centre audit and research [START_REF] Schürhoff | A National network of schizophrenia expert centres: An innovative tool to bridge the research-practice gap[END_REF].

METHODS

The FACE-SZ dataset

Collected data

All patients were assessed by the specialized multidisciplinary team of the Expert centres. A large standardized clinical evaluation was used to determine diagnosis, comorbidities, severity, and other specific variables [START_REF] Schürhoff | A National network of schizophrenia expert centres: An innovative tool to bridge the research-practice gap[END_REF].

Insight was assessed using the French version of the Scale to assess Unawareness of Mental Disorder (SUMD), a clinician-rated instrument [START_REF] Raffard | La SUMD (Scale to Assess Unawareness of Mental Disorder): validation et adaptation française dans une population de patients souffrant de schizophrénie[END_REF]. The SUMD is a standardised rating-scale based on a patient interview. It assesses the current awareness of the 9 following states: 1) a mental disorder, 2) consequences of a mental disorder, 3) effects of drugs, 4) hallucinations, 5) delusional ideas, 6) disorganised thoughts, 7) blunted affect, 8) anhedonia, and 9) lack of sociability. SUMD utilizes three Likert-scale response levels (0: aware, 1: slightly aware/unaware, 2: severely unaware). We used the total SUMD score linearized on a scale of 0 to 100, with 100 representing the highest level of unawareness. The construct validity, the reliability and the external validity of scale have been studied by [START_REF] Michel | Psychometric properties of the abbreviated version of the Scale to Assess Unawareness in Mental Disorder in schizophrenia[END_REF]. Cronbach's alpha coefficients ranged from 0.76 to 0.83 for the 9 items, indicating satisfactory reliability. The inter-rater reliability was also satisfactory, with items ranging from 0.68 to 1.00 [START_REF] Raffard | La SUMD (Scale to Assess Unawareness of Mental Disorder): validation et adaptation française dans une population de patients souffrant de schizophrénie[END_REF].

CT was assessed with the French version of the Childhood Trauma Questionnaire (CTQ), a 25-items questionnaire. All items are answered on a five-point Likert-scale (1: never, to 5: very often). The CTQ provides one total score, and five sub-scores: physical abuse, emotional abuse, sexual abuse, physical neglect, and emotional neglect. The CTQ total score is the sum of all items [START_REF] Bernstein | Development and validation of a brief screening version of the Childhood Trauma Questionnaire[END_REF][START_REF] Paquette | Validation de la version française du CTQ et prévalence de l'histoire de maltraitance[END_REF].

Current symptomatology was assessed with the Positive and Negative Symptoms Scale (PANSS), and its 3 sub-scores: positive, negative, and global psychopathology symptoms. Depressive symptoms were assessed by the Calgary Depression Rating Scale for Schizophrenia (CDSS).

Ethical approval

The study was carried out in accordance with ethical principles for medical research involving humans (WMA, Declaration of Helsinki). The assessment protocol was approved by the relevant ethical review board (CPP-Ile de France IX, January 18th, 2010). All data were collected anonymously. As this study included data from regular care assessments, a non-opposition form was signed by all participants. Approval for access to the system was was obtained from the committee in charge of the safety of computerized databases (CNIL, number DR-2011-069).

Statistical analyses

The data were analysed in a path-analysis using Maximum Likehood estimation with Robust standard errors (MLR) (version 6.1) [START_REF] Muthén | Mplus: Statistical analyses with latent variables[END_REF]. Path analyses from the 5 five subscales of the CTQ, with the clinical variables (CDSS measured depression, and the 3 subscales of the PANSS: positive, negative, and general psychopathology) as mediators, to the score of insight (SUMD) were performed. An iterative procedure allowed the selection of the best-fitting model. The path-analysis started with a saturated model in which all variables were interrelated. Then, non-significant path correlations/associations were gradually excluded until a good-fitting model was reached. The overall model fit was examined using the Chi-square statistic, the Comparative Fit Index (CFI) and the Root Mean Square Error of Approximation (RMSEA). The rules of thumb for determining acceptable model fit were as follows: a non-significant Chi-square test, a CFI value of 0.90 or above, and RMSEA values close to 0.05 or below. All path coefficients and correlations are reported as standardized estimates.

The model examined both direct and indirect associations between the CT subscales and the SUMD. The model also examined the correlations of the 5 CT subscales and putative clinical mediating variables, as well as estimating correlations among the 5 CTQ sub-scales.

A path diagram representation of the model was then drawn: straight single-headed arrows representing regression paths, and curved tow-headed arrows representing correlations.

RESULTS

Characteristics of the sample

A total of 294 community-dwelling stable patients with schizophrenia were included (74 % male), with a mean age at interview of 32.1 years (± 9.8 years). The main sociodemographic and clinical characteristics of the sample are available in Table 1.

-TABLE 1 -

Statistical associations of variables

Here we report only the significant direct associations between i) the different types of trauma and putative mediator variables, and ii) the putative mediator variables and the SUMD. Physical neglect and sexual abuse were associated with higher levels of positive symptoms (physical neglect: β = 0.14, standard-error (SE) = 0.06 and p = 0.02, sexual abuse: β = 0.18, SE = 0.08 and p = 0.03) and of global psychopathology symptoms (physical neglect: β = 0.20, SE = 0.06 and p < 0.01, sexual abuse: β = 0.19, SE = 0.09 and p = 0.03). Physical abuse was associated with a high level of negative symptoms (β = 0.14, SE = 0.05 and p < 0.01) and a low level of depression (β = -0.12, SE = 0.06 and p < 0.05). This is in contrast to emotional abuse, which was associated with lower levels of negative symptoms (β = -0.14, SE = 0.05 and p = 0.04) and raised levels of depression (β = 0.17, SE = 0.08 and p = 0.03).

Emotional neglect was associated with a high level of depression (β = 0.17, SE = 0.08 and p = 0.02). Positive and negative symptoms were associated with low levels of insight, as indicated by a high SUMD scale score: β = 0.21, SE = 0.07 and p < 0.01 for positive symptoms; β = 0.29, SE = 0.07 and p < 0.001 for negative symptoms. The different sub-scores of CT were correlated. We did not find any direct association between the different types of trauma and level of insight. Of the considered mediator factors, none showed a significant mediation between the CT subscales and insight. The other indirect relationships were not significant.

Path-analysis

The model showed a very good fit to the data based on information from each indicator: χ 2 = 3.81 (df = 5) with p = 0.58, CFI = 1.00, TLI = 1.00, and RMSEA = 0.0011.

Considering total (i.e., direct and indirect) relationship, physical abuse and physical neglect were associated with low levels of insight (for physical abuse: total: β = 0.13, SE = 0.06 and p = 0.03, indirect: β = 0.05, SE = 0.03 and p = 0.13; for physical neglect: total: β = 0.14, SE = 0.07 and p = 0.03, indirect: β = 0.10, SE = 0.04 and p < 0.01). Negative symptoms appeared a significant mediator factor between physical abuse and insight (β = 0.04, SE = 0.02 and p = 0.02). Direct significant relations are presented in the Figure 1. -FIGURE 1 -

DISCUSSION

Principal findings

This study utilized a community sample of 294 subjects with schizophrenia showing, for the first time, that CT has an influence on the level of insight. Indeed, physical neglect and abuse were indirectly associated with poor levels of insight. The indirect relationship between physical neglect and poor insight was mediated by negative symptoms. Moreover, this study supports the association between positive symptoms and poor insight, as found in a previous investigation [START_REF] Schrank | Insight, positive and negative symptoms, hope, depression and self-stigma: a comprehensive model of mutual influences in schizophrenia spectrum disorders[END_REF]. The association between depressive symptoms and better levels of insight has previously been shown, being referred to as the "insight paradox" [START_REF] Belvederi Murri | Is good insight associated with depression among patients with schizophrenia? Systematic review and meta-analysis[END_REF]Misiak et al., 2016a). The relationship between negative symptoms and poor insight, which was stronger than the associations of positive symptoms and depression, is less common in the studies investigating the clinical correlates of insight [START_REF] Lysaker | Insight in schizophrenia spectrum disorders: relationship with behavior, mood and perceived quality of life, underlying causes and emerging treatments[END_REF].

Childhood trauma and poor insight

As predicted, some of the considered CT variables, namely physical abuse and neglect, were associated with poor insight. However, contrary to our hypotheses, the level of relationship between CT and poor insight was relatively low (β = 0.14 for physical neglect, β = 0.13 for physical abuse), with negative, but not positive, symptoms mediating this relationship.

Several explanations of this relationship are possible. Poor insight may be linked to a lack of confidence in the outside world, which may be specifically impaired in patients with a CT history [START_REF] Rokita | Early life experiences and social cognition in major psychiatric disorders: A systematic review[END_REF][START_REF] Roux | Mediation Analyses of Insight, Quality of Life, Depression, and Suicidality: Results From the FACE-SZ Cohort[END_REF]. Altered metacognition and theory of mind may also be relevant to such associations [START_REF] Lysaker | Metacognition amidst narratives of self and illness in schizophrenia: associations with neurocognition, symptoms, insight and quality of life[END_REF][START_REF] Pos | Theory of Mind and attachment styles in people with psychotic disorders, their siblings, and controls[END_REF]. Surprisingly, positive symptoms were not identified as a clinical mediator between CT and poor insight. However, as in previous studies, positive symptoms are linked both to poor insight, and to a CT history [START_REF] Misiak | Toward a unified theory of childhood trauma and psychosis: A comprehensive review of epidemiological, clinical, neuropsychological and biological findings[END_REF](Misiak et al., , 2016b)). Over a number of years, previous studies have addressed the relationship between positive psychotic symptoms and the levels of insight. In 2003, a meta-analysis of 40 studies found a significant, but modest, correlation between positive psychotic symptoms and poor levels of insight (effect-size: 0.25, p < 0.001) [START_REF] Mintz | Insight in schizophrenia: a meta-analysis[END_REF]. In a more recent path-analysis considering different clinical dimensions and insight, the relationship between positive psychotic symptoms and poor insight was also significant (r = 0.35) [START_REF] Schrank | Insight, positive and negative symptoms, hope, depression and self-stigma: a comprehensive model of mutual influences in schizophrenia spectrum disorders[END_REF]. In an analysis of the impact of gender on insight, [START_REF] Cobo | Insight and gender in schizophrenia and other psychoses[END_REF] found significant associations between positive symptoms and all items of insight (p < 0.0001), in both gender samples.

Previous studies showed a small association between negative symptoms and poor insight, with an effect-size of -0.23 in one meta-analysis [START_REF] Mintz | Insight in schizophrenia: a meta-analysis[END_REF]. Low insight may be conceptualized as a negative symptom, being a "mental withdrawal" from attempting to understand one's own experience [START_REF] Osatuke | Insight in schizophrenia: a review of etiological models and supporting research[END_REF]. Cognitive dysfunction may be relevant. Indeed, negative symptoms are highly correlated with poor cognitive performances, particularly executive functioning, working memory and episodic memory [START_REF] Millan | Negative symptoms of schizophrenia: Clinical characteristics, pathophysiological substrates, experimental models and prospects for improved treatment[END_REF]. Moreover, among patients with schizophrenia, CT is associated with a higher level of cognitive deficits. [START_REF] Aas | Is cognitive impairment following early life stress in severe mental disorders based on specific or general cognitive functioning[END_REF] showed that, among patients with schizophrenia, early life stress history was associated with impairments in processing speed and working memory.

Moreover, [START_REF] Gil | The association of child abuse and neglect with adult disability in schizophrenia and the prominent role of physical neglect[END_REF] found that functional and social impairment in schizophrenia is related to physical neglect and emotional abuse and neglect, but not with other CT outcomes.

As such, cognitive deficits may also associate with poor insight, including via their association with negative symptoms [START_REF] Koren | The neuropsychological basis of insight in first-episode schizophrenia: a pilot metacognitive study[END_REF][START_REF] Lysaker | Metacognition amidst narratives of self and illness in schizophrenia: associations with neurocognition, symptoms, insight and quality of life[END_REF].

Childhood trauma and clinical dimensions of schizophrenia

The present results show that, among subjects with schizophrenia, those with a history of CT display poorer outcomes. The associations of physical neglect and sexual abuse with positive psychotic symptoms have already been shown [START_REF] Duhig | The prevalence and correlates of childhood trauma in patients with early psychosis[END_REF][START_REF] Mccabe | Relationship between childhood adversity and clinical and cognitive features in schizophrenia[END_REF]. Emotionally adverse experiences, both abuse and neglect, are generally associated with higher levels of depression, and are a significant predictor of suicide in people with schizophrenia [START_REF] Upthegrove | Depression and Schizophrenia: Cause, Consequence, or Trans-diagnostic Issue?[END_REF]. Such data highlights the clinical relevance of the current results. Emotionally adverse experiences have a specific influence on depression, which is associated with higher levels of insight [START_REF] Schrank | Insight, positive and negative symptoms, hope, depression and self-stigma: a comprehensive model of mutual influences in schizophrenia spectrum disorders[END_REF]. Thus, patients with a history of emotionally adverse experiences may be a particularly targeted for suicide prevention. The present results indicate that physical abuse and emotional abuse have contrasting impacts on negative symptoms, with a positive association being evident for physical abuse, whilst an inverse correlation is evident for emotional abuse. Such contrasting effects are also evident for depression, which shows a negative association for physical abuse, and a positive association for emotional abuse. This is consistent with an array of different studies showing that emotional and physical adverse experiences can have differential consequences. Such data would indicate that measures of adversity and trauma subtypes may be more clinically meaningful than a global indicant [START_REF] Morgan | Childhood adversities and psychosis: evidence, challenges, implications[END_REF].

Other work supports this with [START_REF] Baudin | Childhood trauma and psychosis: Beyond the association[END_REF] showing that physical and sexual abuse were specifically associated with a higher risk of psychotic disorders, in comparison to childhood emotional abuse.

Finally, several studies have shown that patients with CT have a poorer treatment response, including a higher proportion of treatment-resistant schizophrenia [START_REF] Hassan | The effect of lifetime adversities on resistance to antipsychotic treatment in schizophrenia patients[END_REF][START_REF] Mondelli | Cortisol and Inflammatory Biomarkers Predict Poor Treatment Response in First Episode Psychosis[END_REF]. A number of studies indicate that early abusive experiences may be associated with alterations in the immune response, which may underlie treatment resistance (e.g., [START_REF] Misiak | Toward a unified theory of childhood trauma and psychosis: A comprehensive review of epidemiological, clinical, neuropsychological and biological findings[END_REF]). The results of the current study indicate that such resistance may also be linked to poor insight, and lower treatment adherence. Results of the present study also suggest that specific treatment of patients with schizophrenia a CT history, including non-pharmacological [START_REF] Selten | Childhood trauma and the treatment for psychotic disorder[END_REF], should improve several clinical outcomes. The screening of post-traumatic stress disorder (PTSD) is important in routine care of patients with schizophrenia [START_REF] Brand | Do trauma-focussed psychological interventions have an effect on psychotic symptoms? A systematic review and meta-analysis[END_REF]. Psychotherapy by written emotional disclosure, may significantly improve insight for patients with schizophrenia and PTSD [START_REF] Swan | Psychological Interventions for Posttraumatic Stress Symptoms in Psychosis: A Systematic Review of Outcomes[END_REF].

Limitations

Several limitations deserve discussion. First, the assessment of CT is retrospective, and this design is not exempt from potential uncontrolled recall bias, particularly among patients with persecutory ideas. However, [START_REF] Fisher | Reliability and comparability of psychosis patients' retrospective reports of childhood abuse[END_REF] have shown that the recall of childhood adversity experiences among individuals with psychosis was stable over time, and not related to the severity of the pathology. Second, the present study analysed cross-sectional data, which preclude causality inference. Thus, although we assumed that the clinical outcomes were mediators between CT and level of insight, we cannot exclude reverse causation: subjects at higher risk for psychosis, e.g., suffering from early neurodevelopmental impairments, and later suffering from more severe disease, could be overexposed to CT experiences (Etain et al., 2017). Moreover, we consider the assessment of insight as reflecting a state for each patient, cross-sectionally. However, insight has been reported as partially variable [START_REF] Ayesa-Arriola | The dynamic relationship between insight and suicidal behavior in first episode psychosis patients over 3-year followup[END_REF], and the relationship between CT and insight may be dynamic. Third, in order to avoid a lack of statistical power in the path-analysis, we considered insight unidimensionally, which is contrary to [START_REF] David | Insight and Psychosis[END_REF]) three-dimensional model of insight: the recognition that one has a psychiatric disorder, the ability to relabel unusual mental events (delusions and hallucinations) as pathological, and the recognition of the positive treatment consequences. Finally, for the same reason, other factors that may impact the relationship between CT and insight, such as cognitive factors, have not been included in the path-analysis. Future studies of relationships between CT and insight should utilize cognitive measures.

Conclusion

Physical abuse and neglect have a significant, but low association with a poor level of insight, and this relationship appeared mediated by the negative symptoms dimension.

Moreover, this study supports some associations between the severity of positive and negative symptoms and poor insight, and between depression and higher levels of insight. Further studies should investigate the impact of age at exposure to CT, and the different dimensions of insight. 1 SUMD was calculated using the mean scores of all items, which were linearized on a scale 0-100.

  Subjects over 18 years-old with schizophrenia or schizo-affective disorder according to DSM-IV-TR have been included through the FACE-SZ (FondaMental Advanced Centre of Expertise -Schizophrenia) network. FACE-SZ is a French national multicentre network of ten schizophrenia expert centres (Bordeaux, Clermont-Ferrand, Colombes, Créteil, Grenoble, Lyon, Marseille, Montpellier, Strasbourg, Versailles) of the FondaMental foundation (https://www.fondation-fondamental.org/). They provide a detailed evaluation and suggest therapeutic interventions. Patients are referred by their general practitioner or psychiatrist. A web application (e-schizo©) is used to record data in a common computerized medical file.

  Abbreviations: CFI: Comparative Fit Index, EA: Emotional Abuse, EN: Emotional Neglect, PA: Physical Abuse; PANSS: Positive And Negative Symptoms Scale, PN: Physical Neglect, RMSEA: Root Mean square of Approximation, SA: Sexual Abuse, sd: standard-deviation; SUMD: Scale to Assess Unawareness of Mental Disorder, TLI: Tucker-Lewis Index.

Figure 1 :

 1 Figure 1: Path-analysis of relationships between childhood trauma and adversities, and the level of insight

Table 1 .

 1 Main sociodemographic and clinical characteristics of the sample of 294 patients Mean (SD) or N (%) Positive And Negative Symptoms Scale; CTQ: Childhood Trauma Questionnaire; SUMD: Scale to Assess Unawareness of Mental Disorder.

	Age


Service Sophie Putman
email: sophie.putman@chru-lille.fr

Henri Migaud

Dominique Saragaglia

Jean-Yves Jenny

Franck Dujardin

Antoine-Guy Hue

Fabrice Fiorenza

Olivier Meric

Paul Bonnevialle

Olivier Méric

Total femur replacement in non-oncologic indications: Functional and radiological outcomes from a French survey with a mean 6 years' follow-up

Keywords: Total femoral replacement, Hip arthroplasty loosening, Knee arthroplasty loosening, Periprosthetic femoral fracture, Chronic periprosthetic infection

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

Introduction

There are few published studies on total femur replacement (TFR) because its indications are rare. Other than malignant diseases, the indications extend to revisions and interprosthetic femur fractures; however the outcomes of these indications have not been well defined. The aim of this retrospective survey was to analyze the complication rate and functional outcomes of these newer indications.

Hypothesis

The morbidity and outcomes after TFR are comparable to those reported in the literature for non-cancer indications.

Material and Methods

Between 1997 and 2016, 29 TFR procedures were done at 6 French teaching hospitals in 15 women and 14 men, average age 68 ± 14 years . The primary indication was degenerative joint disease in the hip and/or knee in 16 cases, mechanical failure of the implant used after tumor resection in 11 cases and femur fracture in 2 cases. The mean number of surgical procedures before TFR was 3.6 (maximum 5) at the hip and 4.5 (maximum 10) at the knee. Six different models were implanted consisting of a rotational hinge knee (except in one case); 20 patients received a dual mobility system and 9 a standard hip replacement bearing.

The femoral shaft was partially conserved 21/29 times and the trochanter 25/29 times.

Results

Five patients suffered a general complication and 12 suffered a local complication (including 4 hematomas and 2 hip dislocations). Eight patients (28.6%) suffered a surgical site infection, although three had a prior infection. Among the 12 patients with a history of infection or progressive infection before the TFR, 9 healed and 3 had the infection continue. At a minimum follow-up of 2 years and mean of 6 years, 23 TFR implants were still in place and not infected; the other 6 had been removed or were infected, including one patient who

Introduction

Total femur replacement (TFR) -in which a diaphyseal stem joins total hip arthroplasty (THA) and total knee arthroplasty (TKA) implants -was performed for sarcoma diffusion by Marcove et al. [START_REF] Marcove | Total femur and total knee replacement. A preliminary report[END_REF] and then validated in cancer-related indications [START_REF] Katznelson | Total femur replacement in sarcoma of the distal end of the femur[END_REF][START_REF] Nerubay | Total femoral replacement[END_REF][START_REF] Sewell | Total femoral endoprosthetic replacement following excision of bone tumours[END_REF][START_REF] Kalra | Total femur replacement: primary procedure for treatment of malignant tumors of the femur[END_REF][START_REF] Sevelda | Total femur replacement after tumor resection: limb salvage usually achieved but complications and failures are common[END_REF]. Buchman had previously published one case of resection-replacement for Paget's disease of the femur [START_REF] Buchman | Total femur and knee joint replacement with a vitallium endoprosthesis[END_REF], followed by a few publications in the same, non-tumor context [START_REF] Huang | Total replacement of the femur with simultaneous total hip and knee joint endoprostheses with plasma sprayed ceramic coating[END_REF][START_REF] Ward | Total femoral endoprosthetic reconstruction[END_REF][START_REF] Freedman | A modular endoprosthetic system for tumor and non-tumor reconstruction: preliminary experience[END_REF][START_REF] Mclean | Femoral replacement for salvage of periprosthetic fracture around a total hip replacement[END_REF]. The role of this procedure is limited, as evidenced by the paucity of publications and the number of reported cases, except for Berend et al. [START_REF] Berend | Total femoral arthroplasty for salvage of end-stage prosthetic disease[END_REF] and Friesecke et al. [START_REF] Friesecke | Revision arthroplasty with use of a total femur prosthesis[END_REF].

In the French scientific literature, Soenen et al. [START_REF] Soenen | Interprosthetic femoral fractures: analysis of 14 cases. Proposal for an additional grade in the Vancouver and SoFCOT classifications[END_REF] reported performing TFR in 3 patients following repeated failure of interprosthetic fractures. Other than in cases of tumor resection, the indications for TFR are rare: septic and mechanical revisions with multiple operations, along with mechanical failure of implants used in tumor resection cases [START_REF] Fountain | The use of total femoral arthroplasty as a limb salvage procedure: the Sheffield experience[END_REF][START_REF] Hoell | Intramedullary and total femur replacement in revision arthroplasty as a last limb-saving option: is there any benefit from the less invasive intramedullary replacement?[END_REF][START_REF] Clement | Total femoral replacement for salvage of periprosthetic fractures[END_REF][START_REF] Amanatullah | Non-oncologic total femoral arthroplasty: retrospective review[END_REF][START_REF] Toepfer | Short to long term follow-up total femoral replacement in non-oncologic patients[END_REF]. The outcomes of TFR are not known in these indications, particularly in France. A retrospective survey was carried out with members of the French Hip and Knee Society (SFHG) about these newer indications to determine 1) the complication rate and 2) the functional outcomes.

We hypothesized that morbidity and outcomes after TFR are comparable to those in the literature for non-cancer indications.

Material and Methods

Inclusion criteria

The inclusion criteria were repeated mechanical failure of ipsilateral THA and/or TKA whether accompanied or not by intra-or interprosthetic fracture; infection with removalimplantation in one or two steps. Failure of arthroplasty (TFR, THA or TKA) implanted for a cancer-related indication in a patient in long-term remission or who is cancer-free was included. A minimum of 2 years' follow-up was required for inclusion in the study. The exclusion criteria were any primary TFR following tumor resection and/or less than 2 years' follow-up.

Patients

Between 1997 and 2016, 29 patients fitting these criteria underwent TFR at various French teaching hospitals: Grenoble (n=4), Lille (n=9), Limoges (n=1), Rouen (n=4), Strasbourg (n=4) and Toulouse (n=7). There were 15 women and 14 men with an average age of 68 ± 14 years [32-85] at the time of TFR. Three patients were ASA 1, 17 patients were ASA 2, 8 patients were ASA 3 and 1 patient was ASA 5 [START_REF] Parker | A new mobility score for predicting mortality after hip fracture[END_REF] The initial femur pathology was a fracture in 2 cases, tumor resection in 11 cases (thus an indication for mechanical failure in a cancer-free patient) and degenerative joint disease in the hip and/or knee in 16 cases. For the 11 patients with a prior tumor, the mean follow-up was 12.4 years (minimum 3, maximum 23). None had clinical or radiological signs of local progression of the tumor. Details of these 29 cases are given in Figure 1. Twenty-seven of the 29 patients had undergone multiple procedures before the TFR; the clinical history of these patients is given in Figure 2. The mean number of procedures at all locations before the TFR was 3.6 (range, 1 to 10). At the hip, the mean number was 3.6 ± 1.8 (maximum 5) and at the knee it was 4.5 ± 2.4 (maximum 10). The reasons leading to TFR are listed in Figure 3.

At the time of TFR, 12 patients had a history of infection on the operated femur in one or both joints, of which 8 were progressive. Three had both knee and hip implants, while nine had a periprosthetic fracture at the THA or TKA.

Surgical technique

The following TFR implants were used: 10 Mega System (Waldemark-Link, Hamburg, Germany), 7 Global Modular Replacement System (Stryker, Pusignan, France) (Figure 4), 7

Mutars (Implantcast GmBH, Buxtehude, Germany) (Figure 5), 2 Modular Endoprosthetic Total System (Stanmore, Borehamwood, UK), 2 Orthopaedics Salvage System (Zimmer-Biomet, Brognard, France) and 1 Askorn (Askorn Medical, Cesson-Sévigné, France). The hip joints had a standard bearing in 9 cases and dual mobility bearing in 20 cases. A metal reinforcement ring was implanted in the acetabulum in 6 cases. At the knee, a rotating hinge implant was used in all cases but one. The femoral shaft was partially conserved in 21 cases and completely resected in 8 cases. The trochanter was conserved in 25 cases and removed in 4 cases. All the tibial implants were cemented.

Assessment methods

The data were collected in a spreadsheet used to capture standard demographic and epidemiology information, in particular the preoperative and postoperative ASA [START_REF] Parker | A new mobility score for predicting mortality after hip fracture[END_REF], Parker and Palmer [21] scores for patients above 65 years of age. The clinical and radiological evaluation carried out at the longest documented follow-up visit (beyond 2 years) was used.

Statistical methods

All statistical tests were performed using the software Statis (version 8.4.6, O. Méricq, Toulouse, France). Qualitative variables were summarized by their counts and percentages, while the numeric variables were summarized by their mean and standard deviation. Student's t-test was used to compare different mean values, while the Chi² test was used with independent qualitative variables when the sample size was more than 5. Fisher's exact test was used when the sample size was less than 5. The significance threshold was set at p < 0.05.

Results

Complications

One patient had an intraoperative fat embolism with regressive cardiopulmonary arrest. In the early postoperative period, four patients suffered a general complication: stroke with neurological sequelae in one patient, pulmonary embolism in one patient and kidney failure related to antibiotics in two patients. The following local complications were documented: hematoma (n=4), hip dislocation (n=2), wound healing disturbance (n=3), implant disassembly (n=1), trochanter detachment in one patient and tibial tuberosity avulsion (n=1). Eight patients (27.6%) suffered a surgical site infection (three of whom had an ongoing infection). Among the 12 patients with a history of infection or on-going infection before the TFR, 9 healed and 3 remained infected, including one who required disarticulation.

The four cases of septic etiology reported by one surgeon (JYJ) all resolved.

Survival

The minimum follow-up was 2 years, with a mean follow-up of 6 ± 4.5 years. In our case series, the patients with a history of infection had the longest follow-up. The median actuarial survival was 18.1 years; at 10 years, 59% of patients were still alive. At the latest follow-up, 23/29 (79.6%) of TFR implants were still in place and not infected, while 6 (20.6%) had been removed or were infected and being treated with long-term antibiotics. The median survival of the 17 TFR cases without history of infection was 15 years; at 10 years, 17/12 (70%) of these patients still had the implants in place and were infection-free.

Functional outcomes

Walking was possible without a cane in 5 patients (17.2%), with one cane in 10 (34.5%), with two canes in 7 (24.1%) and with a walker in 5 (17.2%). Walking was impossible for two patients (6.9%) including the patient who underwent disarticulation.

Active knee flexion (evaluated in 27 patients) averaged 79.4° ± 30.3° [0°-120°]; 17 patients (62.9%) had 90° or more knee flexion, 8 patients (29.6%) had flexion between 40 and 85° while two patients (7.4%) had no flexion. The knee extension deficit averaged 3.7 ± 7° [-20; +10] with a maximum of 20°; 20 patients (74.1%) had no flexion deformity. The average leg length difference was 1.3 ± 2.3 cm (0 to 10 cm); 19 patients (67.8%) had less than 1 cm difference in leg length. The mean final Parker and Palmer score was 6.1 ± 1.9; 6 patients had a score of 8 or more (25 %) and 5 had a score between 1 and 2 (20.8%).

Discussion

This study has three main findings. 1) The indications for TFR are rare, with only 29 cases at 6 French teaching hospitals over a 20-year period. However, we believe the frequency will increase with revision of implants used in tumor resection and aging of the population contributing to revisions for interprosthetic fractures on weakened bone. 2) The frequency of infection-related complications is considerable (28%) with one patient requiring disarticulation.

3) The modest nature of the clinical outcomes is obvious: only 15 patients (58.6%) could walk normally or with a cane after TFR.

Among the six published studies of TFR for non-oncologic indications, two singlecenter studies differentiate themselves because of their sample size (Table 1). Berend et al. [START_REF] Friesecke | Revision arthroplasty with use of a total femur prosthesis[END_REF] reported the outcomes of a cohort of 59 patients operated for periprosthetic fracture or repeated loosening. The Hamburg Endo-Klinik case series reported by Friesecke et al. [START_REF] Soenen | Interprosthetic femoral fractures: analysis of 14 cases. Proposal for an additional grade in the Vancouver and SoFCOT classifications[END_REF] consisted of 100 consecutive patients operated between 1973 and 2000. The indications in our study are identical overall to those of other published studies in terms of age and the number of prior surgeries, evidence of the extreme nature of the TFR indication. The same goes for the clinical outcomes, which are somewhat comparable given the outcome measures used, which essentially show improved patient autonomy.

The rarity of postoperative dislocations in our cohort (2/29 or 6.9%) can be attributed to the nearly systematic use of dual mobility implants; the instability rate in other studies was 25% to 40% (Table 2). Dual mobility implants have been shown to be very useful in the context of tumor resection [START_REF] Philippot | Prevention of dislocation in total hip revision surgery using a dual mobility design[END_REF][START_REF] Philippeau | Dual mobility design use in preventing total hip replacement dislocation following tumor resection[END_REF][START_REF] Schneider | Revision total hip arthroplasty using a reconstruction cage device and a cemented dual mobility cup[END_REF]. In fact, the risk of instability is higher because of gluteal muscle atrophy secondary to multiple surgical procedures. Resection of a larger portion of the femoral shaft leading to direct and unpredictable reattachment of the muscles on the implant, and the uncertain mechanical values of trochanter fixation or even its resection, increase this risk of instability. In most of the cases in our study, the aim was to preserve bone whenever possible (even if more demanding) [START_REF] Lombardi | The shattered femur: radical solution options[END_REF][START_REF] Peters | Intramedullary total femoral replacement for salvage of the compromised femur associated with hip and knee arthroplasty[END_REF]: femoral diaphysis preserved in 21 cases and trochanter in 25 cases.

The postoperative infection rate in our study was in the range of that reported in other published studies (Table 2). It corresponds to that of mega-prostheses in tumor resection surgery [START_REF] Mavrogenis | Infected prostheses after lower-extremity bone tumor resection: clinical outcomes of 100 patients[END_REF]. The highest infection rate was reported in a study with a minimum of 2 years' follow-up [START_REF] Toepfer | Short to long term follow-up total femoral replacement in non-oncologic patients[END_REF] like in the other reported studies. As in our study, more than 2 years' follow-up is recommended to confirm the revision has not resulted in an infection, especially when there is a history of infection or presence of an on-going infection.

The TFR indication was made in 12 patients in our study (41%) who had a history of infection or an on-going infection; this rate is similar to that in other published studies (Table 3). In the studies by Berend et al. [START_REF] Berend | Total femoral arthroplasty for salvage of end-stage prosthetic disease[END_REF] and Frisecke et al. [START_REF] Friesecke | Revision arthroplasty with use of a total femur prosthesis[END_REF], the infection healing rate after TFR was higher than in our study; however, their follow-up was shorter. The four cases of infectiononly etiology belonging to one surgeon (JYJ) all had a good outcome, confirming the advantage of single-stage removal-implantation in a specialized center [START_REF] Jenny | Routine one-stage exchange for chronic infection after total hip replacement[END_REF][START_REF] Massin | Infection recurrence factors in oneand two-stage total knee prosthesis exchanges[END_REF].

The limitations of our study are significant and are mainly related to the diversity of the source of patients, involvement of multiple surgeons, long time span and use of different implants. However, the nearly systematic implantation of a rotating hinge knee implant and the high frequency of dual mobility use at the hip joint contributed to the homogeneity of these cases. Similarly, the extreme nature of the procedures and the high number of prior procedures and infections offer insight into how this procedure will fare in the worst conditions, providing some external validation to our findings.

Conclusion

This study-both in terms of its indications and findings (clinical outcomes and complications observed)-is comparable to the literature and confirms our starting hypothesis. The indications for TFR are rare, but with the increase in the number of patients with ipsilateral hip and knee implants that could fail mechanically or due to infection, this scenario will be increasingly common in the future. When performing TFR for this indication, one should keep in mind the risk of disarticulation in case of failure.

Conflict of interest:

None of the authors have conflicts to declare related to this study. Outside this study, P

  . The mean preoperative Parker and Palmer score was 5.4 [0-9] [21].

  Bonnevialle is a research and teaching consultant for Stryker, S Putman is a research and teaching consultant for Corin-Tornier, H Migaud is a Deputy Editor for Orthopaedics & Traumatology Surgery & Research and is a research and teaching consultant for Zimmer-Biomet, Corin-Tonier, MSD and SERF, JY Jenny has received royalties from B-Braun Aesculap, was a paid consultant for Exatech and received conference financing from FH Orthopaedics, D. Saragaglia has received royalties from B-Braun Aesculap, F. Dujardin is a consultant for SERF. The other authors have no conflict of interest outside this study. FUNDING No funding was received for this study Contributions PB collected the data, operated on patients and wrote the article HM and SP operated on patients, collected data and wrote the article; DS, JYJ, FD and FF operated on patients and collected data AGH and OM collected data.

Table 1 : 4 Hoell

 14 Demographics of the key recently published studies that have more than 10 patients. (in studies 13 and 15, 4 and 2 patients respectively had metastatic indication) Reference Patient inclusion years Sex and mean age (years) total femur replacement (Waldemar-Link) 12 aseptic loosening (8 THA / 3 THA and TKA / 1 TKA) 2 nonunions of interprosthetic fracture Hip: 3.6/ 0-10 Hip and knee: 4 / 2hip arthroplasty, TKA: total knee arthroplasty, M: men, W: Women, N/S: Not specified

Figure 1 :Figure 2 :Figure 3 :Figure 4 :

 1234 Figure 1: Flow chart showing the initial pathology in the 29 patients who underwent total femur

Figure 5 :

 5 Figure 5: Male 47-year-old patient. Distal femur resection in 1990 due to osteosarcoma with

Table 2 :

 2 Outcomes of the key recently published studies (N/S not specified)

	Reference	Range and	Complications	Other complications	Implant	Number of deaths
		mean	Postoperative infection		survival
		follow-up
	Berend et al.	1-13 years	8/59 (13.5%)	5 dislocations (8.4%)	75% at	14
	[12]	m = 4		2 leg length difference	12.5	(23.7%)
		years, 10		1 knee stiffness	years	0 perioperative
		months
	Frisecke et al.	1 month-	12/100 (12%)	7 dislocation (7%)	N/S	3 perioperative
	[13]	11.5 years	(6 early / 6 late)	3 loosening		(3%)
		m = 4.9
		years
	Fountain et al.	1-17 years	3/14 infections (21%)	6 dislocations (42.8%)	N/S	1 at 1 year
	[15]	m = 7.5
		years	2 deep	3 revised successfully / 3
				left in dislocated state
			1 superficial
	Hoell et al. [16]	0.5-7.5	None	1 sciatic palsy	N/S	None
		years
		m = 2.6
	Clement et al.	0.7-12.4	2/19 (10.5%):	1 dislocation	86% at	5% at 1 year
	[17]	m= 5.4	-long-term antibiotics	(reduction/successful)	10 years	58% at 10 years
			-disarticulation
	Amanatullah et	m = 6 ± 5	7/20 (35%)	5 dislocations (25%):	65% at 5	None
	al. [18]	minimum 2		-4 revised	years
		years		-1 reduction (successful)
	Toepfer et al.	m = 6.6	8/18 (44.4%)	5 dislocations (27.7%)	56% at 5	None
	[19]	(2.3-11)		2 stiffness	years
				11 wound healing
				disturbances
	Our study	m = 6	8/29 (27.6%)	2 dislocations (6.9%)	70% at	41% of patients died at
		(2-10)		4 hematomas	10 years
				3 wound healing
				disturbances

Table 3 :

 3 Infection-related complications and infection-related indications from the key recently published studies

	Reference	Complications	Patients with history of infection /
		Postoperative infection and outcome	entire cohort
			Outcome
	Berend et al. [12]	8/49 (16.3%):	14/49 (23.7%):
		-3 removal-reimplantation	-1 recurrence
		(successful)	(disarticulation)
		-1 failure (disarticulation)	-13 healed (92.8%)
		-4 NP
	Frisecke et al. [13]	12/100 (12%)	33/100 (33%):
		-2 failures (disarticulation)	-5 recurrences
		-10 healed	-28 healed (84.8%)
	Fountain et al. [15]	3/14 (21.4%):	12/14 (85.7%)
		-1 good outcome with antibiotics	Outcome N/S
		-2 revision in two phases / healed
	Hoell et al. [16]	0/12	5/12 (41.6%):
			5 healed (100%)
	Clement et al. [17]	2/19 (10.5%):	2/19 (10.5%):
		-1 long-term antibiotics	2 failures (postoperative infection)
		-1 disarticulation
	Amanatullah et al. [18]	7/20: 5 revisions N/S including	10/20 (50%): outcome N/S
		disarticulation / 1 or 2 stage implant
		change
	Toepfer et al. [19]	8/18 (44.4%): 8 healed (1 without	No cases of infection
		changing implant, 4 partial and 3
		complete)
	Our study	8/29 (27.6%): 5 healed / 3 failures (3	12/29 (41.4%):
		long-term antibiotics)	3 chronic infections
			9 healed (75%)

N/S: not specified


Aijing Lu

Eddy Petit

Suming Li
email: suming.li@umontpellier.fr

Yuandou Wang

Feng Su

Sophie Monge

Novel Thermo-responsive Micelles Prepared from Amphiphilic Hydroxypropyl Methyl Cellulose-block-JEFFAMINE Copolymers

Keywords: Hydroxypropyl methyl cellulose, JEFFAMINE, Self-assembly, Micelle, Topology, Thermo-responsive

Abstract:A series of amphiphilic and thermo-responsive block copolymers were synthesized by reductive amination between the aldehyde endgroup of hydrophilic HPMC and the amine group of monoamine, diamine, or triamine JEFFAMINE as hydrophobic block. The resulting diblock, triblock and three-armed copolymers with different hydrophilic/hydrophobic ratios and block lengths were characterized by NMR, FT-IR, DOSY-NMR and SEC. The cloud point (CP) of copolymers was determined by UV-visible spectrometer. Data show that both the geometrical structure and the molar mass of HPMC affect the CP of HPMC-JEF copolymers. The higher the hydrophilic/hydrophobic ratio, the higher the CP of copolymers which is lower than that of HPMC homopolymers. The self-assembly behavior of the copolymers was investigated from dynamic light scattering, transmission electron microscope, and critical micelle concentration (CMC) measurements. Spherical nano-micelles are obtained by self-assembly of copolymers in aqueous solution, and the micelle size can be tailored by varying the block length of HPMC and the geometrical structure. Three-armed HPMC-JEF copolymers present lower CMC and smaller micelle size as compared to linear diblock and triblock ones. MTT assay evidenced the cytocompatibility of HPMC-JEF copolymers, indicating that they could be promising as drug carrier in drug delivery systems.

Introduction

Amphiphilic block copolymers are able to self-assemble in aqueous solution yielding various aggregates such as spherical micelles, rod-like micelles, filomicells, polymersomes, nanotubes, etc. These aggregates have received ever-increasing interest for biomedical and pharmacological applications in drug delivery, [START_REF] Singh | A review on impact of nanomicelle for ocular drug delivery system[END_REF] imaging, [START_REF] Mane | Intrinsically fluorescent, stealth polypyrazoline nanoparticles with large stokes shift for in vivo imaging[END_REF] sensing, [START_REF] Saleem | Synthesis of amphiphilic block and glucose sensing[END_REF] etc. Micelles constitute a dynamic system with permanent exchanges between micelle-forming molecules and free molecules in solution, continuously breaking and re-forming. Some of the micellar systems are responsive to external stimuli such as pH, temperature, ionic strength and light irradiation. [START_REF] Ge | Supramolecular self-assembly of nonlinear amphiphilic and double hydrophilic block copolymers in aqueous solutions[END_REF][START_REF] Xu | Polymeric nanocarriers possessing thermoresponsive coronas[END_REF][START_REF] Cölfen | Double-hydrophilic block copolymers: synthesis and application as novel surfactants and crystal growth modifiers[END_REF][START_REF] Lee | Light-induced reversible formation of polymeric micelles[END_REF][START_REF] Zhao | Rational design of light-controllable polymer micelles[END_REF][START_REF] Wang | Purely salt-responsive micelle formation and inversion based on a novel schizophrenic sulfobetaine block copolymer: structure and kinetics of micellization[END_REF] Thermo-responsivity is one of the most interesting properties of stimuli-responsive polymers. Poly(N-isopropylacrylamide) (PNIPAAm) and poly(N-vinyl caprolactam) (PVCL) are most extensively investigated thermo-responsive polymers which exhibit a lower critical solution temperature (LCST) of approximately 32 °C in aqueous solution. [START_REF] Ramos | Temperature-sensitive nanogels: poly(N-vinylcaprolactam) versus poly(N-isopropylacrylamide)[END_REF] Interestingly, PNIPAAm can be used as hydrophilic shell-forming segments below the LCST and as hydrophobic core-forming segments above the LCST. [START_REF] Wei | Thermo-sensitive polymeric micelles based on poly(N-isopropylacrylamide) as drug carriers[END_REF] Polysaccharides such as cellulose, pullulan, dextran, chitosan, and hyaluronan have attracted growing attention in recent years for uses as biomaterials because of their biodegradability, biocompatibility and inherent bioactivity which can help in improving drug targeting and diminishing inflammatory response. Cellulose is the most abundant biopolymer consisting of linear chains of numerous β(1→4) linked D-glucose units. It has been widely used for applications in fiber, paper, painting and pharmaceutical industries. Nevertheless, cellulose is poorly soluble in water due to strong hydrogen bonding between hydroxyl groups on the glucose from one chain with oxygen atoms on the same or on a neighbor chain. [START_REF] Finkenstadt | Crystal structure of valonia cellulose Iβ[END_REF] Thus, soluble cellulose derivatives have been obtained by chemical modification and used as a hydrophilic building block to prepare amphiphilic copolymers, such as ethyl cellulose [START_REF] Dou | pH-dependent self-assembly: micellization and micelle-hollow-sphere transition of cellulose-based copolymers[END_REF][START_REF] Uraki | Body temperature-responsive gels derived from hydroxypropylcellulose bearing lignin[END_REF][START_REF] Yuan | Amphiphilic ethyl cellulose brush polymers with mono and dual side chains: Facile synthesis, self-assembly, and tunable temperature-pH responsivities[END_REF], hydroxyl propyl cellulose (HPC) [START_REF] Ostmark | Unimolecular nanocontainers prepared by ROP and subsequent ATRP from hydroxypropylcellulose[END_REF], hydroxyl propyl methyl cellulose (HPMC) [START_REF] Ostmark | Unimolecular nanocontainers prepared by ROP and subsequent ATRP from hydroxypropylcellulose[END_REF], hydroxyl ethyl cellulose [START_REF] Hsieh | Nano-sized micelles of block copolymers of methoxy poly(ethylene glycol)-poly(ε-caprolactone)-graft-2-hydroxyethyl cellulose for doxorubicin delivery[END_REF], etc. Some derivatives such as HPC and HPMC exhibit thermo-responsive behaviors. Most modifications exploit the hydroxyl groups on the glucose moieties to prepare graft copolymers. But such reactions are hardly controllable because of the presence of numerous hydroxyl groups along polymer chains. On the other hand, each cellulose chain has a hemiacetal end group which is easily transformed to aldehyde. The latter can serve as a reducing endgroup to build amphiphilic block copolymers by coupling or initiating polymerization. Nevertheless, such reactions have not been widely exploited despite their potential interest due to the limited availability of the reducing-end, the difficulty of finding a common solvent for both blocks, and the eventual necessity to protect the lateral hydroxyl groups. [START_REF] Schatz | Polysaccharide-containing block copolymers: synthesis, properties and applications of an emerging family of glycoconjugates[END_REF] Meanwhile, HPMC oligomers with molar masses between 5K and 20K have been rarely employed as hydrophilic segment to synthesize amphiphilic block copolymers. In our previous work, HPMC-polylactide (HPMC-PLA) block copolymers were synthesized by using a three step procedure: ring-opening polymerization of L-lactide initiated by allyl alcohol, amination reduction of the aldehyde endgroup of low molar mass HPMC, and UV-initiated thiol-ene click reaction. [START_REF] Wang | Synthesis and self-assembly of amphiphilic block copolymers from biobased hydroxypropyl methyl cellulose and poly(L-lactide)[END_REF] JEFFAMINE is a family of polyetheramine composed of poly(propylene oxide) (PPO), poly(ethylene oxide) (PEO), or mixed PPO/PEO blocks with primary amino groups attached to the polyether chain ends. JEFFAMINE M2005 with a PPO/PEO ratio of 29/6 exhibits a LCST of around 30 ℃ in water [START_REF] Agut | Thermoresponsive micelles from Jeffamine-b-poly(L-glutamic acid) double hydrophilic block copolymers[END_REF]. JEFFAMINE exists in the form of monoamines, diamines, and triamines. Thus, amphiphilic block copolymers with different geometrical structures can be obtained combining JEFFAMINE and a hydrophilic block. It has been shown that the geometrical structure of copolymers affects the morphology, stability and size of self-assembled micelles because the hydrophobic and hydrophilic segments lead to complex spatial arrangements during the micellization process [START_REF] Cao | Effect of architecture on the micellar properties of poly (varepsilon-caprolactone) containing sulfobetaines[END_REF][START_REF] Li | Multicompartment micelles from ABC miktoarm stars in water[END_REF]. In this work, HPMC-JEF amphiphilic block copolymers were synthesized from HPMC with different molar masses and JEFFAMINE monoamine, diamine, and triamine by reductive amination.

The self-assembly properties of the resulting diblock, triblock and tri-armed copolymers were investigated from the morphology, size and size distribution, cloud point (CP) and critical micelle concentration (CMC) measurements. MTT assay was performed to evaluate the biocompatibility of HPMC-JEF copolymers as potential drug carrier.

Materials and Methods

Materials

HPMC was kindly provided by Dow Colorcon Limited France, and depolymerized to yield HPMC oligomers with weight average molar mass (Mw) of 7000 (Ð=2.3) and 18000 (Ð=3.3) Daltons, as reported in a previous work. [START_REF] Wang | Synthesis and self-assembly of amphiphilic block copolymers from biobased hydroxypropyl methyl cellulose and poly(L-lactide)[END_REF] JEFFAMINE monoamine M-2005, diamine D-2000, and triamine T-5000 were purchased from Huntsman. Sodium triacetoxyborohydride (NaBH(OAc)3) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich, and used as received.

Synthesis of HPMC-JEF copolymers

HPMC-JEF copolymers were synthesized by reductive amination as reported in literature.

Typically, 0.314 g (0.157 mmol) JEFFAMINE M-2005, 1.0 g (0.143 mmol) HPMC with Mw=7000, and 0.042 g ( 0.198 mmol) NaBH(OAc)3 were dissolved in 20 mL DMSO under stirring. The reaction proceeded at room temperature for 24 h. The product was obtained by dialysis against deionized water for 72 h at room temperature. The dialysis medium was renewed every 12 h. A white flocculent solid was obtained after 48 h freeze-drying. The yield of the reaction was 69.4%.

Characterization

1 H NMR spectra were record on Bruker spectrometer operating at 300 MHz (AMX300) using D2O and DMSO-d6 as solvent. Diffusion ordered spectroscopy (DOSY) NMR was performed on Bruker Avance (AQS600) NMR spectrometer, operating at 600 MHz and equipped with a Bruker multinuclear z-gradient inverse probe head which is able to produce gradients in the z direction with a strength of 55 Gcm -1 . The DOSY NMR spectra were acquired from Bruker topspin software with the ledbpgp2s pulse program. The strength of the pulsed field gradients with respect to maximum 32 increments on a quadratic scale was logarithmically incremented from 2 to 95%. The diffusion sensitive period (Δ) of 200 ms and the gradient duration (δ) of 5 ms were optimized to allow the signals of interest to decrease by a factor of 10-20, in order to keep the relaxation contribution to the signal attenuation constant and ensure full signal attenuation for all samples. MestRe Nova software was used for the treatment of all NMR spectra. Fourier transform infrared (FT-IR) spectra were recorded on a Nicolet NEXUS spectrometer with a DTGS detector at 4 cm -1 resolution. Sixty-four scans were taken for each analysis in the frequency range from 400 to 4000 cm -1 . Sample pellets were prepared by mixing 2-2.5 mg of polymer with 200 mg of spectral grade potassium bromide, followed by compression using a press.

The molar mass and polydispersity of polymers were determined by size exclusion chromatography (SEC), equipped with multi-angle laser light scattering (MALLS) in combination with refractive index (RI) detector. 5 mg sample was added in the mobile phase (10×10 -3 M NaNO3 containing 0.02% NaN3), and stirred for 24 h at room temperature. The solution was filtered using 0.45 µm polytetrafluoroethylene (PTFE) filter (Millipore). The filtrate was injected through a 100 µL loop (Rheodyne injector 7725), and eluted in a TSK-GEL GMPWXL 7.8×300 mm column (TosoHaas Bioseparation Specialists, Stuttgart, Germany) at a flow rate of 0.5 mL min -1 (Waters pump 515). MALLS detector (Dawn DSP, Wyatt Technology Co, Santa Barbara, CA, USA) and RI detector (Optilab Wyatt Technology Co) were used to online determine the absolute molar mass for each elution fraction of 0.01 mL, which enables to calculate the Mw and dispersity (Ð). The refractive index increment (dn/dc) used for calculation was 0.129 mL g -1 for HPMC-JEF copolymers, and 0.137 for HPMC homopolymers. Data analysis was realized using Astra 4 (Wyatt Technology Co.).

The cloud point (CP) of the copolymers was estimated from transmittance changes of 5.0 mg mL -1 solution in the temperature range from 30 to 90 ℃. The solution was stirred for 24 h and kept at 4 °C overnight before analysis. Measurements were made at a wavelength of 500 nm with a Perkin Elmer Lambda 35 UV-visible spectrometer equipped with a Peltier temperature programmer PTP-1+1. The temperature ramp was 0.2 °C min -1 . Temp Lab software was used for data treatment. The critical micelle concentration (CMC) was determined by fluorescence spectroscopy using pyrene as fluorescent probe. The concentration of polymers varied from 1.0×10 -3 to 2.0 mg/ml, and the concentration of pyrene was fixed at 6×10 -7 g/L. The excitation spectra of the micellar solutions were recorded between 300 to 360 nm at an excitation wavenumber of 395. The intensity ratio at 336 nm and 334 nm was plotted versus copolymer concentration. The CMC value was obtained from the cross-over point of the two regression lines.

The size and zeta potential of micelles were determined by dynamic light scattering (DLS) using Anton Paar Litesizer 500 at 25℃. The aqueous solutions of copolymers at 1.0 mg mL -1 were filtered through a 0.45 µm PTFE microfilter. Micelle size changes as a function of temperature from 4 to 60 °C were followed using Zano-ZS (Malvern Instrument) equipped with a He-Ne laser (λ=632.8 nm). Measurements were made after 60 min equilibrium of copolymer solutions at 1.0 mg mL -1 .

Transmission electron microscopy (TEM) was performed on JEOL 1200 EXII instrument, operating at an acceleration voltage of 120 kV. 5 µL micellar solution at 1.0 mg mL -1 was dropped onto a copper grid, stained with phosphotungstic acid, and air dried before measurement.

MTT assay

L-929 cells in logarithmic growth phase were seeded in 96-well plates (Corning Costar, U.S.A.) with a density of 1 × 10 4 cells/mL per well in 100 μL DMEM medium (10% calf serum, 100 μg/mL Penicillin, 100 μg/mL streptomycin). The cells were then placed in a CO2 incubator (NU-4850; NuAire, U.S.A.) at 37 °C under humidified atmosphere containing 5% CO2. After 24 h, the medium was replaced by 100 μL of fresh medium or HPMC-JEF micellar solution at various concentrations (0.25, 0.5, 1.0, 2.0, 2.5 and 5.0 mg/mL) containing 10.0 % calf serum. 100 μL fresh medium was used as the negative control, and 100 μL solution of phenol in water as the positive control [START_REF] Zhang | Evaluation of cytotoxicity of MTAD using the MTT-tetrazolium method[END_REF]. After 24, 48 and 72 h, 20 μL 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, 5 mg/mL) was added. The medium was removed after 6 h incubation, and then 150 μL dimethylsulfoxide (DMSO) was added. The plates were shaken for 10 min. The optical density (OD) was measured by using microplate reader (Elx800; BioTek, U.S.A.) at 570 nm. All experiments were carried out in triplicate. The cell viability was calculated from the OD values of the test sample and the negative control using the following equation.

Relative activity (%) = (ODtest sample ∕ ODnegative control) × 100

The data were expressed as the mean ± SD. Table 1.

The molar mass of copolymers was determined by using aqueous SEC in conjunction with online MALLS and RI detectors. RI detector is most commonly used as concentration detector whose response is proportional to the total solute concentration in the detector cell, whereas the response of MALLS detector depends on the molar mass of the solute. [START_REF] Kostanski | Size-exclusion chromatography-a review of calibration methodologies[END_REF] The molar mass of copolymers was calculated combining with the MALLS and RI curves. Table 1 presents the Mw and dispersity (Ð=Mw/Mn) data of the various copolymers obtained by reaction of HPMC7K and HPMC18K with JEFFAMINE samples M2K, D2K and T5K. The Mw of HPMC7K derived copolymers increases from 6 500 for the diblock copolymer HPMC7K-JEF M2K, to 14 000 for the triblock copolymer (HPMC7K)2-JEF D2K, and to 19 000 for the three-armed copolymer (HPMC7K)3-JEF T5K. Similarly, the Mw of HPMC18K derived copolymers increases from 25 000 for HPMC18K-JEF M2K, to 43 000 and 56 000 for (HPMC18K)2-JEF D2K and (HPMC18K)3-JEF T5K, respectively. These results globally corroborate with the initially designed structure of copolymers, indicating the successful synthesis of copolymers by reductive amination. Nevertheless, there is some discrepancy between the Mw values obtained by SEC and those by theoretical calculation. In fact, GPC is a chromatographic method in which molecules in solution are separated by their size. Thus, HPMC-JEF copolymers with different architectures could have various error range for GPC analysis. Meanwhile, when an RI detector is combined with light scattering, the exact concentration of the sample at each data slice needs to be determined in order to calculate the absolute molecular weight. The key parameter to get exact concentration is the refractive index increment (dn/dc) which is a unique value for a sample-solvent combination.

The dn/dc used for calculation was 0.129 mL g -1 for HPMC-JEF copolymers, and 0.137 for HPMC homopolymers. Copolymers with different architectures and different molar masses may have slight difference in dn/dc, thereby cause some error in SEC results. In addition, HPMC oligomers were obtained by degradation from high molar mass HPMC and have a large molar mass distribution. The dispersity (Ð) is 3.3 for HPMC 18K and 2.3 for HPMC 7K.

And it is known that high-molar-mass species strongly influence the value of Mw [START_REF] Striegel | Modern size exclusion chromatography[END_REF]. In the case of the HPMC-JEF copolymers, the dispersity varies from 1.38 for HPMC18K-JEF M2K to 2.30 for (HPMC18K)3-JEF T5K, which is narrower than the HPMC homopolymers due to dialysis.

Cloud point

HPMC is a thermo-responsive polymer which precipitates out of the solution when the temperature reaches the cloud point due to loss of water or dehydration. [START_REF] Mitchell | The influence of additives on the cloud point, disintegration and dissolution of hydroxypropylmethylcellulose gels and matrix tablets[END_REF] The phase separation leads to a polymer rich phase and a polymer depleted phase. Thus the cloud point can be determined as the polymer rich phase is capable of scattering light. [START_REF] Mohammadpour | HPMC cloud point: exploring hydroxypropylmethyl cellulose behavior in pharmaceutical formulations[END_REF] Figure 4.

The CP of the HPMC-JEF aqueous solutions was determined by transmittance measurements at 500 nm, as shown in Fig. 4. HPMC18K exhibits a CP around 78.0 ℃, whereas HPMC7K has no obvious change in the tested temperature range. Thus, the CP of HPMC homopolymers decreases with the increase of molecule weight. Similar result has been reported in literature in the case of narrow-disperse PNIPAMs. As the Mn,NMR increases from 2.8 to 26.5 KDa, the CP drops from 43.0 to 33.3 °C. [START_REF] Xia | Thermal response of narrow-disperse poly(N-isopropylacrylamide) prepared by atom transfer radical polymerization[END_REF] The CP of HPMC-JEF copolymers is lower than that of HPMC homopolymers in all cases. Meanwhile, the CP of linear diblock and triblock HPMC18K-JEF copolymers was higher than that of HPMC7K-JEF ones. Therefore, the CP of linear JEF-HPMC copolymers is strongly dependent on the hydrophilic/hydrophobic ratio. The higher the hydrophilic/hydrophobic ratio, the higher the CP of copolymers. Similar finding has been reported for P(EO2MA-co-OEGMA) copolymers whose CP could be tuned from 28 ℃ to 90℃ by adjusting the copolymer composition. [START_REF] Zhang | Core-shell magnetite nanoparticles surface encapsulated with smart stimuli-responsive polymer: synthesis, characterization, and LCST of viable drug-targeting delivery system[END_REF][START_REF] Lutz | Point by point comparison of two thermosensitive polymers exhibiting a similar LCST: Is the age of poly(NIPAM) over?[END_REF][START_REF] Lutz | Preparation of ideal PEG analogues with a tunable thermosensitivity by controlled radical copolymerization of 2-(2-methoxyethoxy)ethyl methacrylate and oligo(ethylene glycol) methacrylate[END_REF]. It also has been reported that PLLA-PNIPAAm copolymers exhibit a CP between 32.1 and 32.8 ℃ with the [NIPAAm]/[LA] ratio varying from 2.4 to 6.9. [START_REF] Hu | Synthesis and self-assembling of poly(N-isopropylacrylamide-block-poly(L-lactide)-block-poly(N-isopropylacrylamide) triblock copolymers prepared by combination of ring-opening polymerization and atom transfer radical polymerization[END_REF] The situation of the three-armed (HPMC)3-JEF T5K copolymers is more complicated.

Compared with the linear copolymers, the three-armed (HPMC7K)3-JEF T5K copolymer presents a higher CP (82.9°C) in spite of its higher JEFFAMINE fraction. This finding could be assigned to the topological structures which make the interactions between polymer and solvent different from those of linear copolymers. Honda et al. reported that the CP of amphiphilic poly(butyl acrylate)-block-poly(ethylene oxide) copolymers is strongly affected by the topological structure. [START_REF] Honda | Topology-directed control on thermal stability: micelles formed from linear and cyclized amphiphilic block copolymers[END_REF] An increase by more than 40 °C was observed through the linear-to-cyclic topological conversion. Data for (HPMC18K)3-JEF T5K are not available because it is not fully soluble at 5 g/L. It should be noted that different from the JEF D2K and JEF T5K composed exclusively of PO units, JEF M2K is a thermos-responsive block composed of hydrophobic PO moieties and hydrophilic EO ones with a PO/EO molar ratio of 29/6 (Table S1). In fact, JEF M2K exhibits a CP of 22.6°C at a concentration of 5 g/L (Fig. S2). Two transitions of transmittance corresponding to both components are observed for the diblock copolymers HPMC7K-JEF M2K and HPMC18K-JEF M2K (Fig. 4, Table 1).

Self-assembly of HPMC-JEF copolymers

Micelles of HPMC-JEF copolymers were prepared by direct dissolution method. 10 mg copolymer was dissolved in 10 mL deionized water under stirring overnight. The self-assembled HPMC-JEF micelles consisted of a JEF core surrounded by a HPMC corona. The critical micelle concentration was determined by fluorescence excitation spectroscopy using pyrene as fluorescence probe. Pyrene is highly hydrophobic and tends to migrate into the lipophilic micellar core in the aqueous solution. As shown in Fig. 5, the shift of the fluorescence excitation spectra indicates the process of micellar forming. The CMC value was determined from the two regression lines of the curves of the intensity ratio of I336/334 versus polymer concentration. As shown in Table 1, no major difference is observed between the CMC values of the diblock and triblock copolymers which are in the range of 0.13 to 0.24 g/L. In contrast, the CMC of the three-armed copolymers is much lower.

(HPMC7K)3-JEF T5K and (HPMC18K)3-JEF T5K exhibit a CMC of 0.03 and 0.07 g/L, respectively. In other words, the CMC of three-armed copolymers increases with increasing hydrophilic HPMC block length, which is consistent with literature data. [START_REF] Guo | Preparation of cellulose-graft-poly(varepsilon-caprolactone) nanomicelles by homogeneous ROP in ionic liquid[END_REF] In fact, Guo et al.

reported that cellulose-g-PCL graft copolymers with higher degree of substitution (DS) of PCL present lower CMC value than those with lower DS. The fact that three-armed copolymers exhibit lower CMC than linear diblock and triblock copolymers could be assigned to the difference of the topological structures since star-shaped block copolymers facilitate micellization. Similar results have been found for star-shaped PEG-b-PCL copolymers [START_REF] Kim | Synthesis and micellization of star-shaped poly(ethylene glycol)-block-poly(ɛ-caprolactone)[END_REF] and PCL-b-PDEAS copolymers [START_REF] Cao | Effect of architecture on the micellar properties of poly (varepsilon-caprolactone) containing sulfobetaines[END_REF]. Figure 6.

The size, size distribution, zeta potential and morphology of the micelle were determined by DLS and TEM, and the results are displayed in Table 1 and in Fig. 6. The average size of the micelle with different HPMC and JEF block lengths are in the range from 180 to 320 nm.

The three armed copolymers exhibited the smallest micelle size, whereas the triblock ones exhibited the largest micelle size. These findings could be assigned to the difference of topological structures. It has been reported that linear and star-shaped block copolymers with 3, 4 or 6 arms have significant difference in micelle size. [START_REF] Cao | Effect of architecture on the micellar properties of poly (varepsilon-caprolactone) containing sulfobetaines[END_REF][START_REF] Kim | Synthesis and micellization of star-shaped poly(ethylene glycol)-block-poly(ɛ-caprolactone)[END_REF] On the other hand, HPMC18K derived copolymers exhibit larger micelle size than HPMC7K derived ones, indicating that the micelle size increases with increasing hydrophilic block length. [START_REF] Guo | Preparation of cellulose-graft-poly(varepsilon-caprolactone) nanomicelles by homogeneous ROP in ionic liquid[END_REF] The polydispersity ranges from 0.18 to 0.25, in agreement with narrow distribution of micelle sizes. The morphology of micelles is shown in Fig. 6B. Spherical micelles are observed in all cases. It is also noted that the zeta potential of all micelles is close to zero, in agreement with the neutral nature of copolymers (Table 1). It is also of interest to follow the micelle size changes of doubly thermo-responsive diblock copolymers at different temperatures. As shown in Fig. 7, three phases of micelle size changes are distinguished for HPMC7K-JEF M2K and HPMC18K-JEF M2K diblock copolymers. In the first phase, the micelle size of HPMC7K-JEF M2K increases from 162 nm at 4℃ to 340 nm at 20℃. In the second phase, the micelle size decreases to reach 190 nm at 30 °C. Beyond, the micelle size increases again to reach 380 nm at 60℃. Similar changes are observed for HPMC18K-JEF M2K. The micelle size increase observed in the first and third phases can be assigned to the chain extension of HPMC blocks as the temperature increases.

Similar result has been reported in literature. Yang et al. observed a micelle size increase for PLA-PEG copolymers with increasing temperature from 15℃ to 35℃. [START_REF] Yang | Aggregation behavior of self-assembling polylactide/poly(ethylene glycol) micelles for sustained drug delivery[END_REF] In the second phase from 20 to 30℃, the size deceases because of the phase transition of JEF M2Kblocks.

It has been reported that the JEF core size of JEF M2K-b-poly(L-glutamic acid) copolymer micelles decreases with increasing temperature as a result of dehydration. [START_REF] Agut | Thermoresponsive micelles from Jeffamine-b-poly(L-glutamic acid) double hydrophilic block copolymers[END_REF] Above 60℃, the micelles become unstable and aggregated as both blocks become hydrophobic. In vitro cytotoxicity of biomaterials is generally evaluated by MTT assay. This method is based on the reaction between MTT and mitochondrial succinate dehydrogenases in living cells to form a purple formazan which is soluble in DMSO but insoluble in water. The OD value of formazan-DMSO solution is considered to be proportional to the number of living cells [START_REF] Shen | Biocompatibility of filomicelles prepared from poly(ethylene glycol)-polylactide diblock copolymers as potential drug carrier[END_REF][START_REF] Li | Degradability and cytocompatibility of tricalcium phosphate/poly(amino acid) composite as bone tissue implants in orthopaedic surgery[END_REF]. L-929 cells are a commonly used standard cell line in cyto-compatibility evaluation. The effect of micelle solutions on the growth of L-929 cells is shown in Fig. 8.

MTT assay

The viability of cells is very high in micelle solutions, the relative activity being well above 80% for all samples. With the increase of micelle concentration from 0.25 to 5.0 mg/mL, the cell viability remained almost unchanged. Meanwhile, incubation up to 72 h does not affect the cell viability for all groups. According to the US Pharmacopeia Standards, it can be concluded that HPMC-JEF micelles are not toxic to L-929 cells. It is also noticed that the viability is very low in the positive control.

Conclusion

Amphiphilic and thermo-responsive block copolymers were synthesized by reductive amination between the aldehyde group of hydrophilic HPMC and the amine group of monoamine, diamine, or triamine JEFFAMINE as hydrophobic block. The resulting diblock, triblock and three-armed copolymers were characterized by NMR, FT-IR, DOSY-NMR and SEC. The various copolymers with different hydrophilic/hydrophobic ratios and block lengths self-assembled in water to yield spherical micelles. The geometrical structure of copolymers strongly affects the micelle size of micelles as well as the cloud point. Compared to the diblock and triblock copolymers, three-armed copolymers exhibit lower CMC, smaller size and lower cloud point. Interestingly, HPMC-JEF M2K diblock copolymers present two CP since both components are thermo-responsive. Moreover, the micelle size significantly changes with increasing temperature due to chain extension and/or dehydration. Last but not least, MTT assay showed that the HPMC-JEF micelles present outstanding cytocompatibility, suggesting that these novel copolymers can be safely used as potential drug carrier. Determined from SEC/MALS/RI measurement in water at 5.0mg mL Data are presented as the mean ± sd (n = 3)

3 .

 3 Scheme 1.

Figure 1 .

 1 Figure 1.

Figure 2 .

 2 Figure 2.

Figure 3 .

 3 Figure 3.

Figure 5 .

 5 Figure 5.

Figure 7 .

 7 Figure 7.

Figure 8 .

 8 Figure 8.

1 .Figure 1 . 1 HFigure 2 .

 1112 Scheme 1. Synthesis of HPMC-JEF M2K diblock copolymer by reductive amination Figure 1. 1 H NMR spectra of HPMC7K-JEF M2K in different solvents Figure 2. DOSY NMR spectrum of HPMC7K-JEF M2K copolymer in DMSO-d6.

Figure 3 .Figure 4 .Figure 5 .Figure 6 .Figure 7 .Figure 8 .

 345678 Figure 3. FT-IR spectra of JEFFAMINE (A), HPMC (B) and HPMC-JEF M2K copolymer (C) Figure 4. Plots of light transmittance as a function of temperature for aqueous solutions of HPMC7K-JEF (A) and HPMC18K-JEF (B) copolymers Figure 5. Fluorescence excitation spectra (A) and I336/I334 vs. LogC plots (B) of HPMC7K-JEF M2K diblock copolymer solutions at different concentrations Figure 6. Size distribution (A) and TEM image (B) of HPMC7K-JEF M2K diblock copolymer micelles Figure 7. Micelle size changes of HPMC7K-JEF M2K (A) and HPMC18K-JEF M2K (B) as a function of temperature Figure 8. Relative activity of L-929 cells after 24, 48 and 72 h culture with micelle solutions of HPMC7K-JEF M2K (A), HPMC18K-JEF M2K (B), (HPMC7K)2-JEF D2K (C), and (HPMC7K)3-JEF T5K (D) at different concentrations in comparison with the positive control.

Table 1 .

 1 Characterization of HPMC-JEF block copolymers

				CP b	Size c		Zeta c	CMC d
	Sample	Mw a	Ð a			PDI c
				(℃)	(nm)		(mV)	(g/L)
	HPMC7K-JEF M2K	6500	1.73	42.1/69.0	231.9	0.22	-1.8±0.1	0.24
	(HPMC7K)2-JEF D2K	14000	1.95	78.5	261.1	0.19 -4.21±0.3	0.24
	(HPMC7K)3-JEF T5K	19000	2.0	82.9	183.7	0.24	-3.7±0.2	0.03
	HPMC18K-JEF M2K	25000	1.38	35.1/60.1	298.4	0.18	-7.0±0.3	0.13
	(HPMC18K)2-JEF D2K 43000	2.01	55.7	328.2	0.25	-2.0±0.2	0.24
	(HPMC18K)3-JEF T5K 56000	2.30	-	270.7	0.18	-6.7±0.3	0.07
	a)

Acknowledgements

This work is supported in part by the scholarship from China Scholarship Council (Grant CSC N°201606240124).