P Tio

S Omoumi

Aubry

Alexis Danner

Eléonore Brumpt

Mona Alilet

Grégory Tio

Patrick Omoumi

Sébastien Aubry

Improved contrast for myeloma focal lesions with T2-weighted Dixon images compared to T1weighted images

Keywords: Magnetic resonance imaging (MRI), Multiple myeloma, Dixon sequence, Bone marrow

est destinée au dépôt

Introduction

Multiple myeloma (MM) is characterized by autonomous proliferation of monoclonal plasma cells in the bone marrow. The diagnostic criteria used for the diagnosis of MM are 10% or greater clonal bone marrow plasma cells, or an extramedullary plasmacytoma, plus at least one myeloma-defining event present in the extended calcemia renal anemia bone (CRAB) criteria according to the latest recommendations [START_REF] Rajkumar | International Myeloma Working Group updated criteria for the diagnosis of multiple myeloma[END_REF]. The International Myeloma Working Group (IMWG) has recommended whole body magnetic resonance imaging (MRI) for workup of solitary bone plasmacytoma and patients suspected of having smoldering MM [START_REF] Dimopoulos | Role of magnetic resonance imaging in the management of patients with multiple myeloma: a consensus statement[END_REF][START_REF] Howe | Current concepts in MRI of focal and diffuse malignancy of bone marrow[END_REF].

Conventional T1-weighted spin-echo sequences and short tau inversion recovery (STIR) have been considered as the most useful tools for the evaluation of bone marrow involvement in routine clinical practice [START_REF] Nakatsu | Comparison of short inversion time inversion recovery (STIR) and fat-saturated (chemsat) techniques for background fat intensity suppression in cervical and thoracic MR imaging[END_REF][START_REF] Fleckenstein | Fast short-tau inversion-recovery MR imaging[END_REF]. The Dixon technique is a fat-suppression technique based on chemical shift imaging (CSI) [START_REF] Grande | Fatsuppression techniques for 3-T MR imaging of the musculoskeletal system[END_REF]. Due to differences in size and polarity in the molecules, protons of fat and water experience different magnetic effects leading to a reduced precessional frequency of fat versus water protons. CSI is based on these differences to produce in-phase (IP) and out-of-phase (OP) images. Decreased signal intensity on the OP images relative to IP images indicates the presence of microscopic fat. Water-only images are obtained by adding the IP and OP datasets, while fat-only images are obtained by subtracting the OP dataset from the IP dataset [START_REF] Maas | Uniform fat suppression in hands and feet through the use of two-point Dixon chemical shift MR imaging[END_REF][START_REF] Pezeshk | Role of chemical shift and Dixon based techniques in musculoskeletal MR imaging[END_REF]. The benefits of this method are high signal-to-noise ratio images and simultaneous acquisition of images with different contrasts (IP, OP, fat-only and water-only images) [START_REF] Reeder | Multicoil Dixon chemical species separation with an iterative least-squares estimation method[END_REF][START_REF] Kirchgesner | Fat suppression at three-dimensional T1-weighted MR imaging of the hands: Dixon method versus CHESS technique[END_REF]. Previous studies have shown the non-inferiority or superiority of T2-weighted Dixon sequence over T1-weighted sequence for the detection of bone metastases [START_REF] Maeder | Bone marrow metastases: T2-weighted Dixon spin-echo fat images Can replace T1-weighted Spin-Echo Images[END_REF] or the diagnosis of active and chronic sacroiliitis [START_REF] Özgen | The value of the T2-weighted multipoint Dixon sequence in MRI of sacroiliac joints for the diagnosis of active and chronic sacroiliitis[END_REF].

The purpose of this study was twofold. First, to compare the contrast between spinal MM focal lesions and surrounding bone marrow obtained on T2-weighted Dixon fat-only MR images to that obtained on T1-weighted spin-echo images. Second, to search for correlation between bone marrow fat fraction assessed by T2-weighted Dixon sequence and IMWG myeloma defining events.

Materials and methods

Study population

Local institutional review board approval was obtained and written informed consent was waived. Patients with a newly diagnosed MM based on the updated IMWG diagnostic criteria [START_REF] Rajkumar | International Myeloma Working Group updated criteria for the diagnosis of multiple myeloma[END_REF], and who had undergone a whole spine MRI examination between January 2010 and April 2017 including T1-weighted spin-echo images in the sagittal plane and T2-weighted Dixon images in the sagittal lane with water-only, fat-only, IP, and OP reconstructions were included in this retrospective study. IMWG diagnostic criteria were applied retrospectively to patients who were diagnosed before 2014. Previously treated patients with relapsing disease and no focal MM lesion, or patients with incomplete MRI protocol were excluded. Because of known associations between the following criteria and MM, we excluded patients with: myelodysplasia, history of radiotherapy or chemotherapy. Nineteen patients who previously received specific MM treatment at the time of MRI were not considered for the secondary objective. Figure 1

summarizes patients inclusion.

A total of 39 patients were included. There were 25 men and 14 women with a mean age of 68.8 ± 9.8 [SD] years (range: 49-88 years). Seven patients (7/39; 18%) had no focal MM lesions and no treatment and 32 patients (32/39; 82%) had a total of 112 focal MM lesions. Of these 32 patients, 19 were under treatment for MM. Medical records of patients were reviewed by one author (AD). The following IMWG myeloma defining events were collected: serum calcium level (mmol/L), serum creatinine level (µmol/L), serum hemoglobin level (g/dL), clonal bone marrow plasma cell percentage (%), and uninvolved serum free light chain ratio (≤99 or >100).

MRI protocol

MRI examinations were obtained at 3T in 10 patients (Magnetom Skyra ® , Siemens Healthineers) and at 1.5-T in 29 patients (Magnetom Aera ® , Siemens Healthineers). The entire spine was acquired in two contiguous sagittal stacks with non-enhanced fast spin-echo T1-weighted and three-point fast spin-echo Dixon T2-weighted sequences of the entire spine from the base of the skull to the last sacral piece. Imaging parameters of the sequences are detailed in Table 1.

Image analysis

Image analysis was performed by a musculoskeletal radiologist with more than 15 years experience (SA) by using VuePACS ® software (Version 12.1.5.1156, Carestream Health). Focal MM lesions were defined by a low signal on T1-weighted and a high signal on T2-weighted water-only images and non myelomatous focal lesions such as hemangiomas (characterized by their high signal intensity on T1-weighted images due to fat component), enostosis (which have low signal on every weighting) and cysts (liquid signal) were excluded.

Focal lesions within edematous vertebral spine body due to fracture or degenerative changes were also excluded. For each patient, a maximum of 5 focal MM lesions measuring at least 5 mm in diameter was analyzed and for patients with more than 5 focal lesions, the 5 largest ones were selected. There were 7 patients (7/39 ; 18%) without any lesions, 5 patients (5/39 ; 13%) with 1 MM lesion, 2 patients (2/39 ; 5%) with 2 MM lesions, 9 patients (9/39 ; 23%)with MM 3 lesions, 4 patients (4/39 ; 10%) with 4 MM lesions, and 12 patients (9/39 ; 31%) with 5 MM lesions, yielding a total of 112 focal MM lesions.

Mean signal intensity of focal MM lesions and surrounding bone marrow of the same vertebral body (without focal lesion) were calculated by placing round operator-determined regions of interest (ROI) measuring between 5mm to 15mm in diameter. The ROIs had a mean surface of 82.7 mm 2 ± 75.01 [SD] (range: 19.6 mm 2 -176.6mm 2 ). Reference ROIs were placed on T1-weighted images at the center of each focal lesion and on the same slice within adjacent bone marrow at least 1mm out of the lesion. When present, "Salt & pepper" pattern was not excluded from the analysis of bone marrow. In case of absence of focal lesion, ROIs were placed at the center of first to fourth lumbar vertebral bodies. Then, ROIs were copied and pasted at the same location on all other sequences. The contrast between focal MM lesions and bone marrow was calculated using the following equation:

Results

Mixed model ANOVA showed a significant difference in contrast between images when taking into account patient effect (F [2; 93] = 35.10; P < 0.0001). Tukey's post-hoc test showed a significant difference between each contrast combination ( 2). The type of MRI had no significant effect on the contrast (P = 0.22).

Results of the correlation analysis between fat fraction and biological data are detailed in Table 3. There was no significant correlation between the biological data of the extended CRAB criteria, and fat fraction. No associations were found between fat fraction and uninvolved serum free light chain ratio (P = 0.81). Infiltration and replacement of bone marrow, when present, did not impair the contrast between focal lesions and bone marrow (Figure 3)

Discussion

Our study found that the contrast on T2-weighted Dixon fat-only images was significantly higher than on T1-weighted images, suggesting that T2-weighted Dixon fat-only may be more efficient to detect focal MM lesions than T1-weighted. Recently, Maeder et al. compared T2weighted Dixon fat-only to T1-weighted images for the detection of bone metastatic lesions, and showed that the contrast-to-noise ratio was significantly higher with the T2-weighted Dixon fat-only than with T1-weighted [START_REF] Maeder | Bone marrow metastases: T2-weighted Dixon spin-echo fat images Can replace T1-weighted Spin-Echo Images[END_REF]. They also showed that diagnostic performance was not significantly inferior with T2-weighted Dixon fat-only than that of a standard protocol including T1-weighted and fat-suppressed fluid-sensitive sequences. Another study demonstrated the higher contrast-to-noise ratio of a T2-weighted Dixon sequence compared to T1-weighted images for depiction of the diagnostic signs of active and chronic sacroiliitis [START_REF] Özgen | The value of the T2-weighted multipoint Dixon sequence in MRI of sacroiliac joints for the diagnosis of active and chronic sacroiliitis[END_REF]. Bone marrow is formed of approximately 40% yellow and 60% red marrow in the vertebral bodies during the first decade of life. The fatty component of vertebral bone marrow gradually increases with age [START_REF] Ricci | Normal age-related patterns of cellular and fatty bone marrow distribution in the axial skeleton: MR imaging study[END_REF]. On T2-weighted Dixon fat-only images, hypointense focal lesions are better depicted from high-signal intensity fatty background than on T1-weighted images [START_REF] Howe | Current concepts in MRI of focal and diffuse malignancy of bone marrow[END_REF][START_REF] Lecouvet | Whole-body MR imaging: musculoskeletal applications[END_REF][START_REF] Berg | Magnetic resonance imaging of the bone marrow in hematological malignancies[END_REF]. As in the case of metastases, the T2-weighted Dixon sequence might be an appropriate alternative to the standard protocol (combination of T1-weighted and fatsuppressed fluid-sensitive sequences) to search for spinal focal MM lesions. Nevertheless, Heynen et al showed that T2-weighted Dixon fat-only images had no significantly different interobserver agreement but were less sensitive than T1-weighted images for the detection of hip and pelvic occult fractures [START_REF] Heynen | MRI detection of radiographically occult fractures of the hip and pelvis in the elderly: Comparison of T2-weighted Dixon sequence with T1-weighted and STIR sequences[END_REF]. Due to this specific interest of T1-weighted images, it seems premature to take the opportunity to shorten imaging time by removing the T1weighted sequence from the protocol.

In our study, we did not identify a correlation between fat fraction and biological data of the extended CRAB. In case of a correlation between fat fraction assessed by T2-weighted Dixon sequence and biological data, MRI could have been used as a non-invasive tool to assess the level of clonal bone marrow plasma cells. Unfortunately, we did not demonstrate such a correlation. In our experience, diffuse bone marrow infiltration on T2-weighted Dixon images did not impair the contrast between focal lesions and bone marrow. New sequences have emerged in the diagnosis of MM and treatment follow-up, including the diffusionweighted sequence. Indeed, invasion of the bone marrow by plasmocytes causes restricted water motion [START_REF] Padhani | Diffusion-weighted (DW) and dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) for monitoring anticancer therapy[END_REF]. Therefore, either focal lesions or diffuse tumor cell infiltration of bone marrow appear as hyperintense on high b value diffusion-weighted images (DWI). Bone lesion conspicuity is higher on DWI b=800s/mm 2 than on STIR [START_REF] Pearce | Bone metastases from prostate, breast and multiple myeloma: differences in lesion conspicuity at short-tau inversion recovery and diffusion-weighted MRI[END_REF], and apparent diffusion coefficient (ADC) values have been shown to be useful for assessing disease course in MM [START_REF] Horger | Whole-body diffusion-weighted MRI with apparent diffusion coefficient mapping for early response monitoring in multiple myeloma: preliminary results[END_REF][START_REF] Padhani | Assessing the relation between bone marrow signal intensity and apparent diffusion coefficient in diffusionweighted MRI[END_REF].

In the clinical setting, other modalities such as 18-fluorodeoxyglucose (FDG) PET-CT are useful to detect focal MM lesions [START_REF] Sager | The value of FDG PET/CT in the initial staging and bone marrow involvement of patients with multiple myeloma[END_REF]. MRI and 18-FDG PET-CT have similar performances for the detection of focal MM lesions of the spine and pelvis [START_REF] Fonti | 18F-FDG PET/CT, 99mTc-MIBI, and MRI in evaluation of patients with multiple myeloma[END_REF][START_REF] Walker | Imaging of multiple myeloma and related plasma cell dyscrasias[END_REF]. In the future, the diagnostic value of T2-weighted Dixon whole-body MRI could be compared to 18-FDG PET-CT. Indeed, whole-body MRI has an increasing place in the assessment of MM spread. In a study conducted by Walker et al. involving 611 patients with MM, whole body MRI revealed more focal MM lesions than conventional whole-body x-rays in three of the most common metastatic sites for MM, including spine, pelvis and sternum [START_REF] Walker | Magnetic resonance imaging in multiple myeloma: diagnostic and clinical implications[END_REF]. As compared to whole-body multidetector computed tomography (MDCT), whole-body MRI has a higher sensitivity both for focal and diffuse pattern of bone marrow involvement and will result in upstaging MM. Moreover, the detection rate of extensive disease and the rate of bone manifestations using whole-body-MRI are higher than whole-body MDCT [START_REF] Gleeson | Accuracy of whole-body low-dose multidetector CT (WBLDCT) versus skeletal survey in the detection of myelomatous lesions, and correlation of disease distribution with whole-body MRI (WBMRI)[END_REF].

This study has some limitations. First, we acknowledge that we did not compare the sensitivity of T2-weighted Dixon to that of T1-weighted sequence to detect MM lesions. Such

  Contrast =Lesion signal within ROI -Bone marrow signal within ROI Lesion signal within ROI + Bone marrow signal within ROI Measurements of bone marrow fat fraction were done by placing ROI as large as possible on L1 to L4 vertebral bodies, except where there was a focal MM lesion. The ROIs were located at least 3mm far from the endplates and margins of vertebral bodies to prevent effect of partial volume. The fat fraction was assessed according to the following formula[START_REF] Maas | Dixon quantitative chemical shift MRI for bone marrow evaluation in the lumbar spine: a reproducibility study in healthy volunteers[END_REF] :Fat fraction = Signal IP -Signal OP 2 * Signal IPStatistical analysisQuantitative variables were expressed as mean ± standard deviation [SD] and range for normally distributed data and median and interquartile range for non-normally distributed data. Qualitative variables were expressed as raw numbers, proportions and percentage. We compared the contrast on T1-weighted, T2-weighted Dixon fat-only and T2-weighted Dixon water-only images, taking into account the patient effect due to multiple lesions. We computed the means and standard deviations of contrast of the different lesions of each patient's sequences, in order then to compare the overall averages of variables between patient's sequences.Mixed model analysis of variance (ANOVA) was used to compare contrasts of different sequences, and post-hoc Tukey tests were used to investigate multiple differences.The Wilcoxon rank-sum test was used to search for a potential link between uninvolved serum free light chain ratio and fat fraction. Spearman's correlation test was used to study the correlation between biological data and fat fraction. Wilcoxon rank-sum test was used to search for possible relationship between the type of MRI (1.5T or 3T) and contrast. A P value <0.05 was considered statistically significant.

a

  comparison would have required a complementary study comparing the number of focal MM lesions detected on each sequence. Secondly, it is a retrospective and single-centre study, with the result that our results warrant confirmation by a prospective multicenter study. The size of our sample was too small to allow us to draw any definitive conclusions regarding the potential link between fat fraction assessed by T2-weighted Dixon and the extended CRAB data. Finally, the impact of patient's treatments on the contrast was not assessed. Yet, signal changes of focal MM lesions and of surrounding bone marrow under treatment might likely impact contrast over time. A cohort study with a larger population would be required to overcome this limitation.In conclusion, for the assessment of myeloma focal lesions, T2-weighted Dixon fatonly images allow higher contrast between MM lesions and adjacent bone marrow than T1weighted images, but fat fraction is not correlated with biological data of the extended CRAB.The diagnostic value of the T2-weighted Dixon in MM, and of its association with a T1weighted sequence has to be determined. If the non-inferiority of T2-weighted Dixon sequence compared to other fat-suppressed fluid-sensitive sequences was demonstrated, it could become an all-in-one alternative to the classical association of T1-weighted plus another fat-suppressed fluid-sensitive sequence.

Figure 1 .

 1 Figure 1. Flowchart shows patients inclusion into the study.

Figure 2 .

 2 Figure 2. 63-year-old woman with multiple myeloma. a), T1-weighted image in the sagittal plane shows focal myeloma lesion (large circle) of the fifth lumbar vertebra with low signal intensity. b), On T2-weighted Dixon fat-only image, the focal myeloma lesion has no visible signal intensity. c), OnT2-weighted Dixon water-only image the focal myeloma lesion has high signal intensity. Better conspicuity of this focal lesion on T2-weighted Dixon fat-only image versus T1-weighted image is supported by a higher contrast between the lesion and surrounding bone marrow (0.84 on T2-weighted Dixon fat-only image versus 0.47 on T1weighted image).

Figure 3 .

 3 Figure 3. 81-year-old man with diffuse bone marrow infiltration and multiple focal myeloma lesions. a), On T1-weighted image in the sagittal plane, all myeloma lesions show low signal intensity. b), On T2-weighted Dixon fat-only image all myeloma lesions have no detectable signal. c), On T2-weighted Dixon water-only image all myeloma lesions have high signal intensity. For example, the lesion of the second thoracic vertebral body (large circles) has a contrast equal to 0.37 on T1-weighted, 0.96 on T2-weighted Dixon fat-only and 0.49 on T2weighted Dixon water-only images.

Table 2

 2

). The mean signal intensity of the lesion on T2-weighted Dixon fat-only images was 19.19 ± 27.26 [SD] (range: 1-249), 231 ± 142 [SD] (range: 49-839) on T2-weighted Dixon water-only images and 166 ± 84.91 [SD] (range: 18-442) on T1-weighted images. The contrast was highest on T2weighted Dixon fat-only images (0.63 ± 0.21 [SD]; range: 0.06-0.91), intermediate on T2weighted Dixon water-only images (0.45 ± 0.20 [SD]; range: 0.07-0.8), and lowest on T1weighted images (0.23 ± 0.19 [SD]; range: 0.04-0.87) (Figure

Table 1 .

 1 Magnetic resonance imaging protocols.

Table 2 .

 2 Comparison of contrast of myeloma focal lesions between different three MRI sequences.

Table 3 .

 3 Correlation between biological data of extended CRAB and bone marrow involvement.

			MRI sequence
	Parameter	T1-weighted	T2-weighted Dixon
		1.5 T	3 T	1.5 T	3T
	TR (ms)	550	553	4570	4300
	TE (ms)	11	10	77	107
	Turbo factor	3	3	16	18
	Acquisition time	1 min 38 s	1 min 33 s	4 min 31 s	4 min 07 s
	Slice thickness (mm)	3	3	3	3
	Number of slices	20	21	20	21
	Spatial resolution (mm 2 ) 0.84×0.84	1.04×1.04	0.84×0.84	0.89×0.89
	T indicates Tesla; TR indicates repetition time; TE indicates echo time

Conflicts of interest

The authors have no conflict of interest to declare in relation to this study.


Julien Boudard
email: julienboudard@hotmail.fr

S El Rifaï

D Feuvrier
email: feuvrier.damien@gmail.com

L Obert

F Loisel

Fingertip amputations treated with occlusive dressings Amputation digitale distale traitée par pansement occlusif

Keywords: Fingertip amputation, Second intention healing, Occlusive dressing Mots-clefs : Amputation digitale distale, Cicatrisation dirigée, Pansement occlusif

The aim of this study was to analyze a series of patients who suffered a distal finger amputation and who were treated with occlusive dressings. A retrospective study was done of 19 patients from a single hospital. At the review, an independent examiner evaluated the time required for wound healing, the number of occlusive dressings used, fingertip trophic skin changes, epicritic sensitivity using the Weber two-point discrimination (2PD) test, sensitivity based on the monofilament test, complications, the presence of dysesthesia or cold intolerance, and the QuickDASH score.

The mean follow-up was 12.6 months (6-25). A mean of 3.2 occlusive dressings (3-5) were used per patient, and the mean healing time was 4.3 weeks (4-5). The skin texture, fingertips and nail bed were good or excellent in 18 cases. The 2PD test was good or normal in 16 cases. Eighteen patients were satisfied or very satisfied with the outcome. The mean QuickDASH score was 5.53 (0-20.45).

In the literature, the recovery of tactile sensation is good after use of occlusive dressings (2PD of 2.5 to 4.0 mm). The mean sensitivity reported in various studies is better than that observed after use of a skin flap. While the sample size in our study was small, the functional outcome and appearance were good. Thus we prefer using occlusive dressings in Zone 1 and 2 fingertip amputations, and flaps in zones 3 and 4 to ensure better fingertip viability and sensation.

Résumé

L'objectif de cette évaluation était d'analyser une série de patients ayant présenté une amputation digitale distale traitée par pansement occlusif.

Une évaluation monocentrique rétrospective était réalisée pour une série de 19 patients. Au plus grand recul, un évaluateur indépendant analysait le délai de cicatrisation, le nombre de pansements occlusifs nécessaires, la trophicité pulpaire, la sensibilité tactile épicritique par le test des deux points, la sensibilité au test du monofilament, l'existence de complications, la présence de dysesthésies et d'intolérance au froid, le score QuickDASH.

Dix-neuf patients ont pu être revus au recul de 12,6 mois (6-25). Le nombre de pansements occlusifs par patient était de 3,2 (3-5) avec un délai de cicatrisation de 4,3 semaines (4-5). La trophicité pulpaire était excellente ou bonne dans 18 cas. La sensibilité au test de Weber était normale ou bonne dans 16 cas. Dix-huit patients se disaient satisfaits ou très satisfaits. Le score QuickDASH atteignait 5,45).

Dans la littérature, la récupération de la sensibilité tactile est bonne après pansement occlusif (Weber = 2,5 à 4 mm). La sensibilité moyenne des différentes séries est meilleure que celle observée après lambeaux. L'effectif de notre série est faible, mais nous observons de bons résultats fonctionnels et esthétiques. Notre traitement de choix est représenté par les pansements occlusifs en zones 1 et 2, alors que nous utilisons les lambeaux dans les zones 3 et 4 afin d'apporter une meilleure trophicité pulpaire.

Introduction

The treatment strategy for fingertip amputations depends on the amputation level along the injured finger and the patient's condition, but also on the surgeon's preferences [START_REF] Lasserre | Fingertip reconstruction with occlusive dressing: clinical results and biological analysis of the dressing contents[END_REF][START_REF] Farrell | Conservative management of fingertip amputations[END_REF]. Treatment is a means to provide the missing skin with coverage flaps or to allow spontaneous regeneration of the fingertip though nonsurgical treatment (occlusive dressing, impregnated gauze dressings). This treatment also aims to restore fingertip function: fine sensation, skin stability allowing the fingertips to grasp objects, satisfactory appearance [START_REF] Lasserre | Fingertip reconstruction with occlusive dressing: clinical results and biological analysis of the dressing contents[END_REF].

Over the years, these goals have been achieved using coverage flaps. However, flaps are associated with complications such as necrosis, infection, finger stiffness, reduced sensation, wound healing sequelae, and fingertip instability.

An occlusive dressing prevents scar formation and allows regeneration of the fingertip in zone 1 and 2, or even zone 3 amputations in the Merle and Dautel topography [START_REF] Merle | Amputations d'attente et amputations définitives[END_REF]. Several studies have reported good clinical outcomes [START_REF] Lasserre | Fingertip reconstruction with occlusive dressing: clinical results and biological analysis of the dressing contents[END_REF][START_REF] Farrell | Conservative management of fingertip amputations[END_REF][START_REF] Mennen | Fingertips injuries management with semi-occlusive dressing[END_REF][START_REF] Hoigné | Semi-occlusive dressing for the treatment of fingertip amputations with exposed bone: quantity and quality of soft-tissue regeneration[END_REF][START_REF] Quell | Treatment of fingertip defect injuries with a semiocclusive dressing[END_REF][START_REF] Illingworth | Trapped fingers and amputated fingertips in children[END_REF][START_REF] Fox | Nonoperative management of fingertip pulp amputation by occlusive dressings[END_REF].

The aim of our study was to report on a new series of patients with a distal fingertip amputation who were treated with occlusive dressings and were evaluated continuously over a 4-year period. They were then reviewed by an independent surgeon at the end of the healing phase. We hypothesized that four dressings would be sufficient to regenerate the fingertip.

Patients and methods

This was a retrospective study of cases at a single hospital. Patients who suffered a distal fingertip amputation between 2011 and 2015 and were treated with occlusive dressings were evaluated. Included were patients who suffered a fingertip amputation distal to the distal interphalangeal joint, regardless of which finger was injured, the amputation mechanism or the amputation zone (classification of Merle and Dautel [START_REF] Merle | Amputations d'attente et amputations définitives[END_REF]).

Occlusive dressing application technique

For zone 1, 2, 3 amputations, the dressing was applied in the emergency room after lavage with sterile water, under local anesthesia if necessary. Two semipermeable dressings (Tegaderm®, 3M TM , Maplewood, Minnesota, United States) (Fig. 1) were used to wrap the entire amputation zone and were secured with adhesive down to the proximal interphalangeal joint. No antibiotics or antiseptic agents were used at any time, from the initial treatment until the wound healed [START_REF] Lasserre | Fingertip reconstruction with occlusive dressing: clinical results and biological analysis of the dressing contents[END_REF][START_REF] Hoigné | Amputations de la dernière phalange : régénération par film[END_REF]. The dressing was changed each week during an office visit. In all, three to six dressings were used per patient. The patient was not given any specific instructions on using the amputated and bandaged finger. One week after the dressing was applied, the skin was macerated and whitish in color (Fig. 2). After two or three dressings, the wound gave off an unpleasant, even nauseating, odor. Charcoal or adhesive bandages at the base of the finger were used to reduce this problem [START_REF] Hoigné | Amputations de la dernière phalange : régénération par film[END_REF].

Methods

At the final review, an investigator who was not involved in the initial treatment decision evaluated the time to healing, the number of occlusive dressings used, complications and trophic changes in the reconstructed fingertip. The latter was evaluated relative to the fingertip of the same finger in the contralateral hand as described by Lasserre et al. [START_REF] Lasserre | Fingertip reconstruction with occlusive dressing: clinical results and biological analysis of the dressing contents[END_REF]: "excellent" when both fingertips were identical, "poor" when a defect was present in the reconstructed fingertip that was more than one-quarter compared with the contralateral finger, and "good" between these two extremes. The reappearance of fingerprints was recorded. Epicritic sensation (Weber two-point discrimination test -2PD) was performed and interpreted as described by Moberg [START_REF] Moberg | Objective methods for determining the functional value of sensibility of the hand[END_REF]. The monofilament test was performed with five sizes and interpreted as follows: a response to the 2.83 monofilament was considered normal; a response to the 3.61 monofilament showed a reduction in fine sensitivity, a response to the 4.31 monofilament showed a reduction in protective sensation, and a response to the 4.56 or 5.07 monofilaments showed a loss of protective sensation (Touch-Test TM Sensory Evaluator Instructions). The presence of dysesthesia or cold intolerance and their duration was recorded. The QuickDASH and time to return to work were documented.

Lastly, nail-related complications were evaluated, and the patients were asked at the review visit to rate their satisfaction with the outcome as "very satisfied," "satisfied" or "disappointed."

Statistics

Comparisons of normally distributed data were performed with Student's t test. The threshold for statistical significance (p value) was set at 0.05. An evaluation of whether certain events were related to each other was done using the Chi-square test or Fisher's exact test. The correlation between two quantitative variables was determined with the Pearson correlation test.

Results

Nineteen patients treated with an occlusive dressing for a distal finger amputation between 2011 and 2015 were included. Their mean age was 47.3 years (22-73). The vast majority of the population was male (89.5%). The amputation mechanism consisted of clean transection, crushing, or mutilation. The amputations occurred mainly in zones 1 and 2 (78.9% of cases) (Table 1). The mean follow-up at the review was 12.6 years (6-25).

A mean of 3.2 (3-5) occlusive dressings were used per patient and the mean healing time was 4.3 ± 1 weeks (4-5). The fingertip condition was deemed excellent in 6 cases, good in 12 cases and poor in 1 case. The fingerprints were restored in all patients.

Based on the Weber 2PD and Moberg scale, sensitivity was normal in 15 cases, good in 1 case and discriminatory in the other 3 cases. None of the patients had normal sensation on the monofilament test. Reduction in fine sensitivity was found in 10 cases, reduction in protective sensitivity in 7 cases and loss of protective sensitivity in 2 cases. Thirteen patients continued to have dysesthesia at the review, one patient had altered sensation for 1 month after healing and five patients had no troubles. Four patients had persistent cold intolerance, including two patients who had a zone 3 amputation.

As for complications, nail dystrophy was found in four patients (three hook-nail deformities and one ridged nail), all of whom had a zone 3 amputation. No local or systemic infections occurred that required treatment or surgery.

At the final review, 13 patients were very satisfied with the final outcome while five were satisfied and one was disappointed. The mean QuickDASH score was 5.53 (0-20.45).

The time to return to work in the 13 employed patients was 4.8 ± 4 weeks [START_REF] Lasserre | Fingertip reconstruction with occlusive dressing: clinical results and biological analysis of the dressing contents[END_REF][START_REF] Farrell | Conservative management of fingertip amputations[END_REF][START_REF] Merle | Amputations d'attente et amputations définitives[END_REF][START_REF] Mennen | Fingertips injuries management with semi-occlusive dressing[END_REF][START_REF] Hoigné | Semi-occlusive dressing for the treatment of fingertip amputations with exposed bone: quantity and quality of soft-tissue regeneration[END_REF][START_REF] Quell | Treatment of fingertip defect injuries with a semiocclusive dressing[END_REF][START_REF] Illingworth | Trapped fingers and amputated fingertips in children[END_REF][START_REF] Fox | Nonoperative management of fingertip pulp amputation by occlusive dressings[END_REF][START_REF] Hoigné | Amputations de la dernière phalange : régénération par film[END_REF][START_REF] Moberg | Objective methods for determining the functional value of sensibility of the hand[END_REF][START_REF] Louis | Open treatment of digital tip injuries[END_REF][START_REF] De Boer | The use of silver sulphadiazine occlusive dressings for finger-tip injuries[END_REF][START_REF] Dumontier | Connaissance des complications de la chirurgie des lambeaux pulpaires des doigts longs et information des patients -implications éthiques[END_REF]. None of the variables were significant predictors of the outcome, given the small size of the subgroups.

Discussion

Reality and quality of fingertip regeneration

Our hypothesis that four dressings are needed to regenerate the fingertip was confirmed, although the outcomes were not as good in zone 3 amputations. While occlusive dressings are an effective solution with a long history of use, their use varies greatly between hospitals either due to lack of knowledge or due to fear. We found 17 publications on this topic, 14 of which were case series, including 2 comparative studies, evaluating 382 patients.

The published articles can be divided into pre-2010 and post-2010 for analysis purpose.

Before 2010, and especially in the large studies of more than 200 cases by Mennen and Wiese [START_REF] Mennen | Fingertips injuries management with semi-occlusive dressing[END_REF], the authors described the outcomes after 1 or 2 months of treatment. The Weber 2DP test was 4 mm, the complication rate was very low and there were no infections.

The articles published before 2010 all stressed the nauseating odor.

In 1974, Illingworth [START_REF] Illingworth | Trapped fingers and amputated fingertips in children[END_REF] reported excellent esthetic and functional outcomes in 300 cases of fingertip amputation in children treated non-surgically, even for amputations at the base of the distal phalanx (zone 4).

A few years later, Fox et al. [START_REF] Fox | Nonoperative management of fingertip pulp amputation by occlusive dressings[END_REF] claimed that occlusive dressings were superior to local flaps for zone 1 amputations. The dressing was done using aluminum foil. Louis et al. [START_REF] Louis | Open treatment of digital tip injuries[END_REF] reported very good tactile discrimination in 29 patients treated with occlusive dressings in zone 1. Two-thirds of patients were away from work for only 1 day. In 1981, De Boer and Collinson [START_REF] De Boer | The use of silver sulphadiazine occlusive dressings for finger-tip injuries[END_REF] compared dressings with Fucidine® (31 zone 1 amputations) to occlusive dressings with Flammazine® (26 zone 1 and 3 injuries with exposed bone). They reported excellent fingertip reconstruction, even in the cases of exposed bone (occlusive dressing).

The healing time was the same between groups, but the patients in the occlusive dressing group returned to work faster, as the waterproofness of this dressing allowed early resumption of duties in all working conditions. Quell et al. [START_REF] Quell | Treatment of fingertip defect injuries with a semiocclusive dressing[END_REF] reported good sensitivity and good viability after 42 distal amputations including those with bone loss. Mennen and Wiese [START_REF] Mennen | Fingertips injuries management with semi-occlusive dressing[END_REF] recommended this non-surgical treatment for any distal amputation given the published results (better results for tactile sensation). The appearance of the fingertip was nearly normal.

Starting in 2010, various comparative studies on frostbite were published and ultrasonography was incorporated to evaluate the regenerated fingertip. In 2010, Lasserre et al. [START_REF] Lasserre | Fingertip reconstruction with occlusive dressing: clinical results and biological analysis of the dressing contents[END_REF] were the first to describe the biological composition of the exudate in the dressing.

They found several types of saprophytic and pathogenic bacteria. These potentially aggressive pathogenic species never caused an infection in patients. The types of pathogenic bacteria varied from one week to the next and differed from the normal skin flora.

The second finding was the large amount of angiogenesis-promoting growth factors such as PDGF, VEGF and GF, which were not detectable in the occlusive dressings of the control subjects.

Hoigné et al. [START_REF] Hoigné | Semi-occlusive dressing for the treatment of fingertip amputations with exposed bone: quantity and quality of soft-tissue regeneration[END_REF] were the first to use ultrasonography to measure the percentage of the fingertip regenerated. Nearly 90% of the fingertip was restored after occlusive dressings were used for zone 2 and 3 amputations.

Fingertip sensitivity

In these various studies, the recovery in tactile spatial acuity was good, with mean 2PD ranging from 2.5 to 4 mm. However, there are no data on how these vary by amputation zone. The mean sensitivity reported in the literature is better with occlusive dressings than when flap coverage is used [START_REF] Dumontier | Connaissance des complications de la chirurgie des lambeaux pulpaires des doigts longs et information des patients -implications éthiques[END_REF]. There are no reports of the monofilament test being used.

We have no explanation for why this test found worse sensitivity than the Weber 2PD in our study. The healing time in our study was 4.1 weeks, which is consistent with other published studies, whether using flap coverage or an occlusive dressing (Table 2) [START_REF] Dumontier | Connaissance des complications de la chirurgie des lambeaux pulpaires des doigts longs et information des patients -implications éthiques[END_REF]. However, it is difficult to compare studies given the wide range of situations and extent of fingertip injuries involved. Patients who suffer a zone 3 amputation have the worse outcomes in terms of sensitivity and vitality of the fingertip. This suggests occlusive dressings have limitations in more proximal amputations.

Complications

In our study, complications were observed in patients who suffered a zone 3 amputation: nail dystrophy and ridged nail in a case of dorsal oblique amputation involving the nail bed. We found no infections or fingertip instability. Stiffness was also avoided because occlusive dressings allow early mobilization.

Other studies reported no complications, but they did not always measure sensory disorders and cold intolerance, which we believe are nearly unavoidable, especially in the first few months after healing. These complications occur both after conservative treatment and flap coverage [START_REF] Dumontier | Connaissance des complications de la chirurgie des lambeaux pulpaires des doigts longs et information des patients -implications éthiques[END_REF].

Mechanisms of fingertip regeneration

Fingertip regeneration with a dressing is considered true regeneration [START_REF] Ferguson | Scar-free healing: from embryonic mechanisms to adult therapeutic intervention[END_REF] because no scab forms. Many studies on skin regeneration have been performed on animals such as zebra fish and axolotl, which are models of extremity, limb and tail regeneration [START_REF] Ferguson | Scar-free healing: from embryonic mechanisms to adult therapeutic intervention[END_REF].

In our study, we found growth factors involved in fingertip regeneration, but have no information about their actions or magnitude.

In an animal model, Ferguson and O'Kane [START_REF] Farah | A concise review of common animal models for the study of limb regeneration[END_REF] highlighted the role of various TGFbeta subtypes, ubiquitous growth factors involved in several regenerative and healing processes. These studies open new research avenues and encouraged us to measure the various types of TGF-beta fractions found locally. According to Ferguson and O'Kane, TGF-beta3 is a marker or effector of scar-free healing. This growth factor is found in large quantities in regeneration models, contrary to TGF-beta1 and -2, which cause scarring without regeneration.

Conclusion

For cases of zone 1 and 2 fingertip amputations, healing with an occlusive dressing allows regeneration of the fingertip, with restoration of its shape and dermatoglyphic prints. This regeneration, which is better understood now, occurs with three to four occlusive dressings, thus 1 month of treatment. While certain studies report good cutaneous outcomes in zone 3 and zone 4 amputations, occlusive dressings cannot prevent hook-nail deformity in these zones without another procedure.

Figure legends

Fig. 1. Application of occlusive dressing; the same method is used each time the dressing is changed. In the ideal case, the dressing does not capture the proximal interphalangeal joint to allow movement of the injured finger.

Fig. 2 .Fig. 3 .Fig. 4 .Fig. 5 .

 2345 Fig. 2. Typical appearance of macerated skin after 1 week Fig. 3. Female 46-year-old patient who suffered a zone 1 amputation and required 4 dressings. Appearance at D7, D14 and 3 months. The result was the same at 1 year. Weber = 4 mm, moderate dysesthesia and cold intolerance Fig. 4. Male, 34-year-old patient who suffered a zone 2 amputation and required 4 dressings. Appearance at D0, D21 and 4 months. The result was the same at 1 year. Weber = 4 mm, returned to work after 5 weeks Fig. 5. Male, 39-year-old patient who suffered a zone 3 amputation and required 4 dressings. Appearance at D7, D21 and 4 months. The result was the same at 1 year. Weber = 12 mm, returned to work after 3 weeks, moderate cold intolerance and hook-nail deformity

Table titles Table 1 .

 titles1 Characteristics of the patients included in our case series

Table 2 .

 2 Main published studies on occlusive dressings for fingertip amputation (NR: not reported)

Table 1 .

 1 Characteristics of the patients included in our case series

	Sample size	19 patients
	Sex	2 women, 17 men
	Age	47.3 years [22-73]
	Affected finger	Thumb (2), index (2), middle (11), ring (4)
	Mechanism	4 clean transection, 12 crush, 3 mutilation
	Amputation zone	9 in zone 1, 6 in zone 2, 4 in zone 3

Table 2 .

 2 Main published studies on occlusive dressings for fingertip amputation (NR: not reported)

	Author	Amputation zone	# of	Complications	Time to healing	Results	Discomfort
	# of cases		dressings
			used
	Fox 1977	Zone 1	4	None	4 weeks	Acceptable	Less than 10 days
	22 cases					appearance
	Farell 1977	15 zone 1	NR	2 stiffness	Zone 1: 15 days	Attractive	NR
	21 cases	6 with bone exposure /		4 hypersensitivity		appearance
		zone not listed		3 hypoesthesia
					With bone exposure:
					30 to 45 days
	Louis 1980	Zone 1	3.5	4 cold intolerance + dysesthesia	NR	NR	1 day for 2/3 of
	29 cases			3 cold intolerance only			patients
	De Boer 1981	25 zone 1	NR	None (initial hypersensitivity)	12 days	Good	6 days
	29 cases	4 with bone exposure
	Mennen 1993	NR	2.5	None	20 to 30 days	Nearly	A few days
	200 cases					normal
	Quell 1998	26 zone 1	2 to 8	None	6 to 64 days	Good	NR
	42 cases	8 zone 2
		8 zone 3
	Lasserre 2010	13 zone 1	3.8	None	20 days	Excellent 30%	NR
	19 cases	5 zone 2				Good 70%
		1 zone 3	(2 to 7)		(15 to 35 days)
	Hoigné 2014	Zone 2 & 3	4	1 hypersensitivity at 6 months	6.5 weeks	Good with 90%	NR
	19 cases			1 neuroma		fingertip
				1 nail remainder	(3 to 8 weeks)	reconstructed

Conflict of interest:

Laurent Obert has established relationships with the following organizations: FX solutions, Zimmer, Medartis, Evolutis, Wright, Elsevier. None of the other authors have conflicts of interest related to this work.


Keywords: Antifibrotic, Scaffold, Growth factors, Proof of concept, Amniotic membrane Mots clefs : Anti-fibrosant, Support, Facteurs de croissances, Preuve de concept

Can the amniotic membrane be used to treat peripheral nerve defects? A review of literature La membrane amniotique peut-elle être utilisée pour traiter les anomalies nerveuses périphériques? Une revue de littérature

Introduction

Traumatic peripheral nerve lesions are a genuine public health problem as there are more than 300,000 cases per year in Europe [START_REF] Ichihara | Artificial nerve tubes and their application for repair of peripheral nerve injury: an update of current concepts[END_REF]. The incidence of finger lacerations with nerve injury is up to 6.2/100,000 inhabitants, resulting in a significant cost to society [START_REF] Thorsén | Digital nerve injuries: epidemiology, results, costs, and impact on daily life[END_REF].

Several techniques, such as autologous nerve graft, autograft vein conduits, nerve transfer, collagen or silicone tubes, and neurotubes, have been described for repairing peripheral nerve defects [START_REF] Jardin | Large diameter nerve conduits use in the upper limb: report of four cases and literature review[END_REF]. However, these techniques have drawbacks, [START_REF] Riccio | Nerve repair and regeneration: Biological tubulization limits and future perspectives[END_REF] such as donor site morbidity for autologous grafts, development of fibrosis and limitations on defect size for neurotubes [START_REF] Dunnen | Long-term evaluation of nerve regeneration in a biodegradable nerve guide[END_REF] and vein conduits [START_REF] Boyd | Nerve reconstruction in the hand and upper extremity[END_REF].

One of the goals of peripheral nerve repair is to optimize the healing process in order to prevent neuroma development. Posttraumatic neuromas are a benign, disorganized tissue stemming for an unsuccessful attempt at repairing a peripheral nerve, which results in a "pseudotumor". It appears 1 to 12 months after the injury event. Yülsel et al. believe neuromas develop because of injury to the perineurium [START_REF] Boyd | Nerve reconstruction in the hand and upper extremity[END_REF]. Axons that are not contained in their envelope can escape into adjacent tissues. This phenomenon is accompanied by significant cell proliferation (fibroblasts, Schwann cells) and by blood vessel development.

The result is a disorganized network of axons, which are deviated and curled up [START_REF] Yüksel | Prevention of painful neuromas by epineural ligatures, flaps and grafts[END_REF].

In recent years, the amniotic membrane has had tremendous success in repairing soft tissues, particularly in the ophthalmology and dermatology context [START_REF] Silini | The long path of human placenta, and its derivatives, in regenerative medicine[END_REF], making it a relevant option in nerve repair. Riccio et al. reported encouraging results in humans for repairing posttraumatic nerve defects 3 to 5 cm in length [START_REF] Riccio | The amnion muscle combined graft (AMCG) conduits: a new alternative in the repair of wide substance loss of peripheral nerves[END_REF]. This tissue is mainly composed of types I, III, IV and V collagen, laminin, proteoglycans, elastin and fibronectin [START_REF] Niknejad | Properties of the amniotic membrane for potential use in tissue engineering[END_REF]. It is made up of two layers: the amnion (which is in contact with the amniotic fluid) and the chorion. The amnion is 0.02-0.5 mm thick while the chorion is 1 mm thick. The amnion mainly consists of an epithelium, which is rich in epithelial cells, a basal membrane and a fibroblast layer, which is rich in amniotic mesenchymal stromal cells. The amnion itself is sometimes incorrectly called the "amniotic membrane". The chorion consists of a reticular layer, which is rich in chorionic mesenchymal stem cells, a basal membrane, and a trophoblast, which is rich in trophoblast stem cells.

The amniotic membrane contributes to re-epithelialization and has antifibrotic, antiinflammatory, antiangiogenic, antimicrobial and antiviral properties [START_REF] Dua | The amniotic membrane in ophthalmology[END_REF][START_REF] Baradaran-Rafii | Amniotic membrane transplantation[END_REF]. It can be used as an allograft because the absence of immunogenicity. Furthermore, it contributes to the healing of wounds and has analgesic properties. Its mechanical properties-viscoelasticity due to its high levels of collagen and elastin-and its high levels of several growth factors [START_REF] Koob | Cytokines in single layer amnion allografts compared to multilayer amnion/chorion allografts for wound healing[END_REF] make it a biologically active scaffold well-suited to the challenge of nerve repair [START_REF] Riccio | Nerve repair and regeneration: Biological tubulization limits and future perspectives[END_REF].

We performed a review of literature to establish proof of concept on the use of this highly innovative and promising tissue for treating peripheral nerve defects.

Material and methods

Search strategy

The review of literature encompassed several different sources:

-PubMed using the keywords/MeSH entry terms: "amnion", "amniotic membrane", "angiogenesis", "anti-microbial", "characteristic", "chorion", "epithelialization", "fibrosis", "gap", "growth factors", "use", "nerve". The Boolean operator "and" was used to joint "Amniotic membrane" with "Angiogenesis"; "Antimicrobial"; "Characteristic"; "Epithelialization"; "Fibrosis"; "Growth factors"; "Use"; "Nerve".

Additional articles were added after manually reviewing the reference list of all the included publications.

-The United States clinical trials registry (https://clinicaltrials.gov/ct2/search), using the keywords/MeSH entry terms "amniotic membrane" and capturing completed or on-going clinical trials.

-The book by Jeremy Lim: A Primer on Amniotic Membrane Regenerative Healing.

MiMedx Group, 2015.

-The Google search engine, mainly to search for marketed products.

One of our research group's prior publications describing the clinical use, patents and marketed products was also consulted [START_REF] Gindraux | Human amniotic membrane: clinical uses, patents and marketed products[END_REF].

Selection criteria

For preclinical proof of concept, studies were included if they analyzed amniotic membrane use in animal models of nerve defect or nerve transection.

For clinical trials, studies were included:

-from PubMed if they reported on amniotic membrane use in humans for treating nerve defects.

-from the clinical trials registry when they referred to amniotic membrane use in a rheumatology, orthopedic or trauma context.

In both cases (animal models and clinical trials), only articles in which the amniotic membrane was used as a scaffold were retained. In vitro studies or studies using cells isolated from the tissue were excluded.

For evidence in support of nerve repair/regeneration, the most relevant articles, often the most recent ones and those cited as a reference, were used in our review.

Generally, only articles published in English were retained.

Screening of studies and data extraction

The article selection and data collection was performed independently by two investigators (MB and FG). The title and abstract were selected based on the following question: Is amniotic membrane use in an animal model or clinical study beneficial for treating nerve defects? An article was evaluated in its entirety if the title and abstract pertained to this screening question. Discrepancies between the two investigators were discussed to select the final articles. Tables were generated and used to collect the relevant information.

Results

The PubMed search yielded the following (before 01-OCT-2018):

-Amniotic membrane "and" angiogenesis: 85 articles -Amniotic membrane "and" antimicrobial: 894 articles -Amniotic membrane "and" characteristic: 178 articles -Amniotic membrane "and" epithelialization: 237 articles -Amniotic membrane "and" fibrosis: 348 articles -Amniotic membrane "and" growth factors: 1566 articles -Amniotic membrane "and" use: 456 articles -Amniotic membrane "and" nerve: 204 articles The clinical trials registry search (before 01-OCT-2018) yielded 186 studies.

Amniotic membrane format

The human amniotic membrane, and more specifically the amnion (separated from the chorion), is collected from placenta following Cesarean birth. Tissue from vaginal delivery is not used since it is contaminated by the normal vaginal flora. Several methods exist to store this product: cryopreservation, lyophilization and preservation in dried form [START_REF] Jirsova | Amniotic membrane in ophthalmology: properties, preparation, storage and indications for grafting-a review[END_REF]. Fresh human amniotic membrane, which is sometimes used in animal models, corresponds to tissue that has not been treated after being collected.

In cryopreserved form, the amniotic membrane is packaged in a solution including a cryoprotectant (glycerol or dimethyl sulfoxide -DMSO) and Dulbecco's modified Eagle's medium. The tissue is then stored at -80°C and can be used for up to 1 to 2 years. With this type of preservation, 20% to 50% of the cells survive [START_REF] Laurent | Storage and qualification of viable intact human amniotic graft and technology transfer to a tissue bank[END_REF]. This is the benchmark format in current practice and clinical trials [START_REF] Silini | The long path of human placenta, and its derivatives, in regenerative medicine[END_REF].

In its lyophilized form, the amniotic membrane is quickly frozen at -50 to -80°C, then freeze-dried in a lyophilizer under vacuum. In some cases, the tissue will undergo pretreatment to destroy any residual viable cells; sterilization by gamma irradiation very often follows the lyophilization step. These two additional processing steps offer new hope for ensuring the safety of amniotic membrane from vaginal delivery, thereby increasing the number of placentas collected. This also allows the allograft to be stored in its original format (two layers). In other cases, the chorion (which is in contact with the maternal tissue) is generally separated from the amnion to avoid risk of contamination by the mother's cells. The resulting "amnion-chorion" product is thicker and stronger than amnion alone. Lastly, lyophilization allows the tissue to be stored at room temperature (e.g. in an operating room).

In its dehydrated form, the amniotic membrane is stored at room temperature in a biosafety cabinet and exposed to air for varying lengths of time (12 to 24 hours). This is usually followed by gamma irradiation for sterilization purposes.

Whether the tissue is used in its fresh, cryopreserved, lyophilized or dehydrated form, it may be subjected to de-epithelialization immediately after being harvested in order to remove the epithelial cell layer.

Table 1 lists the features of the amniotic membrane based on its format and suitability for the target clinical indication.

Preclinical proof of concept

From the PubMed search and after the inclusion and exclusion criteria were applied, 15 articles on animal models were analyzed and relevant data were extracted [START_REF] Ozcan | Vascularized nerve tube: an experimental alternative for vascularized nerve grafts over short gaps[END_REF][START_REF] Mohammad | Modulation of peripheral nerve regeneration: a tissue-engineering approach. The role of amnion tube nerve conduit across a 1-centimeter nerve gap[END_REF][START_REF] Mligiliche | Extracellular matrix of human amnion manufactured into tubes as conduits for peripheral nerve regeneration[END_REF][START_REF] Duan | Bridging rat sciatic nerve defects with the composite nerve-muscle autografts wrapped with human amnion matrix membrane[END_REF][START_REF] Ozgenel | Combined application of human amniotic membrane wrapping and hyaluronic acid injection in epineurectomized rat sciatic nerve[END_REF][START_REF] Henry | Improving electrophysiologic and histologic outcomes by photochemically sealing amnion to the peripheral nerve repair site[END_REF][START_REF] O'neill | Preparation and integration of human amnion nerve conduits using a light-activated technique[END_REF][START_REF] Kim | Use of human amniotic membrane wrap in reducing perineural adhesions in a rabbit model of ulnar nerve neurorrhaphy[END_REF][START_REF] Meng | Assessment of processed human amniotic membrane as a protective barrier in rat model of sciatic nerve injury[END_REF][START_REF] Fesli | Enhancement of nerve healing with the combined use of amniotic membrane and granulocyte-colony-stimulating factor[END_REF][START_REF] Fairbairn | Light-activated sealing of nerve graft coaptation sites improves outcome following large gap peripheral nerve injury[END_REF][START_REF] Sadraie | Study of transected sciatic nerve repair by amniotic membrane with betamethasone in adult albino wistar rats[END_REF][START_REF] Lemke | Transplantation of human amnion prevents recurring adhesions and ameliorates fibrosis in a rat model of sciatic nerve scarring[END_REF][START_REF] Hasturk | Stereologic and ultrastructural comparison of human and rat amniotic membrane wrapping for rat sciatic nerve repair[END_REF][START_REF] Marchesini | The amnion muscle combined graft (AMCG) conduits in nerves repair: an anatomical and experimental study on a rat model[END_REF] (Table 2). Among these 15 studies, 10 were published after 2009, evidence of the recent interest in using amniotic membrane for nerve repair. The studies appear to be highly relevant given the number of animals used, the control groups, comparison with other treatments and functional outcomes.

In these animal models, the human amniotic membrane was used in its fresh or cryopreserved form and was very often de-epithelialized. The dehydrated form was used in only two studies [START_REF] Mligiliche | Extracellular matrix of human amnion manufactured into tubes as conduits for peripheral nerve regeneration[END_REF][START_REF] Marchesini | The amnion muscle combined graft (AMCG) conduits in nerves repair: an anatomical and experimental study on a rat model[END_REF]. In most studies, the model used is one where a localized 1-1.5 cm nerve defect or lesion is made in the sciatic nerve of rats.

The amniotic membrane typically measures 1-2 cm and is secured at each end by an epineural suture [START_REF] Ozcan | Vascularized nerve tube: an experimental alternative for vascularized nerve grafts over short gaps[END_REF][START_REF] Mligiliche | Extracellular matrix of human amnion manufactured into tubes as conduits for peripheral nerve regeneration[END_REF][START_REF] Kim | Use of human amniotic membrane wrap in reducing perineural adhesions in a rabbit model of ulnar nerve neurorrhaphy[END_REF][START_REF] Meng | Assessment of processed human amniotic membrane as a protective barrier in rat model of sciatic nerve injury[END_REF][START_REF] Fesli | Enhancement of nerve healing with the combined use of amniotic membrane and granulocyte-colony-stimulating factor[END_REF][START_REF] Sadraie | Study of transected sciatic nerve repair by amniotic membrane with betamethasone in adult albino wistar rats[END_REF][START_REF] Hasturk | Stereologic and ultrastructural comparison of human and rat amniotic membrane wrapping for rat sciatic nerve repair[END_REF][START_REF] Marchesini | The amnion muscle combined graft (AMCG) conduits in nerves repair: an anatomical and experimental study on a rat model[END_REF], photochemical tissue bonding [START_REF] Henry | Improving electrophysiologic and histologic outcomes by photochemically sealing amnion to the peripheral nerve repair site[END_REF][START_REF] O'neill | Preparation and integration of human amnion nerve conduits using a light-activated technique[END_REF][START_REF] Fairbairn | Light-activated sealing of nerve graft coaptation sites improves outcome following large gap peripheral nerve injury[END_REF] or is left unsutured [START_REF] Ozgenel | Combined application of human amniotic membrane wrapping and hyaluronic acid injection in epineurectomized rat sciatic nerve[END_REF][START_REF] Lemke | Transplantation of human amnion prevents recurring adhesions and ameliorates fibrosis in a rat model of sciatic nerve scarring[END_REF]. Only a few studies specified the orientation of the applied amnion: mesenchymal side against the nerve [START_REF] Ozgenel | Combined application of human amniotic membrane wrapping and hyaluronic acid injection in epineurectomized rat sciatic nerve[END_REF] or, more recently, epithelial side [START_REF] Lemke | Transplantation of human amnion prevents recurring adhesions and ameliorates fibrosis in a rat model of sciatic nerve scarring[END_REF][START_REF] Marchesini | The amnion muscle combined graft (AMCG) conduits in nerves repair: an anatomical and experimental study on a rat model[END_REF], after simulation of nerve wrapping in vitro [START_REF] Lemke | Transplantation of human amnion prevents recurring adhesions and ameliorates fibrosis in a rat model of sciatic nerve scarring[END_REF].

The literature search showed that its use as a perineural wrap appears conducive to repairing a nerve lesion or defect (Table 2). First, this increases the number of axons, nerve fibers and myelinated fibers [START_REF] Mohammad | Modulation of peripheral nerve regeneration: a tissue-engineering approach. The role of amnion tube nerve conduit across a 1-centimeter nerve gap[END_REF][START_REF] Mligiliche | Extracellular matrix of human amnion manufactured into tubes as conduits for peripheral nerve regeneration[END_REF][START_REF] Henry | Improving electrophysiologic and histologic outcomes by photochemically sealing amnion to the peripheral nerve repair site[END_REF][START_REF] Meng | Assessment of processed human amniotic membrane as a protective barrier in rat model of sciatic nerve injury[END_REF]. For example, for a 1-cm nerve defect, the number of large-diameter axons is higher when an amniotic membrane is used instead of a silicone tube or nerve autograft [START_REF] Mohammad | Modulation of peripheral nerve regeneration: a tissue-engineering approach. The role of amnion tube nerve conduit across a 1-centimeter nerve gap[END_REF]. It also appears that the diameter of the configured amnion tube greatly affects the repair; a 1-2 mm diameter tube appears ideal [START_REF] Mligiliche | Extracellular matrix of human amnion manufactured into tubes as conduits for peripheral nerve regeneration[END_REF]. The G-ratio, which is defined as the ratio of the axon diameter to the nerve fiber diameter, is improved [START_REF] Henry | Improving electrophysiologic and histologic outcomes by photochemically sealing amnion to the peripheral nerve repair site[END_REF][START_REF] Meng | Assessment of processed human amniotic membrane as a protective barrier in rat model of sciatic nerve injury[END_REF] and the functional recovery, estimated by the Sciatic Functional Index (SFI), is significant [START_REF] Mohammad | Modulation of peripheral nerve regeneration: a tissue-engineering approach. The role of amnion tube nerve conduit across a 1-centimeter nerve gap[END_REF][START_REF] Meng | Assessment of processed human amniotic membrane as a protective barrier in rat model of sciatic nerve injury[END_REF].

From an electrophysiology perspective, Meng et al. showed that amnion combined with nerve autograft helps to increase the amplitude and reduce conduction latency at 8 weeks [START_REF] Meng | Assessment of processed human amniotic membrane as a protective barrier in rat model of sciatic nerve injury[END_REF]. Second, the amniotic membrane has a noteworthy effect on fibrosis and perineural adhesions [START_REF] Ozgenel | Combined application of human amniotic membrane wrapping and hyaluronic acid injection in epineurectomized rat sciatic nerve[END_REF][START_REF] Kim | Use of human amniotic membrane wrap in reducing perineural adhesions in a rabbit model of ulnar nerve neurorrhaphy[END_REF][START_REF] Meng | Assessment of processed human amniotic membrane as a protective barrier in rat model of sciatic nerve injury[END_REF]. Lemke et al. specified that the tissue prevents the recurrence of severe perineural adhesions, reduces perineural and intraneural fibrosis, accelerates nerve structure regeneration and improves recovery of nerve function [START_REF] Lemke | Transplantation of human amnion prevents recurring adhesions and ameliorates fibrosis in a rat model of sciatic nerve scarring[END_REF]. Some authors report the benefits of the amniotic membrane in a vascularized form [START_REF] Ozcan | Vascularized nerve tube: an experimental alternative for vascularized nerve grafts over short gaps[END_REF], or combined with skeletal muscle [START_REF] Marchesini | The amnion muscle combined graft (AMCG) conduits in nerves repair: an anatomical and experimental study on a rat model[END_REF], hyaluronic acid [START_REF] Ozgenel | Combined application of human amniotic membrane wrapping and hyaluronic acid injection in epineurectomized rat sciatic nerve[END_REF], granulocyte colony stimulating factor (G-CSF) [START_REF] Fesli | Enhancement of nerve healing with the combined use of amniotic membrane and granulocyte-colony-stimulating factor[END_REF] or betamethasone [START_REF] Sadraie | Study of transected sciatic nerve repair by amniotic membrane with betamethasone in adult albino wistar rats[END_REF] to potentiate its effect. Lastly, photochemical tissue bonding of the amnion compared to fixation using epineural sutures positively affects axon size, axonal migration, nerve fiber diameter, G-ratio index, muscle mass innervated by the transected then repaired nerve, and myelin sheath thickness [START_REF] Henry | Improving electrophysiologic and histologic outcomes by photochemically sealing amnion to the peripheral nerve repair site[END_REF][START_REF] O'neill | Preparation and integration of human amnion nerve conduits using a light-activated technique[END_REF][START_REF] Fairbairn | Light-activated sealing of nerve graft coaptation sites improves outcome following large gap peripheral nerve injury[END_REF].

Clinical trials

A search on the clinical trials registry reveals that more than 100 clinical studies with human amniotic membrane or its derivatives have been performed or are ongoing. However, we did not find any clinical trials that specifically addressed our research question. Thus we decided to compile the 40 clinical trials related to the musculoskeletal system (surgical and medical pathology) (Table 3): 25 describe treatments for osteoarthritis, rotator cuff disease, osteochondral lesions, tendinitis or low back pain; 15 relate to tendon lesions in the hand or ankle, anterior cruciate ligament injuries, reducing adhesions during total knee arthroplasty and spinal fusion surgery.

We found a single clinical trial on PubMed describing use of the amniotic membrane as a nerve conduit for treating nerve defects. Riccio et al. reported very promising results in a prospective single-center study performed between March 2012 and September 2013 in the Department of Reconstructive Surgery and Hand Surgery, AOU "Ospedali Riuniti" di Ancona, Italy [START_REF] Riccio | The amnion muscle combined graft (AMCG) conduits: a new alternative in the repair of wide substance loss of peripheral nerves[END_REF]. The trial involved 5 patients between 22 and 42 years of age (mean of 33.6 years) who had a median nerve injury at the wrist with a 3-5 cm defect (4 cm on average). The time to surgical nerve repair was 0 to 5 months. Fresh or dehydrated amnion was filled with skeletal muscle harvested from an adjacent area. This sheet was then shaped into a tube and secured with 9-0 epineural suture consisting of two stitches at the back and three stitches in front. They found that sensory and motor functions were restored based on the Lister muscular test, Jamar test and Sakellarides classification: one patient had a sensory result of S3, two were S3+ and two were S4; one patient had a motor result of M3 and four had a motor result of M4. This clinical proof of concept was substantiated by a recently published experimental model [START_REF] Marchesini | The amnion muscle combined graft (AMCG) conduits in nerves repair: an anatomical and experimental study on a rat model[END_REF].

The PubMed search also identified a clinical trial that we chose to include in our analysis: treatment of recurrent ulnar nerve syndrome at the elbow in which the patients were treated with an amniotic membrane nerve wrap sutured on itself with 8-0 suture [START_REF] Gaspar | Recurrent cubital tunnel syndrome treated with revision neurolysis and amniotic membrane nerve wrapping[END_REF]. This retrospective study included 8 patients treated between December 2012 and March 2014.

The average pain level on a visual analog scale was lower by 3.5 points after the procedure than before it; the QuickDASH score improved by 30 points, the grip strength by 25 lbs., or 38% compared with the opposite side. Three patients returned to work full time, one patient was able to go from light duty to a full workload, one unemployed patient began a new job and three remained unemployed. No complications were reported.

Nevertheless, this lack of clinical data means that clinical proof of concept studies on amniotic membrane use for treating nerve defects still need to be done.

Evidence in favor of nerve repair/regeneration

Presence of growth factors

One of the main reasons amniotic membrane is used in tissue engineering is its bioactive nature. In fact, no matter which format is used, this tissue contains a multitude of growth factors [START_REF] Koob | Cytokines in single layer amnion allografts compared to multilayer amnion/chorion allografts for wound healing[END_REF] including neurotrophic factors such as nerve growth factor (NGF), brainderived neurotrophic factor (BDNF), neurotrophin (NT-3), glial-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) [START_REF] Riccio | Nerve repair and regeneration: Biological tubulization limits and future perspectives[END_REF].

However, certain conditions impact the type and amount of growth factors in the tissue:

-Maternal and gestational age: membranes from a full-term pregnancy and from donors under 35 years of age have higher growth factor levels [START_REF] Lopez-Valladares | Donor age and gestational age influence on growth factor levels in human amniotic membrane[END_REF].

-Harvest site: epidermal growth factor (EGF) concentration can vary two-fold depending on where the amniotic membrane is harvested in the placenta [START_REF] Gicquel | Epidermal growth factor variations in amniotic membrane used for ex vivo tissue constructs[END_REF]. This variation in concentration may be related to different epithelial cell morphology and different mitochondrial activity depending on the harvest site [START_REF] Banerjee | Different metabolic activity in placental and reflected regions of the human amniotic membrane[END_REF].

A recent study has shown the region opposite the placenta (reflected region) is most suitable for preventing recurrent adhesions and fibrosis [START_REF] Lemke | Transplantation of human amnion prevents recurring adhesions and ameliorates fibrosis in a rat model of sciatic nerve scarring[END_REF].

-Preservation of both layers (amnion and chorion): the chorion layer contains 82% of the growth factors, except for EGF, of which 20% is contained in the amnion and 80% in the chorion [START_REF] Koob | Cytokines in single layer amnion allografts compared to multilayer amnion/chorion allografts for wound healing[END_REF][START_REF] Mcquilling | Proteomic comparison of amnion and chorion and evaluation of the effects of processing on placental membranes[END_REF].

-Preservation of epithelium in amnion: Fresh amniotic membranes with epithelium are very high in basic fibroblast growth factor (bFGF), EGF, hepatic growth factor receptor (HGFR), keratinocyte growth factor (KGF), keratinocyte growth factor receptor (KGFR), transforming growth factor (TGF) -β1, 2, 3 and tumor necrosis factor (TNF) -α [START_REF] Koizumi | Growth factor mRNA and protein in preserved human amniotic membrane[END_REF].

-Method of tissue preservation: the fresh form has the most growth factors, followed by the cryopreserved form and then the lyophilized form [START_REF] Wolbank | Impact of human amniotic membrane preparation on release of angiogenic factors[END_REF][START_REF] Russo | The effects of different preservation processes on the total protein and growth factor content in a new biological product developed from human amniotic membrane[END_REF][START_REF] Paolin | Cytokine expression and ultrastructural alterations in fresh-frozen, freeze-dried and gamma-irradiated human amniotic membranes[END_REF].

However, this aspect is controversial in the literature.

Antifibrotic, antiinflammatory and pro-or antiangiogenic properties

These growth factors have specific functions. The antifibrotic effect is one of the primary properties that ensures good nerve healing. Fibroblasts are naturally involved in healing and are activated during the process by TGF-β. The antifibrotic effect of the amniotic membrane is achieved by suppression of TGF-β signaling and reduction in the expression of TGF-β1, 2 and 3 isoforms and TGF-β receptor II [START_REF] Jirsova | Amniotic membrane in ophthalmology: properties, preparation, storage and indications for grafting-a review[END_REF]. These antifibrotic properties have been demonstrated in various animal models during peripheral nerve repair (Table 2).

The antiinflammatory effect of the amniotic membrane is guided by inhibition of the expression of proinflammatory cytokines such as bFGF, interferon (IFN) -c, interleukin (IL) -1ɑ, 2, 8, 10, platelet derived growth factor (PDGF) and TNF-β on the damaged ocular surface [START_REF] Jirsova | Amniotic membrane in ophthalmology: properties, preparation, storage and indications for grafting-a review[END_REF].

Lastly, the tissue's mesenchyme has fibroblasts that synthesize proangiogenic growth factors: angiopoietin (Ang) -2, bFGF, EGF, heparin binding epidermal growth factor (HB-EGF), HGF, PDGF-BB, placental growth factor (PLGF) and vascular epidermal growth factor (VEGF) [START_REF] Koob | Angiogenic properties of dehydrated human amnion/chorion allografts: therapeutic potential for soft tissue repair and regeneration[END_REF]. These trigger proliferation of endothelial cells and guide the formation of new blood vessels. Paradoxically, the amniotic membrane can also have an antiangiogenic effect [START_REF] Hao | Identification of antiangiogenic and antiinflammatory proteins in human amniotic membrane[END_REF] through the production of other factors such as tissue inhibitor of metalloproteinase (TIMP) -1, 2 or pigment epithelium-derived factor (PEDF) [START_REF] Hao | Identification of antiangiogenic and antiinflammatory proteins in human amniotic membrane[END_REF][START_REF] Shao | Suppression of corneal neovascularization by PEDF release from human amniotic membranes[END_REF]. The microenvironment in which the allograft is implanted will drive the pathway toward pro-or antiangiogenic activity.

Semipermeable and absorbable scaffold properties

Whether the amniotic membrane is used in its cryopreserved or lyophilized form, it is an excellent biodegradable scaffold with biochemical and mechanical proprieties that make it well-suited to nerve regeneration. It has unique nerve conduit capacities because of its structural properties and specific protein elements such as collagen, laminin and proteoglycans. This tissue has been exploited in patents and marketed products [START_REF] Silini | The long path of human placenta, and its derivatives, in regenerative medicine[END_REF][START_REF] Laurent | Storage and qualification of viable intact human amniotic graft and technology transfer to a tissue bank[END_REF] (Table 4). It has other specific characteristics that are similar to another human tissue typethe induced membrane-and can function as a bioreactor [START_REF] Gindraux | Similarities between induced membrane and amniotic membrane: Novelty for bone repair[END_REF].

As for its degradation, the amniotic membrane (depending on the format used) is still visible when animals are euthanized at 4 weeks [START_REF] Lemke | Transplantation of human amnion prevents recurring adhesions and ameliorates fibrosis in a rat model of sciatic nerve scarring[END_REF], 6 weeks [START_REF] Tao | Implantation of amniotic membrane to reduce postlaminectomy epidural adhesions[END_REF] and 12 weeks [START_REF] Ozgenel | Combined application of human amniotic membrane wrapping and hyaluronic acid injection in epineurectomized rat sciatic nerve[END_REF][START_REF] O'neill | Preparation and integration of human amnion nerve conduits using a light-activated technique[END_REF] after its implantation, but is no longer visible at 4 months (16 weeks) [START_REF] Mohammad | Modulation of peripheral nerve regeneration: a tissue-engineering approach. The role of amnion tube nerve conduit across a 1-centimeter nerve gap[END_REF]. Lemke et al. point out that the membrane can no longer be differentiated from neighboring tissues at 3 months [START_REF] Lemke | Transplantation of human amnion prevents recurring adhesions and ameliorates fibrosis in a rat model of sciatic nerve scarring[END_REF]. Thus, beyond this point in time, we cannot expect the tissue to have a positive impact, although it no longer matters, given the speed of the adhesion process in their study.

Discussion

There is little data on using the amniotic membrane to repair peripheral nerve defects. This literature review describes the exceptional ability of this tissue to regenerate nerves and/or reduce adhesions and fibrosis and serves as proof of concept for the targeted clinical indication. It supplements the similar evaluation done for oral surgery [START_REF] Fénelon | What is the benefit of using amniotic membrane in oral surgery? A comprehensive review of clinical studies[END_REF].

As shown in our review's findings, in addition to its fundamental antifibrotic nature, this bioactive tissue has several growth factors and angiogenic factors that can effectively guide nerve regeneration. In fact, certain growth factors such as NGF, BDNF, NT-3 and TGF-β are involved in the formation of fibrosis [START_REF] Tseng | How does amniotic membrane work?[END_REF]. For example, TGF-β is produced by cells recruited to the site of the local damage. It converts fibroblasts into myofibroblasts to repair the extracellular matrix components. It also regulates the production of enzymes such as matrix metalloproteinases (MMPs) that catalyze the degradation of extracellular matrix. In the case of excessive scarring, myofibroblasts synthesize an abnormally high amount of extracellular matrix components and MMPs [START_REF] Silini | Soluble factors of amnion-derived cells in treatment of inflammatory and fibrotic pathologies[END_REF]. The amniotic membrane contributes to the synthesis of MMP inhibitors, TIMP-1,-2,-3 and -4 [START_REF] Kim | Amniotic membrane patching promotes healing and inhibits proteinase activity on wound healing following acute corneal alkali burn[END_REF], and has an anti-inflammatory action through modulation of cytokines such as IL-6, IL-10 IL-1β, IL-9, IL-17a, IL-22, TNFα, IFNγ and prostaglandins [START_REF] Rossi | Characterization of the conditioned medium from amniotic membrane cells: prostaglandins as key effectors of its immunomodulatory activity[END_REF][START_REF] Pianta | Amniotic membrane mesenchymal cells-derived factors skew T cell polarization toward treg and downregulate Th1 and Th17 cells subsets[END_REF]. In animal models, xenografting of this tissue helps to reduce fibrosis in various contexts: nerve lesions [START_REF] Meng | Assessment of processed human amniotic membrane as a protective barrier in rat model of sciatic nerve injury[END_REF], burns and chronic wounds [START_REF] Tseng | Suppression of transforming growth factor-beta isoforms, TGF-beta receptor type II, and myofibroblast differentiation in cultured human corneal and limbal fibroblasts by amniotic membrane matrix[END_REF], peritoneal [START_REF] Yetkin | Prevention of peritoneal adhesions by intraperitoneal administration of vitamin E and human amniotic membrane[END_REF] or uterine horn adhesions [START_REF] Arora | Controlled comparison of interceed and amniotic membrane graft in the prevention of postoperative adhesions in the rabbit uterine horn model[END_REF] and tendon repair surgery [START_REF] Luo | Effect of human acellular amnion membrane on tendon adhesion in rat[END_REF]. These properties allow the amniotic membrane to act as a scaffold for the development of organized perineural connective tissue, which is a fundamental requirement for good quality axon regeneration.

Our review also highlighted the scaffold properties of this human allograft, particularly strength and resorption, which are compatible with the target clinical indication. While the amnion is no longer clearly distinguishable from other tissues 3-4 months after its implantation in animal models, its resorption rate does not seem to interfere with nerve repair. For clinical use, we theoretically will need to master the physical stability of the membrane so it can prevent adhesion from recurring over time. Along the same lines, we cannot expect consistent production of growth factors by amniotic cells but rather early release of these factors immediately after implantation, from the cells or matrix being degraded [START_REF] Lemke | Transplantation of human amnion prevents recurring adhesions and ameliorates fibrosis in a rat model of sciatic nerve scarring[END_REF]. We propose using a multilayer product (superimposition of several membranes) and/or the original format (both layers: amnion and chorion) to increase its thickness and thus slow its resorption rate to meet this physical stability requirement.

The last element is that this tissue has very little immunogenicity based on the HLA (human leukocyte antigen) system [START_REF] Adinolfi | Expression of HLA antigens, beta 2-microglobulin and enzymes by human amniotic epithelial cells[END_REF]. On one hand, it has been stated that an allograft of amniotic membrane in animals provides a better environment for nerve regeneration than a xenograft [START_REF] Hasturk | Stereologic and ultrastructural comparison of human and rat amniotic membrane wrapping for rat sciatic nerve repair[END_REF]. On the other hand, the foreign body reaction is minimal when fresh human amniotic membrane xenograft is used in animal models [START_REF] Lemke | Transplantation of human amnion prevents recurring adhesions and ameliorates fibrosis in a rat model of sciatic nerve scarring[END_REF][START_REF] Hasturk | Stereologic and ultrastructural comparison of human and rat amniotic membrane wrapping for rat sciatic nerve repair[END_REF]. Thus it is not much of a leap to think that using this tissue will induce little to no foreign body reaction as an allograft in humans. This will also reduce the likelihood of making the fibrosis worse, which sometimes happens when slightly immunogenic neurotubes are used.

Conclusion

The use of amniotic membrane to repair peripheral nerve defects appears very promising based on in vivo animal studies and very early clinical data. This tissue has inherent properties such as bioactivity, immunotolerance and antifibrotic effect that we believe, based on extrapolation, to be very likely to prevent neuroma formation. With the appropriate regulatory approvals, this scaffold could be used in humans, preferably in the lyophilized form consisting of the two original layers (amnion, chorion). The goals would be to 1) preserve as many growth factors as possible, 2) make it easy to handle and store at room temperature in the operating room, and 3) slow down its resorption rate. Its lyophilized form, often supplemented with a virus inactivation process, is theoretically a safer product. Its optimal format appears to be as a tubular membrane, possibly in multiple layers, secured by epineural sutures at both two ends of the nerve defect.

Table titles Table 1 .

 titles1 Summary of the properties of the amniotic membrane in its various formats[8,10- 15,35,37-39]

Table 2 .

 2 Use of human amniotic membrane in animal models for repairing nerve defects or nerve transection.

Table 3 .

 3 Clinical trials (completed or on-going) reporting use of the human amniotic membrane in rheumatology, orthopedic and trauma conditions. Source:

	https://clinicaltrials.gov/ct2/search

Table 4 .

 4 Summary of the marketed amniotic membrane products, including their composition and features.

Table 1 .

 1 Summary of the properties of the amniotic membrane in its various configurations[START_REF] Silini | The long path of human placenta, and its derivatives, in regenerative medicine[END_REF][START_REF] Niknejad | Properties of the amniotic membrane for potential use in tissue engineering[END_REF][START_REF] Dua | The amniotic membrane in ophthalmology[END_REF][START_REF] Baradaran-Rafii | Amniotic membrane transplantation[END_REF][START_REF] Koob | Cytokines in single layer amnion allografts compared to multilayer amnion/chorion allografts for wound healing[END_REF][START_REF] Gindraux | Human amniotic membrane: clinical uses, patents and marketed products[END_REF][START_REF] Jirsova | Amniotic membrane in ophthalmology: properties, preparation, storage and indications for grafting-a review[END_REF][START_REF] Banerjee | Different metabolic activity in placental and reflected regions of the human amniotic membrane[END_REF][START_REF] Koizumi | Growth factor mRNA and protein in preserved human amniotic membrane[END_REF][START_REF] Wolbank | Impact of human amniotic membrane preparation on release of angiogenic factors[END_REF][START_REF] Russo | The effects of different preservation processes on the total protein and growth factor content in a new biological product developed from human amniotic membrane[END_REF]

	Features	Fresh amniotic	Cryopreserved	Lyophilized or
		membrane	amniotic	dehydrated
			membrane	amniotic
				membrane (+
				gamma
				irradiation)
	Need for cold chain after collection	Yes	Yes	No
	and during handling
	Cell viability	+++	++	None
		(controversial)	(controversial)
	Growth factors	Amnion: ++	Amnion: +	Amnion: +
		Chorion: +++	Chorion: ++	Chorion: ++
	Possibility of room temperature	No	No	Yes
	storage (e.g. in operating room)
	Ease of handling and cutting tissue	No	No	Yes
	Use in two-layer configuration	No	No	Yes
	(amnion + chorion)
	Additional gamma irradiation	No	Not usually	Yes

+: moderate; ++: high; +++: very high

Table 2 .

 2 Use of human amniotic membrane in animal models for repairing nerve defects or nerve transection.

	Reference	Animal species	Configuration of human	Nerve insult / type	Main findings
		(strain) / number	amniotic membrane	of nerve
	Ozcan et al. [17]	Rats (Lewis)	Manufactured multilayer	1-cm defect	Vascularized amnion tube achieved nerve regeneration similar to vascularized
		25	amnion tube	Femoral nerve	nerve graft and better than non-vascularized amniotic membrane tube or non-vascularized nerve graft
	Mohammad et al. [18]	Rats (Sprague Dawley) 66	De-epithelialized, dehydrated and gamma-sterilized amnion tube	1-cm defect Sciatic nerve	Advantage of amnion tube: -Better histological results than with nerve autograft or silicone tube
					-Better function in the early stages
	Mligiliche et al. [19]	Rats (Wistar) 24	De-epithelialized and dehydrated amnion tubes of different diameters	0.5-cm defect Sciatic nerve	Advantage of 1-2 mm diameter amnion tube: -Same number of myelinated axons as healthy nerve, but smaller in diameter
					-Gastrocnemius muscle is reinnervated after 9 months
	Duan et al. [20]	Rats (Wistar)	Information not available	1-cm defect	Advantage of composite nerve-muscle autografts wrapped with human amnion
		54		Sciatic nerve	matrix membrane:
					-More neurofilaments in nerve root; neurofilaments are denser and more
					regular
					-More axons; axons are larger in diameter and thicker
	Ozgenel et al. [21] Rats (Sprague-Dawley)	Fresh de-epithelialized	3-cm defect	Nerve wrapping with amnion reduced fibrosis and perineural adhesions
		72	amnion	Sciatic nerve	This action was bolstered by hyaluronic acid injection
	Henry et al. [22]	Rabbits (New Zealand)	Cryopreserved amnion	Transection 5 cm	Amnion fixation on nerve using photochemical tissue bonding better than
		24		from distal end	simple suture
				Peroneal nerve
	O'Neill et al. [23]	Rats (Sprague-Dawley)	Cryopreserved amnion	1-cm defect	Advantage of photochemical tissue bonding of the amnion over epineural
		24		Sciatic nerve	fixation:
					-Better functional outcomes (SFI) at 8 and 12 weeks

-Greater muscle mass in re-innervated gastrocnemius

Acknowledgements:

This work contributes to the COST Action CA17116 International Network for Translating Research on Perinatal Derivatives into Therapeutic Approaches (SPRINT), supported by COST (European Cooperation in Science and Technology). The authors wish to thank Joanne Archambault, PhD and Joan Wallace for English-language assistance.

Conflict of interest:

The authors have no conflict of interest to disclose relative to this article. Kim et al. [24] Rabbits (New Zealand)


Mathieu Gerfaud-Valentin
email: mathieu.gerfaud-valentin@chu-lyon.fr

Florence Chaudot

Pascal Seve

A refractory bicytopenia in a pregnant woman

Keywords: Fanconi anaemia, inherited bone marrow failure syndrome, pregnancy

1-Indication

A 24-year-old woman was referred to our hospital for an anaemia with thrombocytopenia worsening since 3 months at 36 amenorrhoea weeks of her first pregnancy. She had no significant past medical history. However, a macrocytosis (mean corpuscular volume (MCV), 113 fL) investigated one year earlier remained unexplained. Her body mass index was 18. Despite vitamin B12, folates and iron supplementation, and a therapeutic test with corticosteroids, haemoglobin felt to 65g/L (MCV 120fL) and platelets to 17G/L. Laboratory analysis revealed : reticulocytes 50 G/L, haptoglobin 0.64 mg/L (0.54-1.44); haemoglobin electrophoresis : HbA1 92.1% (96-99), HbA2 2.30% (<3.30), HbF 5.6% (<2). Bone marrow aspirate and karyotype were unremarkable. A bone marrow biopsy showed a severe hypocellularity (cellularity <5%) (Fig1.c). The peripheral blood flow cytometry was negative for paroxysmal nocturnal haemoglobinuria.

Transfusions were needed. Six months after the delivery, the cytopenias resolved, without a bone marrow transplant.

What is the diagnosis ?

2-Discussion

The patient's short stature, dysmorphic features, and persistently elevated foetal haemoglobin were consistent with an inherited bone marrow failure syndrome (IBMFs), especially Fanconi Anemia (FA). IBMFs are genetic disorders characterized by hematopoietic aplasia, physical abnormalities, and cancer predisposition (acute myeloid leukemia and solid tumors) [START_REF] Shimamura | Pathophysiology and Management of Inherited Bone Marrow Failure Syndromes[END_REF] . FA is the most frequent IBMFs, in which the most commonly reported physical are short stature, "café-au-lait" spots, pigmentation disorders, thumb abnormalities, microcephaly, microphthalmia, structural renal anomalies, and hypogonadism 1,2 . In the adulthood, these syndromes may be mistaken for acquired aplastic anaemia, hypoplastic myelodysplastic syndromes, or paroxysmal nocturnal haemoglobinuria which remain the main differential diagnoses. However, an early diagnosis is beneficial since it leads to FA-related malignancies screening. Furthermore, it helps to anticipate the management of high-risk pregnancies by a multidisciplinary collaboration to lead to healthy live births despite pregnancy complications related to the underlying disease [START_REF] Gansner | Pregnancy Outcomes in Inherited Bone Marrow Failure Syndromes[END_REF] .

  2 kg/m² (1.48m ; 40kg). The physical examination revealed skin hyperpigmentation ("café-au-lait" spots) of the trunk, left thumb deformity (Fig 1.a) and triangular facial dysmorphia with micrognathia (Fig 1.b).

  The chromosomal breakage test with Mitomycin C attested a defect in DNA repair (43 mitosis on 50 with at least one chromosome break, 17 with ≥ 3 breaks, 4 radial figures). The FANCD2 monoubiquitination test in fibroblasts showed a FA core. Fibroblast DNA screening for FANC gene mutations confirmed heterozygous c.3188G>A/p. (Trp1063*) mutation and heterozygous deletion of the exons 21 and 22 of FANC A genes.

Fig. 1 .

 1 Fig. 1. Dysmorphic features and medullar hypocellularity in a 24-year-old pregnant woman with bicytopenia. a. Left thumb deformity. b. Micrognathia and triangular facial dysmorphia. c. Bone marrow biopsy showing severe bone marrow hypocellularity (< 5%), no megakaryocyte.