L Molimard

M Navarro

S Combreas

R Avril

Paul Philippot

Calmels

B Pierrat

R Oullion

J Molimard

L Navarro

M Combreas

S Avril

R Philippot

P Calmels

Characterisation of in-vivo mechanical action of knee braces regarding their anti-drawer effect

HAL is

Introduction

The knee is the largest joint in the body and supports high loads, up to several times the body weight. It is vulnerable to injury during sport or professional activities, potentially leading to chronic knee instability. This instability is a functional issue to the patient and is characterized by a "wobbly" feeling. Different internal structures take part in joint stabilization by passive (ligaments, capsule) or active (neuro-muscular system and proprioception) action. The most common injury involves the anterior cruciate ligament (ACL) complete or partial rupture: it is involved in 24% of all knee injuries and 59% of ligamentous injuries [START_REF] Bollen | Epidemiology of knee injuries: diagnosis and triage[END_REF]. In the United States,the annual incidence in the general population is approximately 1 in 3500 with 100,000 ACL reconstructions performed each year [START_REF] Gordon | Anterior cruciate ligament injuries, Orthopaedic Knowledge Update: Sports Medicine[END_REF][START_REF] Miyasaka | The incidence of knee ligament injuries in the general population[END_REF]. These conditions are a huge burden on individuals and healthcare systems.

Diagnosis of knee instability involves a discussion with the patient and a clinical examination, usually the Lachman test. It consists in a manual anterior translation of the tibia aiming at putting the ACL in tension. By practice, the examiner is able to grade the laxity by severity [START_REF] Dojcinovic | Instabilités du genou[END_REF]. However, its sensitivity and specificity to detect complete ACL ruptures depends on the experience of the examiner, the patient's body type and the delay between the accident and examination [START_REF] Collette | Objective evaluation of anterior knee laxity; comparison of the KT-1000 and GNRB R arthrometers, Knee surgery, sports traumatology, arthroscopy[END_REF]. In order to reduce this variability, arthrometers were developed. These devices apply an increasing force to induce a postero-anterior drawer and measure the corresponding translation. Displacement-load curves of the healthy and injured knees are compared; ACL rupture is ascertained when differential laxity is higher than a certain threshold. Well-known arthrometer devices are the KT-1000 developed by [START_REF] Daniel | Instrumented measurement of anterior laxity of the knee[END_REF] and the GNRB R [START_REF] Robert | Nouveau système de mesure des laxités sagittales du genou, le GNRB R . application aux ruptures complètes et incomplètes du ligament croisé antérieur[END_REF]. The former is very popular and has been widely studied; its sensitivity to detect complete ACL ruptures is 77% and its specificity 90% (threshold: differential laxity of 3 mm at 130 N). The latter was developed recently; two studies [START_REF] Collette | Objective evaluation of anterior knee laxity; comparison of the KT-1000 and GNRB R arthrometers, Knee surgery, sports traumatology, arthroscopy[END_REF][START_REF] Robert | Nouveau système de mesure des laxités sagittales du genou, le GNRB R . application aux ruptures complètes et incomplètes du ligament croisé antérieur[END_REF] highlighted the superiority of this device compared to similar apparatus. A differential threshold of 3 mm at 134 N is used to determine complete rupture: its sensitivity is 70% and specificity 99%. This arthrometer device is also used to diagnose partial ruptures (threshold of 1.5 mm at 134 N) with a sensitivity of 80% and a specificity of 87%.

Knee braces or orthoses are usually part of the standard therapy for knee instability and are commonly prescribed by physicians and medical practitioners. Their claimed mechanical effects are to support/align the joint and increase proprioceptive input [START_REF] Paluska | Knee braces: current evidence and clinical recommendations for their use[END_REF]. However, very few studies actually show significant actions, from biomechanical studies to therapeutic trials [START_REF] Thoumie | Orthèses de genou. première partie : Évaluation des propriétés physiologiques à partir d'une revue de la littérature. knee orthosis. first part : evaluation of physiological justifications from a literature review[END_REF][START_REF] Thoumie | Orthèses de genou. Évaluation de l'efficacité clinique à partir d'une revue de la littérature. knee orthosis[END_REF]. Mechanical/physiological effects have been emphasized, but the mechanisms of action have been poorly characterized [START_REF] Paluska | Knee braces: current evidence and clinical recommendations for their use[END_REF][START_REF] Thoumie | Orthèses de genou. première partie : Évaluation des propriétés physiologiques à partir d'une revue de la littérature. knee orthosis. first part : evaluation of physiological justifications from a literature review[END_REF][START_REF] Thoumie | Orthèses de genou. Évaluation de l'efficacité clinique à partir d'une revue de la littérature. knee orthosis[END_REF][START_REF] Genty | Place des orthèses en pathologie ligamentaire du genou. revue de la littérature[END_REF][START_REF] Beaudreuil | Clinical practice guidelines for rest orthosis, knee sleeves, and unloading knee braces in knee osteoarthritis[END_REF][START_REF] Chew | Current evidence and clinical applications of therapeutic knee braces[END_REF]. What is more, subjective evaluations of patients highlight a large demand for these products; therefore, their efficiency is still widely discussed among medical experts. In particular, the relative importance of the two principal stabilizing mechanisms is not known:

-joint stiffening by adding supporting structures, e.g. hinged bars secured to the joint by straps and fabric (passive mechanism)

-neuromuscular control enhancement by proprioceptive effect (active mechanism)

As a consequence of these uncertainties, medical practitioners and manufacturers still lack a simple evaluation tool for knee orthoses. A French committee of experts highlighted this problem [START_REF] Ribinik | Évaluation des orthèses de genou et de cheville en pathologie de l'appareil locomoteur. avis d'experts[END_REF] and stated that orthoses must be evaluated by taking both the mechanisms of action and the desired therapeutic effects into account.

Although the passive action of knee braces to prevent a drawer motion has already been characterized on surrogate limbs [START_REF] Beck | Instrumented testing of functional knee braces[END_REF][START_REF] Cawley | Comparison of rehabilitative knee braces[END_REF][START_REF] Liu | Comparison of functional knee braces for control of anterior tibial displacement[END_REF], only one attempt has been made to link these measures to corresponding expected in-vivo actions through numerical modelling [START_REF] Pierrat | Evaluation of the mechanical efficiency of knee orthoses: a combined experimental-numerical approach[END_REF][START_REF] Pierrat | Evaluation of the mechanical efficiency of knee braces based on computational modelling[END_REF], highlighting a passive stiffening much lower than what is brought by intact ligaments. However, a real clinical study is still needed to validate the numerical results and investigate the influence of patient-specific factors such as morphology, injury severity, neuromuscular adaptation and instability feeling control.

This study is aimed at objectively quantifying the joint stiffening action of various commercial braces in-vivo using a GNRB R arthrometer on a number of pathological patients.

Material and methods

The described protocol has been validated by the Ethical Committee of the University Hospital of Saint-Etienne, France.

Patients

A sample of 25 subjects (16 males and 9 females) were selected for this study among hospitalized patients in the orthopaedic and traumatology surgery unit of the University Hospital of Saint-Etienne, France. The inclusion criteria were the following:

-Patients being treated for functional knee instability, either a chronic laxity or after ACL injury, before reconstructive surgery.

-Acute knee anterior instability (positive Lachman drawer test).

-Patients that had been prescribed an arthrometer test in this context.

-Independent walkers.

-Patients who have agreed study conditions and signed the information notes.

Some exclusion criteria were also identified:

-Very recent trauma (e.g. acute sprained knee).

-Other musculoskeletal disease (e.g. fracture, arthrosis...).

Subjects were prospectively and consecutively recruited from February 2013 to July 2013. The characteristics of the whole recruited population are described in Table 1.

Knee braces

Three local orthotic manufacturers agreed to lend their products for this study (Gibaud R , Lohmann & Rauscher R and Thuasne R ). As a wide range of knee braces is available (both custom-fitted and off-the-shelf orthoses), the focus was placed on fabric-based off-the-shelf hinged braces which are the highestselling products of these manufacturers for the studied clinical indication.

The following products were selected for testing (one control brace and one hinged brace for each manufacturer):

• Compression sleeve: Thuasne Genuaction (control brace).

• Fabric hinged orthoses:

-Gibaud GebuGib Stab -Lohmann & Rauscher Ligaction Pro -Thuasne Ligaflex Evolution
They are depicted in Figure 1.

From now on, the names of the braces have been replaced by numbers to de-identify the products and not penalize the manufacturers. The compression sleeve was selected to act as a control brace, because its passive mechanical stiffening action was characterised as negligible [START_REF] Pierrat | Evaluation of the mechanical efficiency of knee orthoses: a combined experimental-numerical approach[END_REF]. Consequently, its effect should not be measured by the arthrometer unless its proprioceptive effect activates muscular stiffening during the tests. The three other braces are similar in design (fabric with hinged rigid bars and straps) but previous work showed slightly different levels of mechanical actions [START_REF] Pierrat | Evaluation of the mechanical efficiency of knee orthoses: a combined experimental-numerical approach[END_REF]. They are prescribed for moderate and serious sprains, chronic laxity or during the rehabilitation process. It is noteworthy that brace #2 slightly differs from the two other braces in the sense that it features supplementary helical straps, and because it had an open design (unlike the two others that were closed cylinders).

ID

Braces were available in different sizes and chosen accordingly to the subject's knee circumference. They were adjusted by the subjects themselves.

GNRB R arthrometer

The GNRB R system is shown in Figure 2. It has already been described and evaluated in the literature [START_REF] Collette | Objective evaluation of anterior knee laxity; comparison of the KT-1000 and GNRB R arthrometers, Knee surgery, sports traumatology, arthroscopy[END_REF][START_REF] Robert | Nouveau système de mesure des laxités sagittales du genou, le GNRB R . application aux ruptures complètes et incomplètes du ligament croisé antérieur[END_REF][START_REF] Lefevre | Validity of GNRB( R ) arthrometer compared to telos TM in the assessment of partial anterior cruciate ligament tears, Knee surgery, sports traumatology, arthroscopy[END_REF].

The usual test protocol is the following. The patient is lying on a table; the lower limb is placed in the support such as the knee is in neutral rotation and the joint line is aligned between the calf support and the thigh support. The ankle and patella are held in place by strapped supports. The tightening of the latter support is controlled by a force sensor to be about 60 N. The patient is asked to relax and an electric actuator exerts an increasing load (up to 250 N) at 11 mm/s through the calf support. A displacement transducer (accuracy of 0.1 mm) measures the relative displacement of the tibia with respect to the patella with a sampling of 5 N, and a displacement-force curve is plotted. This procedure is usually performed once to check if the knee is painful, then three times in a row to check the reproducibility and compute the final response as the mean of these three curves. The maximum force is usually 250 N but may be reduced to 200 or 134 N if the patient feels pain. The usual protocol consists in testing both the healthy and pathological knee to compare the responses and diagnose the degree of laxity. The criterion for the diagnosis of ligament injury is the differential laxity ∆ at 134 N: complete ACL rupture is characterised by ∆ ≥ 3 mm (sensitivity of 70% and specificity of 99% [START_REF] Robert | Nouveau système de mesure des laxités sagittales du genou, le GNRB R . application aux ruptures complètes et incomplètes du ligament croisé antérieur[END_REF]) and partial rupture by ∆ ≥ 1.5 mm (sensitivity of 80% and specificity 87% [START_REF] Robert | Nouveau système de mesure des laxités sagittales du genou, le GNRB R . application aux ruptures complètes et incomplètes du ligament croisé antérieur[END_REF]).

Examples of curves given by this usual testing protocol for subjects 5 and 11 are reported in Figure 3, showing two different grades of injury: a partial tear and a complete ACL rupture.

Preliminary tests have showed no difficulty to perform laxity measurements on this apparatus with any of the knee braces because they all feature a patella opening allowing the patellar support to push directly on this area, and the rigid elements of the braces were not interacting with the securing system of the GNRB R (the hinged bars were free to move, as seen in Figure 2).

Protocol

As the normal diagnosis for these patients involved laxity measurement of both healthy and pathological knee with the GNRB R arthrometer, they were asked to take part of this study and perform 4 supplementary tests with the four different knee braces fitted on their pathological knee. Patients were also asked to grade the braces using a Visual Analog Scale (VAS) regarding two subjective criteria: stabilization sensation (0: no stabilization; 10: complete stabilization) and comfort (0: intolerable; 10: extremely comfortable).

As the internal structures of the knee were reported to have a highly viscoelactic behaviour [START_REF] Bonifasi-Lista | Viscoelastic properties of the human medial collateral ligament under longitudinal, transverse and shear loading[END_REF], one may expect that the repetition of tests would influence the measured laxities. Consequently, the order of the 4 braces to be tested was randomized for each subject.

The testing protocol took place in the following manner:

1. The usual laxity measurements were performed (on both healthy and pathological knees), with maximum forces of 250 N if possible, or lower if the subject experienced pain. 2. The appropriate size of the tested brace was fitted and adjusted by the patient himself, who had to walk around the room to be sure that it was properly tightened and fitted. Special care was taken in aligning the axis of rotation of the hinged bars with the axis of rotation of the knee. 3. The subjective evaluation of stabilization and comfort was performed after a few movements: 20 m walking, one-foot standing and squatting. 4. The subject was asked to place the braced pathological knee on the GNRB R for three consecutive tests (with maximum forces of 250 N or lower).

Steps 2 to 4 were repeated for each of the 4 braces.

A physical therapist experienced in using the GNRB R and in evaluating ACL deficiency performed the procedures.

Data processing

Collected data for each subject consisted in variables reported in Table 1, one averaged GNRB R test curve for the healthy knee and one for the pathological knee, three test curves for each brace and the two VAS scores for comfort and stabilization. The healthy knee was determined to be normal by subjective and objective testing and was used as the control.

Concerning laxity measurements, some patients were excluded from the final data due to outlying measurements:

-Patients with a differential laxity at 134 N lower than 1.8 mm (5 subjects) or higher than 7 mm (2 subjects).

-Patients who experienced pain and could not reach 200 N (1 subject).

-Patients who exhibited test curves with a highly different curvature than others (1 subject).

Consequently, data processing of laxity measurements was performed for 16 subjects (10 males and 6 females).

In order to compare the stiffening levels of the 4 braces, their action has to be decoupled from the inherent stiffness of the knee. For this purpose, the mechanical system could be modelled as three springs in parallel, as depicted in Figure 4: the ACL, the other structures of the knee and the brace. This parallel representation is valid because the displacement of the ensemble of structures is their common displacement. Consequently, the force applied on the ensemble is the sum of their individual forces.

As the arthrometer measures displacements for every 5 N load and produces displacement-load curves, the curves were interpolated using splines and resampled in displacement (every 0.05 mm) to obtain load-displacement curves. This was done to be able to add or subtract the different load responses. Confidence intervals (CIs) on the means were computed using Student's t distribution assuming that values were normally distributed. In this case, the total force F applied to the system is distributed among the springs so that

F = F 1 + F 2 + F 3 .
It was assumed that injured patients had a deficient ACL. Consequently, the following operations were performed for each subject to decouple the responses:

-Contribution of other knee structures: pathological knee curve.

-Contribution of the brace: braced pathological knee curvepathological knee curve.

-Contribution of the ACL: healthy knee curvepathological knee curve.

For comparing datasets between braces (VAS scores, brace effects), multiple comparison tests were performed. Outliers were defined as values larger than q 3 + (q 3 -q 1 ) or smaller than q 1 -(q 3 -q 1 ), where q 1 and q 3 are the 25th and 75th percentiles, respectively. This corresponds to approximately ± 2σ and 95% coverage if the data are normally distributed. The Matlab R R2012 was used to process all data.

Finally, the overall additional stiffness brought by the braces was characterised in terms of k indexes, as introduced in previous works [START_REF] Pierrat | Evaluation of the mechanical efficiency of knee orthoses: a combined experimental-numerical approach[END_REF][START_REF] Pierrat | Evaluation of the mechanical efficiency of knee braces based on computational modelling[END_REF]. This was performed by fitting a 1st order polynomial to the response of the braces for each subject, for displacements between 1 and 5 mm (linear part). The k index is the slope of the fitted line.

Results

Subjective evaluation of stabilization and comfort

The results of the subjective evaluation of the stabilization and comfort sensation of the tested braces is shown in Figure 5. No statistical difference was observed between the three hinged braces. However, the compression sleeve was reported to be statistically less stabilizing than the three braces, and more comfortable than braces #2 and #3. In all cases, a substantial scattering of the scores was observed.

Healthy and pathological knees

Raw test curves of the healthy and pathological knees as given by the GNRB R arthrometer are shown in Figure 6 for the 16 subjects. A high scattering of the curves can be noted. It was attempted to normalize GNRB R measurements by weight, size or knee circumference, but it did not affect the relative standard deviations. Besides, no significant statistical difference was observed between males and females. However, means of healthy and pathological knees were still statistically very different, as indicated by computing the average differential laxities: 3.3 mm at 134 N and 3.8 mm at 250 N.

Intra-and inter-subject variability

Laxity tests with the braced pathological knees were performed three times and results were extracted before being averaged by the GNRB R system, so it was possible to investigate intra-subject variability of these data. It was calculated as the standard deviations of the displacement for the four different braces (the type of brace had no effect on the scattering of the results) at 134 N and 250 N. A mean standard deviation of 2.3 mm was found at 134 N and Inter-subject variability was characterised as the standard deviation between averaged measurement. A mean standard deviation of 15 mm was found at 134 N and 19 mm at 250 N (relative dispersions of 24% and 17% respectively). This high variability shows that GNRB R measurements are very specific to each subject; this observation may be explained by different grades of injury and by the documented variability in mechanical properties of internal structures between individuals [START_REF] Quapp | Material characterization of human medial collateral ligament[END_REF]. It highlights the importance of statistical tests to determine if further results are significant for this number of subjects.

Decoupled contributions of the structures

The mean decoupled contributions of the different structures computed as described in Section 1.5 is shown in Figure 7. The healthy knee response is also shown to have an idea of the relative contribution of each structure. Firstly, the stiffness brought by the compression sleeve was very small, although it was not zero. The load taken by this sleeve increased between 0 and 1 mm drawer and rapidly stabilized around 14 N for higher displacements. The three hinged braces have relatively different responses and may be graded in term of antidrawer efficiency using these measurements: at 5 mm displacement, reaction forces were 27, 37 and 53 N for brace #3, #1 and #2 respectively. The curve profiles were similar with a rapid load increase up to 1 mm drawer, followed by a slower increase. It is noteworthy that reaction forces peaked around 5 mm displacement and slightly decreased thereafter for braces #3 and #1. This was not characteristic of brace #2, although this particularity may be explained by the fact that no high displacements were measured with this brace, so this curve stops before the other ones. Confidence intervals were large and some overlaps are visible, meaning that the responses between some braces were not statistically different (p ≥ 0.95). Only the compression sleeve had a significantly different response for displacements lower than 4 mm.

The load-displacement curve of the ligament exhibited a different profile: its response was more linear, even slightly exponential. Consequently, forces at low displacements were lower than values measured for braces, then largely exceeded these devices for higher displacements, reaching 83 N at 5 mm. This overrun occurred around 0.8 mm for brace #3, 1.6 mm for brace #1 and 2.8 mm for brace #2.

Interestingly, the mean response of the other structures of the knee (obtained with the pathological knees) was found to be very close to the response of the ACL alone, even if the ACL curve was concave while the other curve had a convex profile. This means that the ACL contributed to about half of the overall knee stiffness between 0 and 5 mm anterior displacement.

When comparing the healthy knee responses to the braced pathological knees, it was observed that the braces brought a substantial amount of stiffness compared to the pathological joints. In average, the knees braced with devices #3, #1 and #2 applied an even greater reaction force than the healthy joints for anterior displacements below 0.8 mm, 1.6 mm and 2.8 mm respectively. At 5 mm displacement, the relative load contributions normalized to the average healthy knee were the following (with 95% CI on the means):

-Pathological knee alone: 48 ± 6 % of the healthy knee.

-Pathological knee braced with compression sleeve: 57 ± 8 % of the healthy knee.

-Pathological knee braced with brace #3: 65 ± 7 % of the healthy knee.

-Pathological knee braced with brace #1: 71 ± 8 % of the healthy knee.

-Pathological knee braced with brace #2: 81 ± 9 % of the healthy knee.

Structure effects computed as k indexes.

As the responses were rather linear within the regression domain (1-5 mm), the fits were good and the coefficients of determination R 2 were almost always higher than 0.9. The mean k indexes of the different structures are displayed in Table 2. The ranking of the different devices in terms of efficiency to prevent drawer is the same as previously presented: the compression sleeve exhibited a very low mechanical action, and hinged braces presented large differences. As the standard deviations were high, the p-values were computed for each pair to determine whether differences were significant (p ≤ 0.05). Each pair was significantly different except the following structures: braces #1 and #2 (p=0.27), brace #3 and compression sleeve (p=0.58) and ACL and pathological knee (p=0.75). Braces #1 and #2 were found to bring significantly more stiffness than a simple sleeve. However, the k index of the ACL was found to be twice higher than the index of the most efficient brace. The results did not allow to determine a significant difference between braces #1 and #3, and between braces #1 and #2. Finally, it is noteworthy that no correlation was found between the differential laxity and the k indexes of the braces, meaning that the devices had the same mechanical effect for various degrees of laxity. Subsequently, no device was found to act differently between males and females, and no correlation was found with age or knee circumference. Note that this may only mean that the number of subjects was not high enough to determine significant correlations.

Structure

Discussion

This work was preceded by two other studies that introduced numerical [START_REF] Pierrat | Evaluation of the mechanical efficiency of knee braces based on computational modelling[END_REF] and experimental [START_REF] Pierrat | Evaluation of the mechanical efficiency of knee orthoses: a combined experimental-numerical approach[END_REF] tools to evaluate knee orthoses with a similar approach, so these studies take part in the discussions. Some limitations of the presented methodology have also been identified and are discussed below, but they were taken into account and do not discredit the given results.

Most of the recruited subjects did not usually wear a knee brace. Consequently, they may have adjusted their devices in a different manner as if they were regularly wearing them, and this fact must be taken into account when looking at the VAS scores. It may partly explain the high scattering of the grades, as the subjects did not have a common reference sensation of stabilization and comfort of braces.

No significant relationship was found between subjective stabilization grades and objective stiffening measurements for the 3 different hinged braces. It means that subjects were not able to grade the efficiency of these devices based on their feelings, and that physicians and manufacturers should rely on objective evaluation rather than on patient's feelings about a particular brace. However, VAS scores may still be helpful for comfort assessment. Indeed, an objective methodology to characterise wearing comfort over time has yet to be developed.

It is possible to compare the decoupled responses to the levels of action measured by other evaluation tools as presented in two previous studies [START_REF] Pierrat | Evaluation of the mechanical efficiency of knee orthoses: a combined experimental-numerical approach[END_REF][START_REF] Pierrat | Evaluation of the mechanical efficiency of knee braces based on computational modelling[END_REF]. The numerical study highlighted the influence of brace design on its ability to prevent a drawer motion. The presented braces essentially differed in terms of strap and fabric layout and of fabric stiffness. Brace circumferences may also vary: even if sizes were chosen accordingly to the manufacturer's size charts, a product may size too small and another too big. By selecting a factor range representative of the 4 tested braces, the numerical model predicted efficiency indexes ranging from 1.9 to 3.4 N/mm and corresponding pressure indexes between 2.9 and 7.1 kPa. The exact same braces have also been experimentally tested on the developed surrogate lower limb and the following mean drawer indexes have been measured:

-Brace #1: 1.85 ± 0.36 N/mm.

-Brace #2: 5.49 ± 0.69 N/mm.

-Brace #3: 3.36 ± 0.53 N/mm. The most efficient brace was found to be the same as in the present study. It is noteworthy that indexes computed from arthrometer measurements are higher than those computed from the data measured by the test machine. Some numerical comparison in this study also suggested that testing these braces on a real limb would yield even lower efficiency indexes, which was found here to be incorrect. This may be partially explained by the fact that in-vivo measurements may be slightly influenced by proprioceptive and active reaction of the subjects. Indeed, the GNRB R arthrometer has been reported to slightly underestimate differential laxities compared to a standard technique [START_REF] Jenny | Mesure de la laxité antérieure du genou par des radiographies dynamiques et le système GNRB R . comparaison avec la mesure naviguée peropératoire[END_REF], in this case an intraoperative navigation measurement. This difference might be partially due to involuntary muscle contraction, and to the fact that the anterior motion may be associated to rotation of the joint. The passive state of the limb was indeed subject to caution: several subjects reacted to pain at high loads by contracting hamstrings. Consequently, the measured reaction forces and computed stiffness indexes may be overestimated compared to a complete passive response (e.g. cadaver knee). This assumption is partially confirmed when comparing computed indexes to data available in the literature on cadaver knees: original data from [START_REF] Eagar | A method for quantifying the anterior load-displacement behavior of the human knee in both the low and high stiffness regions[END_REF] allowed to compute mean k indexes of 10.1 and 2.9N/mm for healthy and pathological knees, and 7.1 N/mm for the ACL contribution. Even if the test conditions and specimen were not equivalent, it can reasonably be assumed that there may be non-negligible contribution of active stiffening of the joint in the measures from the GNRB R system. As knee braces have been reported to modify muscle activation of the lower limb [START_REF] Osternig | Effects of prophylactic knee bracing on lower extremity joint position and muscle activation during running[END_REF][START_REF] Ramsey | Electromyographic and biomechanic analysis of anterior cruciate ligament deficiency and functional knee bracing[END_REF][START_REF] Théoret | Study on three-dimensional kinematics and electromyography of ACL deficient knee participants wearing a functional knee brace during running[END_REF][START_REF] Wojtys | Anterior cruciate ligament functional brace use in sports[END_REF], the decoupled responses may also involve indirect active stiffening. An interesting addition to this study would have been to reproduce these experiments while measuring the muscle activity using electromyography (agonist/antogonist contraction/co-contraction).

Besides, another potential bias leading to a slight overestimation of the brace effect was that the brace was not entirely free around the joint, as it would be in a natural standing position; the back fabric fitted between the limb and the support of the GNRB R .

The effect of helical straps of brace #2 was also characterised by the test machine; removing this feature resulted in a decrease of the k index by a factor 2. Besides, this feature also had an effect of preventing the drop in reaction force for displacements higher than 8 mm, and the comparison with the present study shows that this feature definitely helped reducing the drawer.

Altogether, the characterised in-vivo efficiencies of these fabric-based braces as measured in this study give more optimistic results than what had been measured using a surrogate limb as testing device. Theses knee braces were found to effectively replace the mechanical role of the ACL up to anterior displacements of 1-4 mm, and even provided a corrected stiffness of 81% of the healthy knee for the most efficient brace at 5 mm, whereas the relative stiffness of the pathological knee was only 57%. [START_REF] Eagar | A method for quantifying the anterior load-displacement behavior of the human knee in both the low and high stiffness regions[END_REF] showed that this value of 5 mm corresponds to the mean anterior displacement at which the mechanical behaviour of the joint changes from a low stiffness region (7.3 N/mm) to a high stiffness region (35.7 N/mm) because of the tensioning of the ACL. Consequently, the brace was able to compensate for a deficient ACL in the low stiffness region only. This is in agreement with the literature [START_REF] Branch | Controlling anterior tibial displacement under static load: a comparison of two braces[END_REF]. More efficient brace designs should ideally mimic the non-linear mechanical behaviour of the ligament and induce an increase of the stiffening level as the anterior displacement increases, instead of quickly reaching a maximal load as seen in Figure 7. This can be done by operating on both passive and active mechanisms. The most straightforward technique is to optimize brace designs in order to maximize their passive stiffness, by using standardised surrogate limbs as testing devices. However, joint stability also comes from muscle activation. [START_REF] Wojtys | A gender-related difference in the contribution of the knee musculature to sagittal-plane shear stiffness in subjects with similar knee laxity, The Journal of bone and joint surgery[END_REF] used an arthrometer to measure sagittal-plane shear stiffness of passive and active knees. Passive knees exhibited a mean stiffness of 18.7 N/mm (men) and 19.3 N/mm (women) while active limbs resulted in stiffer joints: 70.9 N/mm (men) and 40.7 N/mm (women). That is, the stabilizing effect of an optimised brace could be enough to compensate for a deficient ACL if the muscles are recruited as the main active stabilizers, as proposed by [START_REF] Wojtys | Anterior cruciate ligament functional brace use in sports[END_REF]. Consequently, novel methods have yet to be developed to understand and evaluate the effect of brace design on its ability to stimulate the active recruitment of the musculature to participate in maintaining joint stability. It is also important to note that a high patient-specificity was found in the results, characterised by a large scattering, and the given conclusions are based on mean tendencies. However, the best brace was not always the same: it was brace #1 in 4 cases, brace #2 in 7 cases, brace #3 in 4 cases and the compression sleeve in 1 case. Consequently, a patient-specific approach is strongly desirable when actually selecting a brace for a given subject.

Conclusions

The ability of three commercial fabric hinged knee braces to effectively compensate for a deficient ACL was tested in-vivo using a GNRB R arthrometer on 16 patients. It was determined that based on subjective feelings, subjects were not able to predict which brace would be the most efficient on themselves, highlighting the importance of objective testing procedures for these devices. Subsequently, it was found that these braces effectively replaced the mechanical role of the ACL for low displacement -low force, i.e. within the low stiffness region of this structure. Finally, some results suggested that braces may stimulate an indirect stiffening mechanism (stabilizing contraction of muscles), which should be further investigated as it might prove to be a major part of their efficiency in active dynamic situations. This study was part of a global approach in evaluating these devices which will hopefully lead to the introduction of efficiency-labels directed at manufacturers, for the benefit of the patients. Further studies should also consider a patient-specific approach.

Figure 1 :

 1 Figure 1: Panel of knee braces selected to be tested: Thuasne Genuaction (a), Gibaud GenuGib Stab (b), Lohman & Rauscher Ligaction Pro (c) and Thuasne Ligaflex (d).

Figure 2 :

 2 Figure 2: GNRB R system.

Force

  h o lo g ic a l (b) Complete ACL rupture

Figure 3 :

 3 Figure 3: Response curves as given by the GNRB R device: (a) partial tear with differential laxity of 0.9 mm at 134 N (subject 5); (b) complete ACL rupture with differential laxity of 3.7 mm at 134 N (subject 11).

Figure 4 :

 4 Figure 4: Schematic illustration of the braced knee mechanical system as three springs in parallel, representing the stiffness brought by the brace, the ACL and the other knee structures.In this case, the total force F applied to the system is distributed among the springs so that F = F 1 + F 2 + F 3 .

Figure 5 :

 5 Figure 5: Box plot of the VAS score of the subjective evaluation of stabilization sensation and comfort of the different braces (25 subjects). On each box, the central black mark is the median, the edges of the box are the 25th and 75th percentiles and the whiskers extend to the most extreme data points not considered outliers. Stars show statistically different means between braces (p ≥ 0.95) and plus signs the outliers.

Figure 6 :

 6 Figure 6: Displacement-load test curves given by the GNRB R arthrometer of healthy and pathological knees for the 16 subjects. Bold lines represent the means and dashed lines the 95% CIs for the means.

4. 2

 2 mm at 250 N. Both these values corresponded to a relative dispersion of 3.8%, which indicated a good reproducibility of the tests for each patient.

Figure 7 :

 7 Figure 7: Mean decoupled load-displacement curves of the following structures for the 16 subjects: the compression sleeve, the three hinged braces, the ACL, the other structures of the knee and the healthy knee (as reference). Bold lines represent the means and dashed lines the 95% CIs for the means.

Table 1 :

 1 Subject characteristics with measured laxities of healthy and pathological knees using the GNRB R arthrometer. * Subjects not included in the final results due to outlying GNRB R measurements.

							Laxity at	Laxity at
		Sex Age	Height (cm)	Weight (kg)	Knee circ. (cm)	134 N, healthy	134 N, pathol.
							knee (mm)	knee (mm)
	1 *	M	26	184	98	39	2	5.2
	2	F	21	159	54	35	4.4	6.2
	3 *	M	44	177	75	38	4.8	6.5
	4	M	20	182	80	39	4.4	7.2
	5 *	F	26	167	53	33	3.5	4.4
	6	M	26	167	60	35	3.9	7.5
	7	F	17	163	57	37	4	9.2
	8	M	28	174	68	35	5	7.8
	9	F	34	169	69	40	4.7	10.1
	10	M	20	173	78	37	3.6	5.9
	11	F	29	171	56	36	4.8	8.5
	12	M	22	184	75	40	5.2	8.2
	13	M	23	178	63	37	3.8	8.3
	14	M	25	184	96	43	4.9	8.2
	15 *	M	28	175	65	36	7.6	8.8
	16 *	M	15	180	65	38	4.8	13.5
	17 *	M	13	171	56	36	5.8	11
	18	M	24	183	92	42	4.7	7.4
	19	F	43	164	74	37	4.3	6.2
	20	M	18	175	72	42	6.3	9.3
	21 *	F	26	160	61	37	3.6	11.5
	22 *	M	48	159	88	41	3.4	4.9
	23 *	F	59	165	70	42	4.4	5.7
	24	M	29	182	76	39	5.1	10.2
	25	F	18	169	56	34	4.4	6.6
	av.		27	173	70	38	4.5	7.9

Table 2 :

 2 Mean k indexes computed for the different structures of N subjects after removing the outliers.

		Mean k index (N/mm)	Standard deviation (N/mm)	N
	Compression sleeve	1.2	3.0	15
	Brace #1	5.5	2.8	14
	Brace #2	7.1	4.3	15
	Brace #3	2.0	2.5	15
	ACL	13.9	3.6	12
	Pathological knee	14.3	4.8	16
	Healthy knee	28.3	5.1	14

Acknowledgements

This work was funded in part by the ANRT (Association Nationale de la Recherche et de la Technologie) and the following orthotic manufacturers: Thuasne R , Gibaud R and Lohmann-Rauscher R .


Leticia Carro

Elodie Hablot

Thibaud Coradin
email: thibaud.coradin@upmc.fr

Hybrids and biohybrids as green materials for a blue planet

Keywords: bio-hybrids, encapsulation, bioplastics, biofuel cells, biosensors, bioremediation

There is an urgency to identify novel technological answers to the decreasing availability of important resources together with increasing accumulation of pollution. Among the many ways materials science can contribute to these issues, the enhanced use of renewable resources, the optimal production of alternative energies and the improved monitoring/cleaning of contaminated environments can be identified as area where the sol-gel technology has, and will have, a major role to play. In this short review, we more specifically illustrate recent developments in biohybrid chemistry applied to bioplastics, biofuel cells, biosensors and bioremediation.

Introduction

« The Earth is blue like an orange » wrote the french poet Paul Eluard in 1929. Almost one century later, our planet is starting to be covered with shades of gray. Two main threats are well-identified, greenhouse gas effect and environmental pollution, with their cortege of deleterious impacts on climate, biodiversity, human health… Official estimations indicate that these dangers are mainly of anthropogenic origin, and mostly related to energy production [START_REF] Popescu | Anthropogenic air pollution sources[END_REF].

These activities currently consume for most part non-renewable resources, of both organic and inorganic nature.

As an answer to the predicted shortage of fossil and mineral stocks, an acceptable short-term solution is to decrease our resource consumption rate by developing more energy/atom-effective technologies and improving waste management/recycling. However, in a longer-term perspective, there is a clear need to identify substitution strategies based either on virtually unlimited (sunlight, wind, seas oceans) or so-called renewable resources (mainly of vegetal origin). Indeed, doubts can be raised about the possibility for nearly 7 billion human beings to live without impacting negatively on the atmo-and biosphere. This is wellillustrated by the issues that are currently raised by crop-based biofuel production [START_REF] Smith | [END_REF].

The possible role of the sol-gel processes in contributing to a more eco-responsible form of material synthesis following the principles of Green Chemistry has been recently reviewed [3]. The application of soft chemistry routes to the preparation of inorganic/hybrid devices for energy production or storage is also commonly described [START_REF] Klein | Conventional energy sources and alternative energy sources and the role of sol-gel processing, in : Sol-gel processing for conventional and alternative energy[END_REF]. The importance of the sol-gel technology for pollution assessment and remediation has been less explored [START_REF]Sol-Gel Methods for Materials Processing: Focusing on Materials for Pollution Control[END_REF].

In this short review, we would like to present some recent achievements in these three areas, i.e. green materials, energy and environmental monitoring/remediation, that involve the sol-gel technology, especially focusing on biohybrids that associate natural systems, being biomolecules or whole cells, with inorganic materials and suggest some perspectives in these area.

Sol-gel based bioplastics

Polymers associated with sol-gel materials are of great interest in many applications [START_REF] Mammeri | [END_REF]7].

Replacing conventional polymers by bio-based and biodegradable polymers would add to these materials the proposition of a reduced material carbon footprint and minimize our dependence on fossil/petro resources [8,9]. Biodegradable polymers issued from plants that have been associated with sol-gel materials include polysaccharides, proteins and vegetable oils [START_REF]Nanocomposites with biodegradable polymers[END_REF].

In particular, many polysaccharides, such as pectin [START_REF] Agoudjil | [END_REF], xanthan gum [12], carageenan [13], guar gum [14] and alginic acid [15] have been considered for the synthesis of hybrid composites and nanocomposites to be used in biotechnological, biomedical and environmental applications. Cellulose-and chitin/chitosan-based biohybrids associated with silica are described in more details afterwards to illustrate the diversity of strategies and materials that have been reported in this area.

Cellulose and cellulose derivatives

The preparation of SiO 2 /cellulose nanocomposites has been reported as displaying promising properties for different potential applications. These nanocomposites are prepared either by deposition of morphologically well-defined SiO 2 nanoparticles at the fiber surface via polyelectrolytes assembly or via the synthesis of SiO 2 nanoparticles using tetraethoxysilane (TEOS) hydrolysis in the presence of cellulose fibres [16]. Both methodologies induced a significant decrease in the water uptake capacity of cellulose fibres. Silica-modified cellulosic aerogels have also been prepared [17]. They consist of two interpenetrating networks of cellulose and silica, prepared from shaped cellulose solutions by (1) regenerating (reprecipitating) cellulose with ethanol, (2) subjecting the obtained shaped alcogels to sol-gel condensation with TEOS as the principal network-forming compound, and

(3) drying the reinforced cellulose bodies with supercritical carbon dioxide. An open-porous network structure was obtained when silicification was performed under mild acidic conditions.

Bacterial cellulose (BC) hydrated membranes also present advantages for the preparation of new organic-inorganic hybrids due to its nanometer-scale reticulated structure [18][19][20][21]. BC-silica hybrids were prepared from BC membranes and TEOS in neutral pH conditions at room temperature. The resulting water-free translucent membranes exhibited improved mechanical properties and thermal and UV resistance [18,21]. Homogeneous membranes were obtained containing up to 66 wt% of silica spheres, 20-30 nm diameter, that were clearly attached to cellulose microfibrils (Fig. 1) [20]. Cellulose derivatives, such as cellulose acetate (CA) and carboxymethyl cellulose (CMC) were also associated with TEOS or silicates to obtain nanocomposites (Fig. 2) [22,23]. Of particular interest is the demonstration that the biodegradation of CA/SiO 2 membranes by Thricoderma harzianum fungi was not modified by the presence of the inorganic phase [22].

Direct modification of cotton fibers by deposition of SiO 2 /TiO 2 multilayer films for outdoor applications also illustrate the versatility of the sol-gel based mineralization of natural materials [24]. In a first step, SiO 2 particles were deposited on the surface of the cotton fibers using hydrolyzed TEOS. This first layer allows the protection of the fibers against photocatalytic decomposition by the second layer of TiO 2 nanoparticles deposited using relatively mild temperature conditions (100°C) by a sol-gel method. This method led to dense and uniform multilayer films.

Chitin and chitosan

Chitin and chitosan have been widely investigated in the preparation of new hybrids materials via sol-gel process. As an example, a silica network was obtained by controlled hydrolysis of tetraethyl orthosilicate using deacetylated chitin as an organic counterpart [25].

The resulting polymer hybrids were homogeneous, transparent, film-forming glassy materials that can be obtained in a wide composition range. Similar systems were prepared through a sol-gel process starting from alkoxysilane and chitosan [26]. Further functionalization was performed through in situ hydrolysis-condensation reaction of methyltrimethoxysilane (MTMS) and vinyltrimethoxysilane (VTMS) in the reaction medium, leading to transparent and hydrophobic silica-chitosan hybrids that can find application in the optics industry.

Membranes were also prepared by in situ crosslinking of chitosan and formation of chitosansilica hybrid membranes with γ-glycidoxypropyltrimethoxysilane as a crosslinking agent [27].

The formed chitosan-silica hybrid materials exhibited improved thermal stability and low degree of swelling in water.

Porous chitosan/silica hybrids microspheres were prepared by impregnation of preformed polysaccharide beads with silica precursors, and considered for use in heterogeneous catalysis [28]. More recently, chitosan-silica hybrid microspheres were obtained in a one-step process using a simple microfluidic method [29]. Bovine serum albumin was chemically grafted on the obtained microspheres and the presence of silica led to an enhancement of both mechanical stability and protein loading capacity of the microspheres.

One key advantage of these approaches is that non-gelling polymers can be obtained in a gel form by generating silica in situ via sol-gel processing [30]. This can be done by using a silica precursor, tetrakis(2-hydroxyethyl)orthosilicate (THEOS), that is fully water-soluble and compatible with the biopolymer. The gelation is caused by mineralization of carbohydrate macromolecules, which strengthen them and allow their cross-linking [31,32].

Biohybrid chitosan-SiO 2 materials have been described for many applications, including biosensors [33,34] and sorbents [35,36]. Chitosan-silica hybrid membranes for bone repair were also described and evaluated in vitro [37][38][39]. Recently, it was possible to obtain hybrid nanofibers by electrospinning chitosan, polyethylene oxide with tetraethoxysilane and 3-glycidyloxypropyltriethoxysilane as silica precursors, exhibiting promising properties for guided bone generation [40].

Bio-hybrid materials for energy

In the field of eneregy, sol-gel routes to oxides as well as to hybrid materials allow to control the nanostructure of the material and/or to develop efficient interfaces. For lithium batteries, nanostructured systems using TiO 2 /LiCoO 2 electrodes have been studied [41].

Mesoporous forms of these materials showed improved properties thanks to short lithium/electrons distance within the pore walls. Mesoporous materials may also be useful for capacitors design [42], as well as in photovoltaic systems, for instance by confining conjugated polymers within the mesoporosity of photoactive metal oxides, such as TiO 2 [43] The design of fuel cells is another area where the sol-gel technology is of particular interest. For instance, it allows the elaboration of nanostructured ceramic thin layers for high temperature solid oxide fuel cells (SOFC) design [44]. In the field of proton exchange membrane fuel cells (PEMFC), working at low temperature, nanocomposite, mesoporous and hybrid materials have been studied [45]. One particularly interesting approach is the use of ionic liquids as solvents to address the requirement for high humidity conditions, as demonstrated for phosphonated silica particles dispersed in a styrene-acrylonitrile resin in the presence of 1-ethyl-3-methyl-imidazolium-bis (trifluoromethanesulfonyl) imide [46]. Biofuel cells, that use a biological activity as a source of energy, also constitutes an interesting area to which sol-gel chemistry can contribute [47]. Indeed, from the pioneer works of Avnir [48] and Carturan [49], the possibility to immobilize enzyme and living cells inside silica gels while preserving, if not enhancing, their biological activity has been largely demonstrated [50,51]. Indeed, as bioelectrodes are targeted, it is important that the sol-gel matrix exhibit electronic conduction properties. However, silica is a very poor electronic conductor. This issue has been addressed earlier for the design of enzymatic electrochemical devices, via the incorporation of carbon particles and/or conductive polymers [52]. The additional use of mediators allowing the transfer of the electron from the enzyme to the material has also been described, leading to complex electrodes with high performances [53].

On this basis, silica gels incorporating carbon nanotubes (CNT) or graphene were found suitable for current generation based on the enzymatic glucose conversion into oxygen [54,55].

In contrast, the design of microbial fuel cells are much more challenging as it requires the development of a conductive matrix in conditions where the targeted organisms maintain their activity. As far as living cells are concerned, two options are being studied: (i) hydrogenproducing species, mainly cyanobacteria and micro-algae and (ii) electroactive organisms, especially bacteria [56]. The first approach has the main advantage that these organisms survive in a rather large range of pH, ionic strength or even temperature, enlarging the possible conditions for gel synthesis. Moreover, some of them possess a so-called exopolysaccharide (EPS) that constitute a convenient barrier to avoid detrimental interactions not only between the silica internal surface and the cell membrane but also with other solid additives that may be present to insure conductivity (Fig. 3) [57]. Finally, their viability has been shown to be preserved over months period in silica [58]. However they have two major drawbacks. First they are photosynthetic organisms that require access to visible light. This is in contradiction with the incorporation of conductive species that absorb strongly in this wavelength domain. Second, H 2 itself should be converted into protons and electrons, requiring an additional component or compartment in the biofuel cell device. Therefore, at this time, only the first step of the process, i.e. hydrogen production by silica-immobilized photosynthetic organisms has been achieved [59]. Using Synechocystis sp. cyanobacteria, the production rate was found comparable for immobilized cells and cells in suspension after optimization of silica precursors and additives [60].

The second option, i.e. the use of electroactive bacteria, has opposite advantages and drawbacks. On the one hand, bacteria do not require light to survive and they can produce electrons, although some molecular shuttle may be necessary to transport charge to the electrode surface. On the other hand, they are more sensitive to physico-chemical conditions and lack the protective EPS coating. The earliest report in this area was based on the use of a sol-gel process to deposit a silica layer on a biofilm of Shewanella oneidensis bacteria formed on a conductive carbon surface [61]. However, a true tridimensional encapsulation was only achieved recently using silica-filled graphite felt [62]. The viability of immobilized Escherichia coli bacteria was preserved in the sol-gel matrix (Fig. 4) and their electroactive metabolites could be used for current production using the conductive carbon network.

Noticeably, the formation of silica-CNT materials in cytocompatible conditions, i.e. using an alcohol-free process at pH 7, was also reported, that appear promising for this application [63].

Bio-hybrid materials for environment monitoring and remediation

Silica gel presents great thermal stability and high surface area that contains many hydroxyl reactive groups. These groups provide adequate biding sites for the attachment of different organic compounds, so that silica gel surface can be easily functionalized. Through these chemical modifications new hybrid materials with high selectivity to retain pollutant compounds can be obtained that are currently evaluated in natural water treatment. For instance, 4-amino-2-mercaptopyrimidine was recently chosen for anchorage onto the silica surface due to the biding potential by amine and thiol groups for Cu(II) sorption [64]. Silica gels modified with EDTA and DTPA were investigated to remove Co(II) and Ni(II) from polluted water [65]. Some aliphatic amines were also immobilized onto silica gel to test their capacity to eliminate Cu(II) and Fe(III) from natural waters [66]. A great deal of papers describing Hg(II) removal from hybrid mesoporous materials are available [67,68]. More simply, N-pyrazole could be anchored on a silica gel surface for mercury elimination [69]. A green chemistry route to efficient Hg(II) sorbent was also described using gels obtained from natural silica functionalized with the amino acid L-cysteine [70]. In parallel, some natural products such as tannins were also immobilized over silica beads in order to enhance the material capacity to remove Cr(III) from aqueous solution [71]. Moreover, some current researches are based on the addition of some inorganic compounds to the sol-gel host in order to obtain composites materials with improved capacities for pollutant removal. For instance, hydroxyapatite, a good example of porous material with high metal affinity, has been associated with zirconia, leading to materials that combine affinity for cations and anions (Fig. 5) [72].

In contrast, there are only a few works concerning the sol-gel encapsulation technology applied to bioremediation processes. In enzymatic-based process, an interesting example is provided by an immobilized recombinant dye-decolorizing peroxidase [73]. This enzyme exhibits a very specific tertiary structure that could not be preserved by any traditional immobilization techniques, except for its adsorption in mesoporous silica. Regarding cell immobilization processes, some reports exist about the use of dead biomass either adsorbed on or trapped in silica [74,75]. Considering living organisms, microbial mats were immobilized on silica gel. The resulting silica mats particles were used for the bioremediation and bioreduction of uranium-contaminated waters [76]. Some sol-gel matrices have been functionalized with bacteria such as Bacillus sphaericus JG-A12 for the successful biosorption of uranium and copper [77]. Burkholderia sp and Stereum hirsutum have been trapped in silica gels for chromium and malachite green removal, respectively [78,79]. Nostoc calcicola immobilized in silica-coated alginate beads were shown to have high metal accumulation properties, the silica coating enhancing the stability of the beads upon recycling [80]. Yeast cells were immobilized in zirconia for 4-chloro-2-methylphenoxyacetic acid degradation but the viability rate of encapsulated cells was very poor [81]. Inorganic hosts prepared by the freeze-drying technique were shown to be suitable for Rhodococcus ruber immobilization and phenol degradation. These materials are promising but they are macroporous and allow internal cell division so that clogging and leaching can be expected on the long-term [82,83].

The capture and degradation or conversion of green house gases (GHG) and volatile organic compounds (VOC) using sol-gel based materials is another area of intensive research [84,85]. For instance, several porous materials incorporating aminated molecules were evaluated for CO 2 sorption and storage [86,87]. A promising alternative relies on the use of immobilized carbonic anhydrase enzymes [88]. Interestingly, Favre et al. showed that the pH modification induced by the enzymatic conversion of CO 2 could be used to activate the gelation of silica precursor solutions [89]. Indeed entrapped photosynthetic organisms may in principle also be used for CO 2 transformation into useful products, such as biofuels, [58] but the possibility to develop sol-gel based photobioreactors with long-term stability and on a large scale is still to be demonstrated.

Noticeably, a material that interacts with pollutants should in principle be also used as a sensor provided that the corresponding binding and/or transformation event is associated with a measurable signal. For instance, mesoporous silica materials modified with metal ligand and/or dyes combine the ability to simultaneously trap and detect heavy metals [90,91]. In the case of biohybrid materials, polysaccharides can indeed be used for sorption purposes but are hardly capable of transduction [92]. In contrast, many enzymes are able to transform organic pollutants, a reaction that can be detected by UV-visible absorption, fluorescence spectroscopy and electrochemical methods [93]. In this context, a biosensor based on the coencapsulation of urease and acetylcholinesterase (AChE) in the presence of a fluorescent dye in silica gels was described. Whereas AChE was useful for pH monitoring, the inhibition of urease activity by metal ions could be used for their detection and quantification [94]. Cellbased biosensors involving the sol-gel technology have also been described [95]. In a simple approach, the metal depletion of solutions in contact with silica gels containing dead algae can be followed by electrochemical measurements [96]. Considering living cells, most sensors are based on the use of photosynthetic organisms [97]. Modification of chlorophyll fluorescence or photosynthesis yield using the pulse amplitude modulated (PAM) fluorimetry technique can be used to directly monitor the impact of pollutants on cell behavior allowing the development of fiber-optic based biosensors [98,99]. Raman spectroscopy was also recently found useful to image the influence of toxic species on the production of carotenoid pigments (Fig. 6) [57]. Recently, hybrid materials consisting of algae-containing alginate beads trapped in a silica matrix were found suitable for the detection of the atrazine herbicide [100]. Such an approach is all the more interesting as previous works demonstrate both the possibility to tailor the silica gel optical properties to optimize algae survival [101] and the ability of the alginate network to be involved in the metal binding [80,102].

Perspectives

The examples provided in this short review illustrate the benefits of the sol-gel process in terms of designing more robust and complex (in terms of composition, structure and properties) materials than their bio/organic counterparts through a large diversity of processes.

Considering the environmental impact of sol-gel chemistry, one should emphasize that, although metal alkoxides cannot be considered as "green chemicals" due to their harsh conditions of preparation and associated alcohol content, the main advantage of the soft chemistry approach relies in low temperature/low pressure conditions that are a source of significant energy saving compared to the traditional ceramic technology. As far as the resulting materials are concerned, silica is certainly a specific case because of its intrinsicallybenign ecological impact. Other metal oxides that are currently often prepared by the sol-gel process, such as Al 2 O 3 , ZrO 2 , TiO 2 or V 2 O 5 , may raise more environmental concerns. Interestingly, while the ecological impact of manufactured oxides is raising large interest from the point of view of living organisms (eco-toxicity), more efforts must be made to elucidate their life cycle, including biodegradation issues.

Another key advantage of the sol-gel process is the possibility to convert liquid systems into solids, providing easily handled and recoverable materials while preserving the structure and reactivity of the immobilized objects. This can be useful for storage purposes or for transferring homogeneous reactions into heterogeneous ones, as already well-known in catalysis but also for developments in biotechnology. Hence there is still a great deal of efforts to be done to evaluate and optimize the association of inorganic precursors with industrial by-products or wastes as well as with biomolecules or living organisms.

Needless to say, the urgency of the situation is a strong driving force for novel technological developments that fully integrate sustainability issues. We hope that this short

Figure captions

Fig. 1 SEM images of bacterial cellulose membranes (left) and BC-silica (66wt.%) composite (right). Reproduced from [20] with kind permission from Springer Science+Business Media. Reproduced from [23] with permission from The Royal Society of Chemistry (RSC).

Fig. 2

 2 Fig. 2 (a) Optical micrograph of SiO 2 /carboxymethyl cellulose CMC solutions/gels with

Fig. 3

 3 Fig. 3 SEM (a) and TEM (b) images of encapsulated Klebsormidium flaccidum algal

Fig. 4

 4 Fig. 4 Time evolution of fluorescent E. coli bacteria in graphite-silica electrodes as monitored

Fig. 5

 5 Fig.[START_REF]Sol-Gel Methods for Materials Processing: Focusing on Materials for Pollution Control[END_REF] Zirconia-hydroxyapatite nanocomposites combining anionic and cationic sorption

Fig. 6

 6 Fig. 6 Raman imaging of encapsulated Klebsormidium flaccidum algal cells : reconstructed

Figure 6

 6 Figure 6

Acknowledgments L. C. thanks University of A Coruña for funding her stay in Paris. T. C. acknowledges financial support from the PEPS «Biofilms Biocapteurs Biopiles pour une Ingénierie Environnementale » program of Centre National de la Recherche Scientifique.

review demonstrates that the sol-gel technology will play a major role in this direction in the nearest future.