Rose G Long

Alexander Bürki

Philippe Zysset

David Eglin

Dirk W Grijpma

Sebastien Bg Blanquer

Andrew C Hecht

Dr James C Iatridis
email: james.iatridis@mssm.edu

Peter W Leni

Mechanical Restoration and Failure Analyses of a Hydrogel and Scaffold Composite Strategy for Annulus Fibrosus Repair

Keywords: annulus fibrosus repair, annular closure, intervertebral disc herniation, spine biomechanics, intervertebral disc degeneration

Unrepaired defects in the annulus fibrosus of intervertebral discs are associated with degeneration and persistent back pain. A clinical need exists for a disc repair strategy that can seal annular defects, be easily delivered during surgical procedures, and restore biomechanics with low risk of herniation. Multiple annulus repair strategies were developed using poly(trimethylene carbonate) scaffolds optimized for cell delivery, polyurethane membranes designed to prevent herniation, and fibrin-genipin adhesive tuned to annulus fibrosus shear properties. This three-part study evaluated repair strategies for biomechanical restoration, herniation risk and failure mode in torsion, bending and compression at physiological and hyper-physiological loads using a bovine injury model. Fibrin-genipin hydrogel restored some torsional stiffness, bending ROM and disc height loss, with negligible herniation risk and failure was observed histologically at the fibrin-genipin midsubstance following rigorous loading. Scaffold-based repairs partially restored biomechanics, but had high herniation risk even when stabilized with sutured membranes and failure was observed histologically at the interface between scaffold and fibrin-genipin adhesive. Fibrin-genipin was the simplest annulus fibrosus repair solution evaluated that involved an easily deliverable adhesive

Introduction

Lower back pain affects up to 70% of people in their lifetimes [START_REF] Andersson | Epidemiological features of chronic low-back pain[END_REF] and is the leading cause of global disability [START_REF] Vos | Years lived with disability (YLDs) for 1160 sequelae of 289 diseases and injuries 1990-2010: a systematic analysis for the Global Burden of Disease Study[END_REF][START_REF] March | Burden of disability due to musculoskeletal (MSK) disorders[END_REF]. Defects of the annulus fibrosus (AF) can result in herniation of intervertebral disc (IVD) material and compression of the nerve root, inducing back or leg pain. Discectomy is the most common and effective surgical procedure at reducing leg pain related to IVD herniation [START_REF] Asch | Prospective multiple outcomes study of outpatient lumbar microdiscectomy: should 75 to 80% success rates be the norm?[END_REF][START_REF] Weinstein | Surgical versus nonoperative treatment for lumbar disc herniation: four-year results for the Spine Patient Outcomes Research Trial (SPORT)[END_REF][START_REF] Gray | Population-based trends in volumes and rates of ambulatory lumbar spine surgery[END_REF]. It consists of removing extruded and loose nucleus pulposus (NP) tissue through an incision in the AF. However, both small and large defects in the AF can lead to altered biomechanics and increased degeneration risk [START_REF] Elliott | The effect of relative needle diameter in puncture and sham injection animal models of degeneration[END_REF][START_REF] Iatridis | Role of biomechanics in intervertebral disc degeneration and regenerative therapies: what needs repairing in the disc and what are promising biomaterials for its repair?[END_REF][START_REF] Michalek | Height and torsional stiffness are most sensitive to annular injury in large animal intervertebral discs[END_REF][START_REF] Masuda | A novel rabbit model of mild, reproducible disc degeneration by an anulus needle puncture: correlation between the degree of disc injury and radiological and histological appearances of disc degeneration[END_REF]. A systematic review of the discectomy literature indicated that discectomy procedures have up to 27% risk of re-herniation requiring further surgery, and the prevalence depends on the size of the annular defect and the amount of material removed [START_REF] Mcgirt | A prospective cohort study of close interval computed tomography and magnetic resonance imaging after primary lumbar discectomy: factors associated with recurrent disc herniation and disc height loss[END_REF][START_REF] Watters | An evidence-based review of the literature on the consequences of conservative versus aggressive discectomy for the treatment of primary disc herniation with radiculopathy[END_REF]. There also remains a relatively high risk of degeneration and persistent low back pain in longer-term follow-up, particularly if excess IVD material was removed to lower the reherniation risk. While discectomy procedures are very effective at reducing leg pain, there are opportunities to improve this procedure with AF repair techniques that reduce the rate of reherniation, improve biomechanics and can potentially be part of a disc regeneration strategy. Annular defects result in loss of NP pressurization and AF integrity. A functional AF repair strategy must restore IVD height, neutral zone characteristics, and torsional biomechanics to the healthy condition without risk of herniation under the high physiological loads occurring in the spine [START_REF] Iatridis | Role of biomechanics in intervertebral disc degeneration and regenerative therapies: what needs repairing in the disc and what are promising biomaterials for its repair?[END_REF]. Current AF repair strategies include sutures, plugs, adhesives and hydrogels [START_REF] Guterl | Challenges and strategies in the repair of ruptured annulus fibrosus[END_REF]. Suturing the IVD during a discectomy procedure adds substantial complications to the procedures and extends the operation time. Suturing did not restore the intradiscal pressure of IVDs with different annular defects in sheep [START_REF] Ahlgren | Effect of anular repair on the healing strength of the intervertebral disc: a sheep model[END_REF], suggesting suturing alone provided inadequate biomechanical repair. An alternative to manual sutures was a commercially available suture delivery system, the Xclose Tissue Repair System (Anulex Technologies, Minnetonka, MN). The Xclose device was designed to strengthen the suturing procedure and simplify the suturing process via an elongated suture delivery system allowing arthroscopic access. In a two year prospective, randomized control trial there was no difference in functional or disability outcomes between microdiscectomy performed with and without XClose sutures, although there was a significant reduction in re-herniation surgery observed in a subset cohort with predominant leg pain at 3 and 6 months [START_REF] Bailey | Prospective, Multicenter, Randomized, Controlled Study of Anular Repair in Lumbar Discectomy[END_REF]. The reduction in re-herniation rates was promising for a sutured AF repair, but no data was reported on disc height, biomechanical restoration of the motion segment, or MRI signal intensity providing no evidence that this technique would successfully prevent accelerated degeneration following discectomy procedures. An annulus closure plug had favorable biomechanical results in vitro but showed deformation and herniation after 6 weeks in an in vivo goat model [START_REF] Bron | Biomechanical and in vivo evaluation of experimental closure devices of the annulus fibrosus designed for a goat nucleus replacement model[END_REF]. Alternately, Barricaid is a device resembling a shield that is secured with an anchor into the adjacent inferior vertebral body to reduce the risk of tissue expulsion following a discectomy procedure (Intrinsic Therapeutics, Inc., Woburn, MA). In an in vitro test of 6 IVDs, no herniation was seen after more than 100,000 cycles of dynamic bending, indicating a low risk of herniation, however, the IVD height loss due to loading was not decreased [START_REF] Wilke | Can prevention of a reherniation be investigated? Establishment of a herniation model and experiments with an anular closure device[END_REF] indicating limited biomechanical restoration. Barricaid demonstrated a reduced risk of facet degeneration [START_REF] Trummer | Protecting facet joints post-lumbar discectomy: Barricaid annular closure device reduces risk of facet degeneration[END_REF], suggesting AF repair strategies capable of retaining NP pressurization following discectomy are likely to have improved outcomes. While several mechanical annular repair strategies exist, they all complicate discectomy procedures, several have risk of herniation, and none completely restore IVD biomechanics.

The broad aim of this study was to evaluate the repair of large AF defects using a composite repair strategy involving fibrin-genipin (FibGen) hydrogel adhesive to seal the AF defect, a poly(trimethylene carbonate) (PTMC) space-filling scaffold capable of cell delivery, and a polyurethane membrane to prevent herniation. Design criteria we considered necessary for an effective AF repair strategy included low risk of herniation, restoration of IVD biomechanics to the healthy (intact) condition, ease of delivery, and the potential to be functionalized to deliver cells or drugs in order to promote long-term healing or regeneration. This three part biomechanical study assessed the performance of multiple composite repair strategies using these components evaluated under multiple degree of freedom biomechanical testing using bovine caudal IVD injury models. Part 1 applied a torsional stiffness test to evaluate whether the repair restored AF integrity since mechanical integrity of annulus fibrosus integrity is most sensitive to torsion [START_REF] Michalek | Height and torsional stiffness are most sensitive to annular injury in large animal intervertebral discs[END_REF][START_REF] Michalek | Needle puncture injury of the rat intervertebral disc affects torsional and compressive biomechanics differently[END_REF]. Part 1 tested the hypothesis that the composite repair strategy with scaffolds would best restore biomechanics to intact levels but the adhesive alone would have the lowest herniation risk. Part 2 selected the most promising repairs from Part 1 and after procedure refinement, evaluated them for herniation risk and biomechanical restoration in axial compression and two bending degrees of freedom. Bending was evaluated since it can rigorously test for herniation risk. Part 2 tested the hypothesis that the FibGen adhesive alone and the composite repair with suturing augmentation could restore biomechanics and disc height loss without risk of herniation. Part 3 used the most successful repair strategies from Parts 1 & 2 and evaluated mechanical restoration using a cruciate-style defect to be more representative of the variety of defects found clinically following herniation and discectomy procedures [START_REF] Watters | An evidence-based review of the literature on the consequences of conservative versus aggressive discectomy for the treatment of primary disc herniation with radiculopathy[END_REF] (rather than biopsy defects) and to evaluate if FibGen could adhere to irregularly-shaped defects. Part 3 used an angle control with moment limits test. in 3 rotational degrees of freedom (i.e., torsion, flexion-extension & lateral bending). Part 3 was performed at the University of Bern (rather than Mount Sinai). Part 3 tested the hypothesis that the FibGen can seal a cruciate-style defect and restore the biomechanical behaviors to intact levels in bending, flexion-extension, and axial rotation.

Materials and Methods

Material selection

FibGen involved a genipin-crosslinked fibrin gel with a formulation previously tuned to match the shear properties of the native AF tissue [START_REF] Guterl | Characterization of Mechanics and Cytocompatibility of Fibrin-Genipin Annulus Fibrosus Sealant with the Addition of Cell Adhesion Molecules[END_REF]. The space filling PTMC scaffolds consisted of 5,000g/mol oligomers crosslinked with stereolithography to ensure a precise structure that mimicked the complex architecture of the AF collagen bundles which are oriented in an angle-ply fashion that evolve from ±30° in the inner AF to ±45° in the outer AF [START_REF] Blanquer | Development of poly(trimethylene carbonate) network implants for annulus fibrosus tissue engineering[END_REF]. Two scaffold geometries were evaluated: a truncated cone shape and a cylindrical shape. A polyurethane membrane was designed to prevent extrusion of IVD tissue and provide a barrier between the IVD and the adjacent sensitive nerve. The polyurethane membrane was adhered to the native AF tissue using FibGen adhesive as well as suturing.

Animal model, dissection and storage

Bovine coccygeal IVDs are large and comparable to human lumbar IVD [START_REF] Demers | Value and limitations of using the bovine tail as a model for the human lumbar spine[END_REF][START_REF] Oshima | The use of coccygeal discs to study intervertebral disc metabolism[END_REF], easily available and well-studied. Motion segments (bone-IVD-bone) were harvested from skeletally mature bovine tails obtained from a local abattoir (Green Village Packing, Green Village, NJ). Following dissection, IVDs were wrapped in saline soaked tissue and frozen at -20°C until testing. On the day of testing, all specimens were thawed for 3 hours in 1L PBS at 37°C and warm to touch, then potted in polymethylmethacrylate (PMMA). No discs underwent additional freeze-thaw cycles in order to decrease the change in biomechanics due to freeze-thaw cycles [START_REF] Tan | Cumulative multiple freeze-thaw cycles and testing does not affect subsequent within-day variation in intervertebral flexibility of human cadaveric lumbosacral spine[END_REF]. To minimize variability in injury and repair procedures, a single biomedical engineer performed injury and repair procedures for each Part (as described in Sections 2.3-2.5) following training by a spine surgeons and extensive practice on all techniques.

Part 1: Biomechanical restoration and herniation risk in torsion of three repair strategies

Thirty bovine coccygeal IVDs were distributed to five groups (n=6): Intact, Injured, FibGen, Cylindrical (scaffold), and Conical (scaffold) (Figure 1). The Intact specimens did not have a defect. The Injured group had a 5 mm biopsy punch defect through the posterolateral AF with removal of approximately 30% of the nucleus pulposus (173±84 mg) with rongeurs. In the FibGen group, the injury was repaired by filling the defect with FibGen adhesive and covering the FibGen with the polyurethane membrane. The Cylindrical group specimens were repaired by filling the defect partially with FibGen adhesive and inserting a cylindrical scaffold (diameter=5 mm, length=7 mm). The Cylindrical scaffold was designed to match the width of the bovine annulus fibrosus which is about 5 mm [START_REF] O'connell | Comparison of animals used in disc research to human lumbar disc geometry[END_REF]. The porous scaffold was filled with FibGen and covered with membrane cut into a circle (diameter=8 mm). The Conical group was prepared identically to the Cylindrical group but involved a scaffold with a truncated cone shaped scaffold (diameter= 4/6 mm, length 10 mm) with the larger diameter to be seated at the innermost portion of the AF to create a press-fit, and was longer than the Cylindrical scaffold since it was designed to be seated within the nucleus pulposus. For all the repair groups, the FibGen adhesive was prepared as previously described [START_REF] Guterl | Characterization of Mechanics and Cytocompatibility of Fibrin-Genipin Annulus Fibrosus Sealant with the Addition of Cell Adhesion Molecules[END_REF]. Briefly, 40.5 μL of thrombin (100 U/mL) was mixed with 20.25 μL of genipin (400 mg/mL in DMSO) and 226.8 mL PBS. This was mixed 1:4 with fibrinogen (200 mg/mL) upon injection using a dual barrel syringe with mixing tip.

All specimens were submerged in PBS with protease inhibitor (cOmplete, Roche Diagnostics, Indianapolis, IN, USA) and allowed to set at 37°C overnight before testing. Specimens were tested using a Bionix Servohydraulic system (MTS, Eden Prairie, MN, USA) capable of one translational and three rotational degrees of freedom. Each specimen was subjected to several rounds of rotations of 20 cycles each, increasing incrementally from physiological to hyper-physiological levels (±19°) at 0.1 Hz with axial displacement fixed after a 60 N preload in compression was applied (Figure 1) to allow identification of rotations and loads that induced herniation. Herniation was monitored with a camera throughout the loading and was defined by extrusion of the repair beyond the surface of the IVD. Torque range and torsional stiffness were calculated using custom Matlab (Mathworks, Natick, MA, USA) code with the torque range calculated as the total torque developed in the 2 nd to last cycle of each round and torsional stiffness calculated as the average of the top 10% rotation of both loading directions. Torsional stiffness and torque range were calculated for each sample until the end of the test and only presented up to the point of extrusion. Herniation was observed visually with photography, and in most samples (75%), was coincident with an abrupt reduction of axial force, indicating a loss of nucleus pressurization. The torque, rotation and nominal axial stress at herniation were measured. Nominal axial stress was calculated as the maximum axial force just before herniation divided by undeformed cross-sectional area of the IVD. This nominal axial stress was developed as a reactive stress due to applied compression, fixing of the disc height and applied torsion. While failure occurred due to excess torsion, these values were calculated to give an indication of the nominal axial stress when that failure occurred during torsional loading.

Part 2: Biomechanical restoration and herniation risk prevention in bending of two repair strategies

Part 2 was designed to test herniation behaviors and herniation prevention of refined repairs using a repeated measures study design with three groups: Injured, Conical and FibGen (n=6) (Figure 2). Part 2 repairs were improved from Part 1 repairs by the following: i) the membrane over the Large Scaffold repair was cut to be square-shaped to allow suturing, one at each corner, in order to better prevent expulsion and herniation of the scaffold; and ii) the FibGen repair did not have a membrane since it was considered unnecessary due to low risk of herniation and minimal contribution of the membrane observed in Part 1.

Each specimen was subjected to a moderate loading protocol in Intact, Defect and Repaired conditions (Figure 2). Specimens were x-rayed to evaluate orientation, alignment and IVD height to ensure centrality with respect to the circular pots and alignment with the cephalocaudal axis. Diameter was found by averaging four caliper measurements at different locations around the disc assuming a circular cross-section. The IVD height was calculated using a custom written Matlab (Mathworks, Natick, MA, USA) code that averaged the distance between cubic functions fit to each endplate. The moderate loading protocol consisted of twenty cycles each of compression from 0.06 MPa to 0.50 MPa at 0.1 Hz, and off injury axis and on injury axis bending to ±8° at 1.5°/sec (Figure 2). The nominal axial stress of 0.50 MPa was chosen because it approximates sitting [START_REF] Wilke | New in vivo measurements of pressures in the intervertebral disc in daily life[END_REF][START_REF] Sato | In vivo intradiscal pressure measurement in healthy individuals and in patients with ongoing back problems[END_REF] and would represent the axial loading the repair must withstand after a discectomy procedure. The ±8° bending amplitude falls within the human lumbar motion segment physiological ranges of 8-13° in flexion, 1-5° in extension, and 3-11° in lateral bending [START_REF] Lee | Does lumbar disk degeneration increase segmental mobility in vivo? Segmental motion analysis of the whole lumbar spine using kinetic MRI[END_REF][START_REF] Nagel | Quantification of continuous in vivo flexionextension kinematics and intervertebral strains[END_REF][START_REF] Pearcy | Axial rotation and lateral bending in the normal lumbar spine measured by three-dimensional radiography[END_REF][START_REF] Pearcy | Three-dimensional x-ray analysis of normal movement in the lumbar spine[END_REF][START_REF] Li | Segmental in vivo vertebral motion during functional human lumbar spine activities[END_REF]. Force, displacement, torque and rotation around each axis were recorded through the test. IVD height change was calculated by dividing displacement at the end of the loading by the baseline IVD height determined with a pretest x-ray. Torque range was calculated as the total torque developed in the full range of bending in the 2 nd to last cycle to allow for viscoelastic equilibrium [START_REF] Johannessen | Trans-Endplate Nucleotomy Increases Deformation and Creep Response in Axial Loading[END_REF]. The dynamic stiffness was calculated as the slope of a line fit to the final 20% deformation of the force-deformation curve and torque-rotation curve in each loading direction so as to approximate the linear region. The stiffnesses of each loading direction for both bending regimes were averaged. The defect and repaired condition stiffness values were normalized against the matching intact condition. After the last moderate testing cycle, the nonherniated specimens (6 FibGen & 1 Conical) were subjected to one of two rigorous testing protocols. The first 3 FibGen samples underwent a high magnitude failure test where on injury axis bending magnitude was increased successively to specimen failure. However, the test machine load cell limit (±10 Nm) was reached as bending angles reached ~±10.5° without observing any FibGen herniation. As a result, the remaining samples (3 FibGen & 1 Conical) underwent a rigorous cyclic failure test consisting of 2 hours of on injury axis bending to ±8° at 5°/sec resulting in ~1000 cycles. Herniation was monitored visually with photography.

All samples were fixed for histological analysis following testing. The failure mode of the repair was also assessed by imaging the interface between the repair and the native tissue using methacrylate embedded specimens histological sections with Picrosirius Red/Alcian Blue staining under brightfield microscopy as described [START_REF] Walter | Form and function of the intervertebral disc in health and disease: a morphological and stain comparison study[END_REF][START_REF] Gruber | An improved staining method for intervertebral disc tissue[END_REF].

Part 3: Biomechanical restoration in pure bending of FibGen with cruciate defect

Part 3 was designed to test mechanical restoration under a repeated measures flexibility test protocol with samples tested in three rotational degrees of freedom under three conditions: Intact, Injured and Repaired (n=7) (Figure 3). Important differences from Part 2 testing involved: i) a flexibility test was used in order to allow greater rotational motion; ii) a different and clinically relevant cruciate-style discectomy defect was used; and iii) testing was performed at the Institute for Surgical Technology and Biomechanics at the University of Bern. The cruciate injury was created using a No. 11 scalpel blade with a depth of approximately 8mm. The FibGen repair was allowed to set for four hours instead of overnight in order to approximate the timing of the first movement of a patient. The membrane was an octagon shape measuring about 15mm×15mm with no suture.

Samples were tested in intact, injured, and repaired conditions. The test consisted of three cycles in flexion-extension, lateral bending and torsion at a constant angular motion of 1°/s up to a torque limit of 1.5 Nm or a rotation limit of 20°. No axial compression was added (Figure 3). The moment and rotation were recorded through time. The range of motion (ROM) was calculated as the angular motion within the load range of ±1.5 Nm for flexionextension and torsion and ±0.5 Nm for lateral bending during the third loading cycle (Figure 3).

Statistical Analysis

For Part 1, a Kruskal-Wallis test (non-parametric 1-way ANOVA) with Dunn's Multiple Comparison test was used to compare the torque range and stiffness between the groups and differences between nominal axial stress, maximal rotation and maximal torque. In Part 2, a Friedman's test (non-parametric, repeated-measure ANOVA) with Dunn's Multiple Comparison Test was used to compare the torque range and compressive and bending stiffness ratios between the three groups. The Friedman's test with Dunn's Multiple Comparison testwas used to test the effect of the injury on disc height loss compared to matched intact samples of all groups (n=18) and to test the effect of repair compared to matched injured samples within each treatment group (n=6). In Part 3, Friedman's test (nonparametric, repeated-measures ANOVA) with Dunn's Multiple Comparison test was used to compare the ratios of injured/intact to repaired/intact. All quantitative data are presented as mean ± standard deviation. Significance was based on α = 0.05 level and all statistical analysis was performed using GraphPad Prism v. 5 (GraphPad Software, San Diego, California, USA).

Results

Part 1 Results: All repair groups partially restored biomechanics and FibGen had low herniation risk

All repair groups had stiffness and torque range values that were not different than the intact condition, suggestive of partial restoration to intact levels although injured was not significantly different than intact either (Figure 4B). The Intact group exhibited a shift in the shape of the Torsional Stiffness-Angle curve at 19°, and this may reflect injury initiation. No significant differences in torque range were detected between the repaired, intact and and injured group (Figure 4C).

As torsion angles increased, the repairs of the Conical and Cylindrical groups herniated, as demonstrated with an abrupt drop in nominal axial stress (Figure 5A). Herniation occurred for 100% of the Cylindrical scaffold and 83% of the Conical scaffold herniating compared to just 33% of the FibGen group. The FibGen group repairs withstood higher average rotations, torques and nominal axial stresses than either the Cylindrical or Conical groups, but this was only significant compared to the Conical group for max rotation (Figure 5B). No difference in the average maximum torque or nominal axial stress was observed between the Cylindrical, Conical and FibGen groups. Due to the slightly greater biomechanical restoration of the Conical scaffold group and lowest herniation risk of the FibGen group, both FibGen and Conical scaffold groups were retained and refined for Part 2 biomechanical evaluations.

Part 2 Results: FibGen had low herniation risk in compression and bending while Conical Scaffold had high herniation risk

FibGen repair showed low risk of herniation in bending and compression with no samples herniating in moderate or rigorous testing. The scaffold repair had a high risk of herniation with 5 out of 6 samples herniating during the moderate testing procedure (Figure 6B) and 1 sample herniating during the rigorous testing. Rigorous testing involved rotation angles of 10.7±4.7° on-axis bending for the high magnitude failure tests and ~1000 cycles of ±8° onaxis bending for the cyclic failure tests. The FibGen repair significantly reduced the disc height loss during moderate loading compared to matched Intact and Defect conditions. The disc height loss of the Conical repair group was not significantly different than the matched Intact and Defect conditions (Figure 6C), likely due to the high rate of observed herniation of the scaffold. There was no significant change in torque range, or bending and compressive stiffness ratios between the three groups.

The mechanism for scaffold extrusion was observed on histology to be failure between the FibGen and the PTMC scaffold for the scaffold groups (Figure 6A). FibGen remained adhered to the AF tissue, although FibGen showed some cracking on histology when observed after undergoing rigorous loading that also appeared to induce cracks in the native disc tissue.

Part 3 Results: FibGen partially restored biomechanical behaviors without herniation in bending flexibility tests

The cruciate-style AF injury resulted in significantly increased ROM compared to the Intact condition in flexion-extension, lateral bending and torsion with mean increases of 5.7%, 4.0% and 17.1%, respectively (Figure 7B-D). The FibGen repair restored the ROM to values significantly lower than Injured levels in flexion-extension and not significantly different than Intact levels in lateral bending. For torsion, FibGen did not decrease ROM from the injured condition to intact levels, consistent with the incomplete restoration of torque range in torsion seen in Part 1.

Discussion

A successful AF repair strategy remains an important unmet clinical need, and has the potential to reduce long-term degeneration following herniation and discectomy procedures. This comprehensive biomechanical study evaluated multiple AF repair strategies using FibGen, an adhesive sealant, a PTMC space-filling scaffold capable of delivering cells, and a polyurethane membrane designed to prevent herniation. A 3-part biomechanical study was performed involving distinct repair configurations, defects, and loading conditions in order to robustly assess if these materials could meet our design criteria. Part 1 found all repair strategies had values for torsional stiffness and torque range that were greater than injured but lower than intact groups, although there were minimal differences between repair groups and no groups had significant differences. FibGen had little herniation risk and the Conical scaffold group had the largest values for torsional stiffness so both repair strategies underwent continued evaluation in Part 2 studies, however, the conical scaffold repair was refined by adding sutures to the membrane to better prevent herniation. In Part 2, FibGen significantly reduced disc height loss and had the greatest values for compression stiffness ratio and both axes of bending stiffness ratio, although this was not significant. The Conical scaffold retained a high herniation risk even with the sutured membrane so was not used for further evaluation. In Part 3, FibGen adhesive filled the irregularly shaped cruciate-style defects and ROM values were lower than the Injured condition for all degrees of freedom, and this was significant for flexion-extension.

FibGen was demonstrated to be capable of filling cylindrical and cruciate-style AF defects. FibGen reduced IVD height loss and partially restored biomechanical behaviors of injured IVDs in multiple loading degrees of freedom, specifically flexion-extension ROM in bending and disc height loss after compression and bending. FibGen had little herniation risk and herniated only in some samples and only at hyper-physiological rotation angles. FibGen was also previously demonstrated to have surgically relevant gelation times of about 15 minutes[add guterl et al 2013]. The FibGen formulation in this study was tuned to mechanically match AF shear behaviors of the AF [START_REF] Schek | Genipin-crosslinked fibrin hydrogels as a potential adhesive to augment intervertebral disc annulus repair[END_REF], but this isotropic hydrogel is unlikely to additionally match tensile and compressive behaviors of the anisotropic and fiber-reinforced AF. As a result, it is not surprising that torsional biomechanical behaviors were not completely restored to the intact condition. Furthermore FibGen remained adhered to the tissue and some evidence of FibGen cracking was observed on histology following rigorous loading. Therefore, FibGen enhancements to increase failure strength are suggested by this data to improve biomechanical performance. However, FibGen was previously shown to allow cell integration in vitro and in vivo and had degradation rates that were much longer than fibrin, suggesting potential for repair [START_REF] Guterl | Characterization of Mechanics and Cytocompatibility of Fibrin-Genipin Annulus Fibrosus Sealant with the Addition of Cell Adhesion Molecules[END_REF][START_REF] Schek | Genipin-crosslinked fibrin hydrogels as a potential adhesive to augment intervertebral disc annulus repair[END_REF][START_REF] Likhitpanichkul | Fibrin-genipin adhesive hydrogel for annulus fibrosus repair: performance evaluation with large animal organ culture, in situ biomechanics, and in vivo degradation tests[END_REF]. It is not possible to determine if the current formulation of FibGen is sufficient without future in vivo testing.

PTMC scaffold groups exhibited non-significant increases in torsional stiffness of the injured IVD although herniation occurred well within physiological ROM, which is less than approximately ±4° since rotation is limited by the facet joints [START_REF] Pearcy | Axial rotation and lateral bending in the normal lumbar spine measured by three-dimensional radiography[END_REF][START_REF] Li | Segmental in vivo vertebral motion during functional human lumbar spine activities[END_REF][START_REF] Xia | In-vivo motion characteristics of lumbar vertebrae in sagittal and transverse planes[END_REF]. Histology demonstrated failure occurred between the FibGen and PTMC scaffold so that FibGen adhesive did not adequately adhere to the scaffolds. The high rate of herniation observed for both scaffold geometries was likely due the hydrostatic pressure of the nucleus pushing the scaffold outward creating high shear stresses at the interface between the scaffold and FibGen. The polyurethane membrane was not sufficient to prevent scaffold expulsion under loading even when reinforced with sutures. The sutured polyurethane membrane in this study was visually observed to stretch and allowed the scaffolds to extrude even at low physiological loading levels. Consequently, it is unlikely that improved suturing methods (e.g., the Xclose Tissue Repair System which can tighten suture knots and reduce risks associated with suturing in close proximity to neural structures) would sufficiently reduce the high herniation risks. Furthermore, the Barricaid system has been shown to prevent NP herniation during rigorous biomechanical testing [START_REF] Wilke | Can prevention of a reherniation be investigated? Establishment of a herniation model and experiments with an anular closure device[END_REF], and could be a useful augmentation to resist NP pressurization and reduce herniation risk of AF repair biomaterials described. Future optimization of the PTMC formulation, scaffold structure, and scaffold repair strategies that better prevent herniation are required, and additional optimization may be warranted since organ culture results indicate that activating the scaffold with human bone marrow derived mesenchymal stromal cells resulted in upregulation of anabolic genes and down regulation of catabolic genes [START_REF] Pirvu | A combined biomaterial and cellular approach for annulus fibrosus rupture repair[END_REF].

Different test groups and loading conditions were used in Parts 1-3 due to the evolving refinements and demands of the AF repair strategies. However, not all biomaterials were able to be compared separately and some factors may interact preventing conclusions on specific biomechanical roles of each biomaterial component. For example, FibGen was not always evaluated with and without a membrane and the different scaffold geometrics differed in shape and length. The lack of significant effects of injury on torsional stiffness and torque range in Part 1 may be due to the inter-animal differences in biomechanics since torsion is commonly reported to be strongly affected by AF injury [START_REF] Vos | Years lived with disability (YLDs) for 1160 sequelae of 289 diseases and injuries 1990-2010: a systematic analysis for the Global Burden of Disease Study[END_REF][START_REF] March | Burden of disability due to musculoskeletal (MSK) disorders[END_REF][START_REF] Michalek | Height and torsional stiffness are most sensitive to annular injury in large animal intervertebral discs[END_REF][START_REF] Michalek | Needle puncture injury of the rat intervertebral disc affects torsional and compressive biomechanics differently[END_REF]. This study used n=6, 6 & 7 for Parts 1, 2 & 3, respectively, so there is limited statistical power on some of the tests to detect smaller effects. Results demonstrated that the stiffness and the torque range of the repaired specimens in Part 1 were higher than injured condition and lower than intact condition, but not significantly, suggestive of reliable but insignificant pattern. Interspecimen variability was reduced with repeated measures study designs in Parts 2 & 3. While there is likely to be an effect of loading history, it does not influence our conclusions since our conclusions are between groups with a repeated measures design and the same loading history for all groups. No significant effects of injury were detected in Part 2 which we attribute to the limited nucleus pulposus tissue removal which more rigorously induced implant failure but also likely retained tension in the surrounding AF fibers that were not injured. However, significant effects of injury were detected in Part 3 studies. The Part 1 & 2 tests tested herniation risk at greater bending and torsion angles than the commonly reported rigorous eccentric loading test of Heuer and colleagues [START_REF] Heuer | Biomechanical evaluation of conventional anulus fibrosus closure methods required for nucleus replacement[END_REF], although our testing involved far fewer loading cycles and fatigue loading experiments is a relevant future investigation.

Conclusions

Discectomy procedures are highly effective at addressing acute pain due to herniation, yet risks of accelerated degeneration, reherniation, and diminished long-term success rates indicate there is an unmet clinical need for an effective AF repair strategy. An ideal AF repair strategy must have a low herniation risk, restore IVD biomechanics, and be easily deliverable during surgical procedures. Of the repair strategies evaluated in this study FibGen best met these design criteria because it had low herniation risk and was easily deliverable, but FibGen only partly restored disc biomechanical behaviors. The PTMC scaffold and polyurethane membrane had attractive design characteristics for AF repair, but when used as part of a composite repair strategy, FibGen was not sufficiently strong to prevent herniation of the PTMC scaffold even when reinforced by a sutured polyurethane membrane. Additional repair strategy development and biomaterial optimizations are required to prevent herniation of the PTMC scaffold and improve biomechanical performance of FibGen. However, complete biomechanical performance may not be necessary for IVD repair, and future large animal studies may be warranted to determine if FibGen can remain in place and adequately seal the IVDs under high levels of spinal loading in vivo until the AF can sufficiently repair itself. All repair strategies partially restored torsional stiffness and torque range to intact levels. (A) Torque-Rotation curves were used to calculate (B) torsional stiffness. No significant differences in torsional stiffness between groups were detected. Data presented as mean ± standard deviation. Torsional stiffness and torque range were calculated for each sample until point of extrusion so that results are only presented up to 5° for Cylindrical and Conical groups. Maximum levels of (B) nominal axial stress, (C) rotation and (D) torque were calculated at the cycle just prior to herniation. Data presented as mean ± standard deviation. *p<0.05 compared to FibGen. # Means for FibGen were calculated using values at the maximum test rotation angle for the 4 FibGen samples that did not herniate so that these data underrepresent failure properties for FibGen.

Figure 1 .

 1 Figure 1. Part 1 Study Design Torsion tests to determine biomechanical behaviors and herniation risk of 3 repair strategies. (A) Samples were distributed to five groups: Intact, Injured with a cylindrical biopsy punch defect, FibGen, Cylindrical scaffold and Conical (truncated cone) scaffold. Both scaffold groups also had FibGen adhesive and polyurethane membrane. (B) Individual components of the repair strategies. (C) Torsion stiffness test protocol increased torsion angle for each 'round' of 20 loading cycles. (D) Torsion angle magnitudes increased to super-physiological levels.

Figure 2 .

 2 Figure 2. Part 2 Study Design Moderate and rigorous bending stiffness tests to determine biomechanical behaviors and herniation risk of 2 repair strategies. (A) Samples were tested Intact, Injured with a cylindrical biopsy punch defect, and after being Repaired with either FibGen or a composite repair with Conical Scaffold. (B) Specimens were subjected to a bending stiffness test protocol. (C) Bending angle was applied on-axis or off-axis with the injury.

Figure 3 .

 3 Figure 3. Part 3 Study DesignMulti-axial flexibility tests were applied to determine biomechanical behaviors of FibGen repair. (A) Samples were tested Intact, Injured with a cruciate-style defect and Repaired with FibGen. (B) Flexibility testing was performed using a constant angular velocity to a moment controlled protocol limit.

Figure 4 .

 4 Figure 4. Part 1 Results. Torsional biomechanics

Figure 5 .

 5 Figure 5. Part 1 Results. Herniation biomechanics FibGen herniated at larger rotations and torques than the scaffold based repairs and all herniated at super-physiological levels. (A) Nominal axial stress recorded during torsional loading with abrupt decrease of axial force concurrent with herniation (indicated by arrow).Maximum levels of (B) nominal axial stress, (C) rotation and (D) torque were calculated at the cycle just prior to herniation. Data presented as mean ± standard deviation. *p<0.05 compared to FibGen. # Means for FibGen were calculated using values at the maximum test rotation angle for the 4 FibGen samples that did not herniate so that these data underrepresent failure properties for FibGen.

Figure 6 .

 6 Figure 6. Part 2 Results. Herniation characterization, disc height & biomechanics FibGen did not herniate and restored disc height while the Conical scaffold group herniated and did not restore disc height. (A) Photos and histological sections of sagittal bovine IVDs sections following repair and moderate loading for Conical samples and moderate and cyclic failure loading for FibGen. Scale bars are 1000 μm in mosaic image and 50 μm in magnified areas. For Conical group, failure occurred between Conical scaffold and FibGen (arrows, top). For FibGen group, FibGen remained adhered to the AF tissue with failure occurring by cracking of the FibGen mid-substance (black arrow) and fissuring of native disc tissue (white arrow) (bottom) (B) Axial stress and bending angle during moderate testing for characteristic sample with times of herniation for 5 of the conical scaffolds indicated (∨).(C) Disc height loss was greatest for the specimens in Defect condition and was restored for FibGen. (D) Axial and bending biomechanical results for Injured Control, FibGen and Conical scaffold groups. FibGen had the largest stiffness ratio values for all testing modes, but no significant biomechanical differences were observed with group for any stiffness ratio value. Data presented as mean ± standard deviation. *p<0.05.

Figure 7 .

 7 Figure 7. Part 3 Results. Multi-axial range of motion ROM was significantly increased in the Injured group compared to Intact for all degrees of freedom. FibGen had ROM values lower than for Injured in all degress of freedom but this was only significant in flexion-extension (A) Moment and angle curves were used to calculate the ROM as the total angular motion between ±1.5Nm moment for each of the 3 rotational degrees of freedom for (B) flexion-extension, (C) lateral bending and (D) torsion. Data presented as mean ± standard deviation. *p<0.05, **p<0.01.

Acta Biomater. Author manuscript; available in PMC 2017 January 15.

Acknowledgments

The research reported in this publication was supported by grants from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (R01AR057397) and National Institute of General Medical Sciences; Traineeship (T32 GM062754) of the National Institutes of Health and a Collaborative Research Partner Program grant on Annulus Fibrosus Rupture from the AO Foundation, Davos, Switzerland. The authors gratefully acknowledge the technical contributions and helpful discussions from Keegan Cole, Sarah Elsharkawy, Dr. Stephen Ferguson, Warren Hom, Damien Laudier, Diana Litsas, and Dr. Morakot Likhitpanichkul, Philip Nasser, Carine Rognon.

The providers of financial support for this study had no involvement: in the conduct of the research and/or preparation of the article; in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

Abbreviations


Karl Henga-Botsikabobe

Alfred Ngomanda

Richard Oslisly

Charly Favier

Serge Muller

Laurent Bremond

Henga-Botsikabobe Karl

Ngomanda Alfred

Oslisly Richard

Favier Charly

Bremond Laurent

Modern pollen-vegetation relationships within tropical marshes of Lopé National Park (Central Gabon)

Keywords: modern pollen assemblages, marshes, sediments, Congo Basin, savanna, lowland rainforest

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

Introduction

The savannas on the edge of the forest block of the Congo Basin as well as those enclosed are probably remnants of the "forest crisis" that occurred during the third millennium BP [START_REF] Maley | A catastrophic destruction of African forests about 2,500 years ago still exerts a major influence on present vegetation formations[END_REF] or relict of Last Glacial Maximum climate conditions [START_REF] Oslisly | Premiers résultats pluridisciplinaires d'une stratigraphie vieille de plus de 40000 ans du site de Maboué 5 dans la réserve de la Lopé au Gabon[END_REF]. The causes of forest destruction have been the under heavy debate recently, with arguments for a human [START_REF] Bayon | Intensifying Weathering and Land Use in Iron Age Central Africa[END_REF][START_REF] Garcin | Early anthropogenic impact on Western Central African rainforests 2,600 y ago[END_REF][START_REF] Bayon | The roles of climate and human land-use in the late Holocene rainforest crisis of Central Africa[END_REF] or climatic origin [START_REF] Ngomanda | Seasonality change and the third millennium BP rainforest crisis in southern Cameroon (Central Africa)[END_REF][START_REF] Maley | Late Holocene forest contraction and fragmentation in central Africa[END_REF]. Increasing the number of fossil records and their spatiality is necessary to better understand this event, but is made difficult due to the scarcity of lakes with sediment accumulation spanning the last thousand years in the region. It is likely that the basin's geological and hydrological characteristics, combined with tropical forest ecological dynamics, do not permit lake maintenance over several thousand years.

In Gabon, an enclave of savanna in the forest has the particularity of sheltering many lowlands that have preserved organic matter in sedge marshes [START_REF] White | A Guide to the Vegetation of the Lopé Reserve[END_REF], disseminated in the savanna itself, as well as in the surrounding forest. This area lies in the Lopé national Park (LNP), where the savanna is kept open by annual fires for decades by the park managers [START_REF] Jeffery | Fire management in a changing landscape: a case study from Lopé National Park, Gabon[END_REF]) but also probably for centuries or millennia, as a consequence of traditional landscape management [START_REF] Oslisly | The history of human settlement in the middle Ogooué valley (Gabon) Implications for the environment[END_REF]. The numerous marshes, of few hundred square meters each, are not fed by rivers and preliminary studies showed that they accumulated sediments for several thousand years [START_REF] Oslisly | La relation homme/milieu dans la réserve de la Lopé (Gabon) au cours de l'Holocène; les implications sur l'environnement[END_REF] and recorded local deposition conditions [START_REF] Obame | Le rôle de l'évolution des paysages holocènes dans le comblement des bas-fonds du Parc national de la Lopé, moyenne vallée de l'Ogooué au Gabon[END_REF]). To our knowledge, no equivalent environmental archives were ever used for paleoecological reconstructions in West or Central Atlantic Africa nor in lowland tropical Africa. The most similar studies probably come from marshes in the Brazilian Cerrados, where pollen assemblages have been analyzed over sequences of several thousand years [START_REF] Ledru | Late Quaternary history and evolution of the cerrados as revealed by palynological records[END_REF].

The purpose of this study is to assess the potential of the marshes for paleoecological investigations, by comparing pollen assemblages at the surface of 23 marshes and vegetation surveys along transects from sampling areas. On a larger scale, the spatial forest/savanna structure is addressed using satellite data.

The main questions addressed in this article are:

1-What type of pollen signal do these small marshes record? Specifically, is it a local or regional signal? 2-Is the forest-to-savanna ratio properly recorded by commonly used arboreal/nonarboreal pollen ratio? 3-Are the floristic composition of the Guinean-Congolese forest and the subcategories often used in pollen-based vegetation reconstructions such as "mature forest", "secondary forest" or "pioneer forest" recorded?

Material and methods

Geographical and botanical setting

The study area is located in the northern part of the Lopé National Park (LNP), where a savanna-forest mosaic stands as approximatively 320 km 2 enclosed open area in the Guineo-Congolian tropical moist forest formation (1000km 2 for the savannas of the middle Ogooue valley) [START_REF] White | The vegetation of Africa[END_REF]. The annual rainfall in this part of the park is around 1500 mm, with two dry seasons: the longer spans about three/four months from mid-May to mid-September, the shorter is about one/two-months long in January-February. The low tree cover of the savannas is mainly composed by shrubs of Crossopteryx febrifuga, Psidium guineense, Sarcocephalus latifolius and Bridelia ferruginea. Marshes occupying the topographical depressions are mainly dominated by the tall Cyperaceae Rhynchospora corymbosa [START_REF] White | A Guide to the Vegetation of the Lopé Reserve[END_REF]. Most marshes are permanent and fed by groundwater resurgences; the others are only rain fed and dry out during the dry season. We selected 23 marshes, in an area of 45 km 2 , along a gradient from closed forest to plain savanna (Figure 1; names of the marshes in italic in the text).

Table 1. Characteristics of the marshes

Vegetation survey

Botanical nomenclature follows the International Code of Nomenclature for algae, fungi and plants [START_REF] Turland | International code of nomenclature for algae, fungi, and plants (Shenzhen Code) adopted by the Nineteenth International Botanical Congress[END_REF]) and Family level is updated according to APG III (APG 2009) and APG IV [START_REF] Chase | An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG IV[END_REF].

Botanical surveys were carried out around the 23 marshes in September 2016. Most plants were identified to species, but some to genus and a few to family, due to the absence of distinctive character at this period of the year. The selected marshes were either in an open savanna environment, partially against the forest or entirely in the forest. We conducted two different botanical survey protocols in savanna and forest.

In the savanna surrounding each marsh, three 100 m-long transects were defined in different directions from the marsh margin. The margin was established visually based on the soilsurface quality (from black in wet parts to brownish in the marsh border) and on the transition from hydrophytic vegetation, with Rhynchospora corymbosa, to dry land vegetation. In seven 5 x 5 m quadrats placed every 15 m along each transects, botanical surveys were performed according to [START_REF] Braun-Blanquet | Plant sociology. The study of plant communities[END_REF], using abundance-dominance coefficient from 5 (plants covering more than 75% of the surveyed area) to 1 (plants covering a small area of the surveyed (<5%), R for rare specimens and + for specimens present one time. Abundancedominance is a general estimate of density and recovery rate (top-down projection of plant air elements) of different species in the inventory square [START_REF] Braun-Blanquet | Plant sociology. The study of plant communities[END_REF]. The semiquantitative values obtained were translated into quantitative data that can be used for vegetation survey analysis. The median of each classes has been used to obtain a percentage for each species (Class + (present) = 0.1%; R (rare)=1%; 1=2.5%; 2=15%;3=37.5%;4=62.5%;5=87.5%).

When forest patches were against the marsh area, three 250 m-long transects in forest originating from marsh margin were performed. In three 50 x 10 m quadrats placed every 100 m in each transect, all trees with a diameter at breadth height (dbh) greater than 10 cm were identified and counted.

Two Correspondence Analyses (CA) were performed on the median of classes for the savanna surveys and on the number of tree species for forest surveys. Due to the different protocols used in savanna and forest, we were not able to make a single analysis of the whole dataset.

Analyses were done using the CA function in the FactoMineR package of R [START_REF] Lê | FactoMineR: An R Package for Multivariate Analysis[END_REF].

Remote-sensing data analysis

A 0.5 m resolution WorldView-2 images acquired on 07/13/2016 (id 10300100595CDA00 and 1030010058399300) downloaded from Esri Satellite available through Qgis. Relying on the clear visual difference between forest and savanna in the image, we performed an automatic pixel classification into "forest" and "savanna" with Qgis (QGIS Development Team 2015). We then computed the proportion of savanna and forests in circular buffer zones centered on each marsh with radii 20 m, 50 m, 100 m, 200 m, 300 m, 500 m and 700 m.

Pollen sampling, extraction and counting

Surface marsh sediment were sampled for pollen analysis in March 2015. Sampling method was based on [START_REF] Wright | The use of surface samples in quaternary pollen analysis[END_REF]. In each of the 23 marshes, about 50 subsamples (the uppermost centimeter of sediment) were randomly collected over the whole surface of the marsh.

Subsamples were homogenized and put in a plastic bag to obtain one sample per marsh.

Pollen extraction was conducted on 1 cm 3 of sediment following Faegri and Iversen protocol [START_REF] Faegri | A contribution to deciphering the meaning of AP/NAP with respect to vegetation cover[END_REF]. A minimum of 400 pollen grains (excluding Pteridophyte spores) was counted per sample with an optical microscope at X630 magnification. The identification was based on the pollen collection of Tropical Africa at the "Institut des Sciences de l'Evolution de Montpellier (ISEM, Université Montpellier https://data.oreme.org/observation/pollen), and on African atlases of pollen and spores [START_REF] Bonnefille | Atlas des pollens d'Éthiopie principales espéces des forêts de montagne[END_REF][START_REF] Ybert | Atlas de pollens de Côte d'Ivoire[END_REF][START_REF] Sowunmi | Pollen of Nigerian plants: II woody species[END_REF][START_REF] Gosling | Atlas of the tropical West African pollen flora[END_REF]. The nomenclature of pollen taxa was based on [START_REF] Vincens | African pollen database inventory of tree and shrub pollen types[END_REF].

Pollen taxa were attributed to five ecological class according to the classification of [START_REF] Vincens | African pollen database inventory of tree and shrub pollen types[END_REF] for Africa, and locally specified according to [START_REF] White | A Guide to the Vegetation of the Lopé Reserve[END_REF] For representation diagram, pollen percentages were calculated on the sum of all determined pollen, including hydrophytic taxa (e.g. Cyperaceae) but excluding spores. The whole pollen sum is used as in modern pollen-vegetation relationships studies (e.g. [START_REF] Vincens | Pollen-rain -vegetation relationships along a forest -savanna transect in southeastern Cameroon[END_REF].

Pollen concentrations have been calculated by adding Lycopodium tablets during the chemical treatment [START_REF] Stockmarr | Tablets with spores used in absolute pollen analysis[END_REF]. Pollen diagrams were created with the Rioja package of R [START_REF] Juggins | Rioja: analysis of quaternary science data version[END_REF]. The most common taxa were defined as the taxa present in a minimum three samples and with more than 2% of the pollen sum in at least one sample.

A Correspondence Analysis was performed on the total pollen sum (excluding undetermined pollen and spores) and according to different options to refine interpretations: on the most common taxa defined above, on pollen from terrestrial plants by excluding those attributed to the Marsh class, and finally on Forest taxa only. Analyses were done using the CA function in the FactoMineR package of R [START_REF] Lê | FactoMineR: An R Package for Multivariate Analysis[END_REF].

Representation indices

After establishing a correspondence between surveyed plants and identified pollen taxa, we computed indices of Association (A), Under-representation (U) and Over-representation (O)

for the main taxa present in plant and pollen datasets [START_REF] Davis | Pollen frequencies reflect vegetation patterns in a great basin (U.S.A.) mountain range[END_REF]. These indices are based on presence/absence data in vegetation and pollen from the 23 marshes and allow qualifying the relationship between vegetation and pollen. They are defined as follows:

𝐴 = 𝐵 0 𝐵 0 + 𝑃 0 + 𝑃 1 𝑈 = 𝑃 1 𝐵 0 + 𝑃 1 𝑂 = 𝑃 0 𝐵 0 + 𝑃 0
and expressed as percentages, where: B 0 is the number of samples in which both the pollen type and the associated plant are present; P 0 is the number of samples in which the pollen type is present but not the associated plant; and P 0 is the number of samples in which the pollen type is not present but the associated plant is. We also follow the classification used by [START_REF] Amami | Modern pollenvegetation relationships within a small Mediterranean temporary pool (western Morocco)[END_REF] with Strongly-Associated taxa: A>65% ; Associated taxa: 50%<A<65% ;

Weakly Associated taxa: low A values and relatively high values for U (under-represented taxa)

or O (over-represented taxa).

Results

Floristic distribution

Canonical Analysis applied on the 439 botanical survey quadrats in savanna reflects a gradient of plant distribution from quadrats closest to the marsh (Q1 and Q2; 0-30m) to the furthest in the savanna (Figure 2; Panel A). The two first axes express 13.3% of the total dataset inertia.

In the species representation, the first axis opposes species from marshy areas with high frequencies for species like Aspilia africana, Hyparenia sp., Hypoxis angustifolia, Imperata cylindrica, Ipomoea sp1., Ourenia lobata, Paspalum sp., Rhynchospora corymbosa, Sauvagesia

Pollen diagram

158 pollen taxa were identified in surface soil samples. Most of the pollen taxa (115 taxa) are produced by forest trees. The "Savanna" vegetation is mainly represented by 27 taxa, the "Marsh" by 8 taxa and the "Marsh Border" by 8 taxa.

The most common pollen taxa (>2%, present in more than 3 samples) of surface samples are presented in a pollen diagram according to the five ecological classes and ranging according to the first axis of a CA performed on the whole pollen assemblages (Figure 4; detailed pollen diagram in supplementary file. Sup. X and CA in Sup x).

The pollen diagram shows that marshes with the highest degree of openness (marshes within savanna) show low percentages of "Savanna" taxa in their pollen assemblages, with variation between ca. 5 and 30%. The highest values of the "Savanna" taxa are obtained in marshes within forest-savanna mosaic with values between ca. 20 and 40%, but do not exceed 50%.

"Forest front" pollen taxa are registered in all the 23 pollen assemblages with close values, between ca. 1 and 5% and do not exceed 10%.

"Forest" pollen taxa are registered with the highest values in all marshes within savanna, between ca. 70 and 80%, but do not exceed 90%. Their values vary between ca. 60 and 70%

and do not exceed 80% in the marshes within forest-savanna mosaic. The marshes within forest (Lopé 2 and Tortue-foret) have similar percentages to those respectively in forestsavanna, and in savanna.

"Marsh" taxa are better registered in marshes within forest-savanna mosaic than those in savanna or forest, with values between ca. 10 and 35%, without exceeding 40%.

In all marshes, pollen assemblages are dominated by Poaceae and Cyperaceae, with unusual correlation of occurrence. The percentages remain low, respectively 4-27% and 1-33% for all the marshes.

Pollen diversity

Several Correspondence Analysis (CA) were performed on the whole pollen assemblages, marsh pollen excluded, on the forest taxa, on the forest taxa without rare taxa (superior to 2% in almost three samples) and with Tortue-foret excluded (because an over dominance of Uapaca pollen in the assemblage) to refine the interpretation of the results. Figure 4

Indices of vegetation representation in pollen assemblages

The Davis indices calculated for the 33 main pollen taxa indicate that 9 taxa are Strongly Associated, 2 Associated, 0 Under-represented and the majority (20 taxa), Over-represented.

Table 2. Davis indices calculated for the main 33 pollen taxa: is the number of samples in 𝐵 0 which both the pollen type and the associated plant is present;

is the number of samples 𝑃 0 in which the pollen type is present but not the associated plant; and is the number of 𝑃 1 samples in which the pollen type is not present but the associated plant is associated (A), under-represented (U) and over-represented (O).

Pollen concentrations

Pollen concentrations were calculated first on the pollen sum (total concentration), second on the regional pollen excluding Cyperaceae, Poaceae, other hydrophytes and spores (regional concentration), and third on forest taxa only (forest concentration). Total concentration varies from 36,996 (Yao) to 274,821 (Vitex), regional concentration from 16,288 (Yao) to 127,780 (S05), and forest concentration from 12,006 (Yao) to 94,173 (S05).

There is no trend in concentrations, either in terms of distance from the forest (forest concentration), in terms of marsh size (regional concentration) or in terms of the permanent or temporary nature of wetlands (total concentration). There is also no correlation with the mean sediment accumulation rate calculated for 6 marshes, for which we have radiocarbon dates (K. Henga-Botsikabobe & L. Bremond, unpublished data).

Discussion

Savana pollen representation

Arboreal Pollen grains versus Non-Arboreal Pollen grains (AP/NAP ratio) has long been used to characterize the structure of the vegetation by simplifying complex pollen assemblages [START_REF] Faegri | A contribution to deciphering the meaning of AP/NAP with respect to vegetation cover[END_REF]. In tropical areas, as in temperate regions, this ratio has been confronted and discussed with quantified vegetation cover [START_REF] Vincens | Pollen-rain -vegetation relationships along a forest -savanna transect in southeastern Cameroon[END_REF]Favre et al. 2008). In fossil samples, the NAP grains are mainly Poaceae pollen and a common interpretation is to link Poaceae pollen abundance and presence of savanna around the site (e.g. [START_REF] Maley | Late Holocene forest contraction and fragmentation in central Africa[END_REF]. In lake fossil records, the Poaceae that grow along the shoreline can mask the regional forest signal and lead to a misinterpretation of the real openness of the environment [START_REF] Bush | On the interpretation of fossil Poaceae pollen in the lowland humid neotropics[END_REF], but it has never been observed that fossil sediments from a savanna region contain very little Poaceae pollen grain. In lake Kamalété, in the South/East part of the Lopé reserve, the study of [START_REF] Ngomanda | Vegetation changes during the past 1300 years in western equatorial Afnrca: a highresolution pollen record from Lake Kamalete, Lope Reserve, Central Gabon[END_REF] shows that Poaceae pollen grains were well recorded and preserved (>60% of the pollen sum) in the sediments during the last 1400 years. Also, [START_REF] Lebamba | Modern pollen rain in savanna and forest ecosystems of Gabon and Cameroon, Central Atlantic Africa[END_REF] observed that modern soil assemblages from the savanna surrounding our studied marshes present more than 60% of Poaceae pollen grains in the assemblages. These studies are consistent with previous works [START_REF] Ybert | Emissions polliniques actuelles dans la zone du contact forêt-savane en Côte d'Ivoire[END_REF][START_REF] El Ghazali | Modern lowland pollen spectra and contemporary vegetation in the eastern Sahel Vegetation Zone, Sudan[END_REF][START_REF] Vincens | Pollen-rain -vegetation relationships along a forest -savanna transect in southeastern Cameroon[END_REF][START_REF] Julier | The modern pollenvegetation relationships of a tropical forest-savannah mosaic landscape, Ghana, West Africa[END_REF], which consider tropical savanna Poaceae as strong pollen producers, and their wind-mediated pollen as well dispersed and usually overrepresented in pollen assemblages.

The surface samples of our study zone surprisingly reveal very weak percentages of Poaceae pollen grains that never exceed 25%. The studies of [START_REF] Ngomanda | Vegetation changes during the past 1300 years in western equatorial Afnrca: a highresolution pollen record from Lake Kamalete, Lope Reserve, Central Gabon[END_REF] and [START_REF] Lebamba | Modern pollen rain in savanna and forest ecosystems of Gabon and Cameroon, Central Atlantic Africa[END_REF] clearly prove that the very low proportions of Poaceae pollen found in our samples are not due to low pollen production by Poaceae species from the savanna of La Lopé (mainly Anadelphia afzeliana, Hyparrhenia diplandra, Panicum nervatum and Schizachyrium platyphyllum; [START_REF] Jeffery | Fire management in a changing landscape: a case study from Lopé National Park, Gabon[END_REF]). Moreover, in our surface samples, Poaceae are well correlated with Cyperaceae (Pearson test: r=0.78; p<0.01), which suggests that they originate from local helophytic vegetation, and not from savanna. The discrepancy between previous results and our data should be due to both the marshy nature of our study sites and the spatial scale of pollen trapping, inadequate to record the savanna vegetation. The spatial scale represented by pollen assemblages has long been investigated. It is dependent on pollen productivity and dispersion for each plant species, including its mode of primary and secondary dispersal and its ability to be dispersed. [START_REF] Jacobson | The selection of sites for paleovegetational studies[END_REF] proposed that the relative weights of local (< 20 m), extra-local (< a few hundred meters) and regional vegetation (from a few hundred meters to about 30 km) in the pollen assemblage strongly vary with the size of the site. Here, the studied wetlands are small, very often less than 100 m in diameter, which should reduce the proportion of the regional pollen influx. In addition, they are not open water bodies, but herbaceous marshes without streams that feed them.

Herbaceous plants should block the runoff, further reducing extra-local supply. Pollen assemblages will therefore be strongly influenced by local vegetation and partially by regional one. The extra-local vegetation will therefore be masked by these two components. The study of [START_REF] Jacobson | The selection of sites for paleovegetational studies[END_REF] was quite theoretical, but later Sugita (1994) simulated pollen loading in relation to distance-weighted plant abundance around the sedimentary basin and specified that pollen loading and proportion in large lakes record the spatial distribution pattern of vegetation as homogeneous rather than patchy. This confirms our interpretation, especially since in Sugita's model the height of trees producing pollen grains is not considered. Trees in the galleries in the savanna rarely exceed 25 m in height, while trees in the mature forest can occasionally exceed 65 m [START_REF] White | A Guide to the Vegetation of the Lopé Reserve[END_REF]. The production of pollen from tall trees against Poaceae pollen produced at less than one meter-high will amplify the phenomenon of masking the recording of savanna Poaceae in the deposits. Our samples therefore reflect a truncated composition of the extra-local vegetation. The regional composition is represented by something like to 35-55% of the pollen count, which is consistent with the estimates of [START_REF] Jacobson | The selection of sites for paleovegetational studies[END_REF].

Pollen diversity: marshes, savanna, forest

Axis 1 of the CA indicates that there is a gradient from the most open environments to those where the forest is closer. The assemblages that explain this distribution seem however to be counter-intuitive when referring to the usual interpretations of pollen patterns in tropical Africa [START_REF] Maley | Vegetation dynamics, palaeoenvironments and climatic changes in the forests of western Cameroon during the last 28,000 years B.P[END_REF]. Indeed, Fabaceae Caesalpinioideae, usually considered as a marker of mature forest, are better recorded far from the forest front, and pioneer taxa such as Annonaceae and Lophira alata are more present in the sites close to the forest bloc. These observations, based on pollen percentages, are confirmed by pollen concentrations of forest trees that show no trend towards distance from the forest. This implies that the recorded gradient clearly does not translate the nature of extra-local vegetation (savanna or forest). It should be rather related to the mosaic nature of Lopé landscapes that include savannas, gallery forests and forest stands. In the open part of the studied zone, there are tree species associated to the presence of marshes and streams such as Anthocleista spp. (Loganiaceae), Berlinia spp. (Fabaceae Caesalpinioideae), Calancoba glauca (Flacourtiaceae), Mitragyna ciliata (Rubiaceae) and Nauclea vanderguchtii (Rubiaceae), found quite close to rivers and streams, or Homalium (Flacourtiaceae) generally found in gallery forests close to savanna [START_REF] White | Herbaceous vegetation in different forest types in the Lopé Reserve, Gabon: implications for keystone food availability[END_REF]. In the southern samples, closed to the forest, there are species of the colonizing forest front, including Aucoumea klaineana (Burseraceae), a very weak pollen producer, Lophira alata (Ochnaceae) and some species which are able to colonize savanna, such as Elaeis guineensis (Arecaceae), Klainedoxa gabonensis (Irvingiaceae), Uvariastrum pierreanum (Annonaceae) and Xylopia spp. (Annonaceae) [START_REF] White | Herbaceous vegetation in different forest types in the Lopé Reserve, Gabon: implications for keystone food availability[END_REF].

The analysis of the botanical statement performed in the forested area closed to the studied marshes is difficult to compare with the results of the pollen assemblages because the gallery forest was not enough sampled. Statements are missing in open environments because galleries and forest "bosquets" were not against the marsh area. However, we can note a roughly similar distribution, with a distribution from swamps in open areas to those close to the forest bloc. Vegetation in Lopé NP is complex due to the mosaic of forest types, 27 according to [START_REF] White | Herbaceous vegetation in different forest types in the Lopé Reserve, Gabon: implications for keystone food availability[END_REF], and the botanical surveys did not permit to differentiate some of them.

In contrast, the botanical statements performed in the savanna surrounding the marshes reflected a clear distribution of plants from the swamp border to further in the savanna.

Swampy species are opposed to inland savanna species. This distribution cannot be directly recorded by the assemblages of surface pollen because of the sampling method which consisted in obtaining a homogeneous assemblage over a surface area of several hundred square meters and not a transect as [START_REF] Amami | Modern pollenvegetation relationships within a small Mediterranean temporary pool (western Morocco)[END_REF] done. Thus, it is important to identify "indicator taxa" of the different environments and especially the presence of savanna around the sites. [START_REF] Janssen | Stevens Pond: A Postglacial Pollen Diagram from a Small Typha Swamp in Northwestern Minnesota, Interpreted from Pollen Indicators and Surface Samples[END_REF][START_REF] Janssen | Problems in the recognition of plant communities in pollen diagrams[END_REF] and [START_REF] Amami | Modern pollenvegetation relationships within a small Mediterranean temporary pool (western Morocco)[END_REF] emphasized the importance of "indicator taxa" to characterize a particular community. Our results, both quantitative, based on Davis indices, and qualitative, based on pollen diagrams, indicate that no pollen correctly records the main herbaceous and shrubby savanna species, namely Crossopteryx febrifuga, Sarcocephalus latifolius, Poaceae, Psidium guineense and Bridelia frerruginea. Marsh border species, such as Ascolepis capensis and Hypoxidaceae, are more sensitively recorded. These taxa are associated with an opening of the environment around the marsh and probably a lower annual permanent humidity condition.

Fossil assemblage interpretations

Our results from marsh surface sediments show that soil samples (e.g. [START_REF] Lebamba | Modern pollen rain in savanna and forest ecosystems of Gabon and Cameroon, Central Atlantic Africa[END_REF] are not good analogues for marsh pollen records. Tropical savanna marshes are rare because of the very strong evapotranspiration in these environments. A constant supply of water during dry seasons is necessary to allow the accumulation of organic matter. To our knowledge, there are no equivalent fossil deposits that have been studied in Africa. In Brazil, some palm swamps, so-called "vereda", are growing in the savanna-like vegetation commonly known as "cerrado" and were studied for their pollen fossil contain [START_REF] Salgado-Labouriau | Late Quaternary vegetational and climatic changes in cerrado and palm swamp from Central Brazil[END_REF]). This study do not really investigate the relationship between present vegetation and pollen deposits, but the obtained results clearly do not show any distortion similar to the one we observe in our sites, particularly with regard to Poaceae. In [START_REF] Ledru | Late Quaternary history and evolution of the cerrados as revealed by palynological records[END_REF], some surface samples from cerrados can however register less than 20% of Poaceae, but the published data does not allow identifying the exact environmental conditions of the study. Thus, the interpretation of fossil data from the marshes where we studied the contemporary deposits will have to be done very finely, considering these limitations. Fossil assemblages may have to be analyzed by adding them to the Canonical Analyses performed on modern data in order to differentiate riparian forest assemblages from those of mature forests as done by [START_REF] Montade | A new modern pollen dataset describing the Brazilian Atlantic Forest[END_REF].

Conclusion

The results of the present work on pollen assemblages from Gabonese marsh surface sediments are quite complex because they are often counter-intuitive. The low proportion of Poaceae in the assemblages is explained but challenges the simplistic interpretation that consists in considering this percentage as a degree of openness of the environment. These results do not affect previous calibration results performed on soil surfaces and applicable to lake sediments. However, they are essential for interpreting sedimentary sequences from marshes.

The very high diversity of forest pollen taxa is not affected by the taphonomy of the deposits, which nevertheless makes it possible to finely qualify the major changes in plant diversity that may have occurred in the forests of the Congo Basin over the past few millennia.

To conclude, anywhere in the world "when you hear hoofbeats, think of horses not zebras"; here, in Lopé, it is always the zebras you find.

  erecta and Schizachyrium platyphyllum, and species more related to inland savanna zone such as Andropogon sp., Crossopteryx febrifuga and Kyllinga echinata. The second axis also opposes species from marshy areas, as Fern (unidentified), Clappertonia ficifolia, Desmodium sp., Phyllanthus sp. and Triumfetta sp., to more inland savanna species, as Andropogon sp., Crossopteryx febrifuga and Kyllinga echinata. The second lower panel (B) of Figure 2 where Quadrat numbers are plotted reveals an opposition between Q1 plus Q2 and the other quadrats presenting a higher homogeneity. The two first axes of the Canonical Analysis applied on the 101 botanical survey quadrats in forest are presented Figure 3. The axes express 28.3% of the total dataset inertia. They oppose marshes close (Citron, Paurosa, S08) or within the forest (Lopé2 and Tortue-foret) to others in more open areas as Vitex, S06, S15 and S16 associated to gallery forest (Figure 3). The first group is mainly explained by the frequency of Aucoumea klaineana, Maprounea membranacea, Variastrum sp. and Xylopia quintasii, compared to Ceiba pentandra, Elaeis guineensis and Irvingia grandifolia for the second group.

  presents selected CA with rare taxa and Tortue-foret excluded (A: all taxa and B: only forest taxa). The distribution of marshes and taxon are quite the same for the two CA. Marshes in open areas (S04, S08, S03, S12 and S01, S02) are opposed from those in more closed habitats (Citron, Paurosa, Vitex, Yao and Lopé2, Tortue-Savane). The distribution is mainly explained by the high frequency of Cassia-type, Dialium, Euphorbiaceae, Fabaceae Caesalpinioideae, Flacourtiaceae, Macaranga-type, Olacaceae, Pontederiaceae, and for the second group by Annonaceae, Celtis, Cyperaceae, Laccosperma, Lophira alata-type, Poaceae and Uapaca. The gradient from marshes in open areas to closed habitats along the first axis is well visible in the map of Figure 4C where value of the first axe of the CA (B) has been plotted with a color scale.

Figure 1 .

 1 Figure 1. Study area and location of sampled marshes

Figure 3 .

 3 Figure3. Canonical Analysis applied on the 101 botanical survey quadrats in forest. Panel A: Distribution of the plant species according to axes 1 and 2. Color gradient illustrate the contribution of each species to the CA according to the squared cosine (cos 2 ). Panel B: Distribution of the marsh where transects where performed according to axes 1 and 2.

Figure 4 .

 4 Figure 4. Pollen diagram of the most common grains counted in the marsh surface samples.Samples are ranging according to the first axe of an CA performed on the whole pollen assemblages and taxa ranging according to their ecology affinity.

Figure 5 .

 5 Figure5. Canonical Analysis applied on the most common pollen grains. Panel A: Distribution of the taxa according to axes 1 and 2. Color gradient illustrate the contribution of each taxa to the CA according to the squared cosine (cos 2 ). Panel B: Distribution of the sampled marshes according to axes 1 and 2. Panel C: Mapping of Axe 1 of the CA with a color gradient for each marshes according to their location and position towards the forest-savanna mosaic.

Acknowledgement

We thank ANPN (Agence Nationale des Parcs Nationaux) and IRD (Institut de Recherche pour le Développement) for having financed part of the project. We thank Judicael Lebamba for constructive discussions and Jean Maley for introducing KHB to palynology. We are grateful to Laure Paradis for extracting satellite data and mapping the field of study.