Dr Vusala Ibrahimova

Dr Hang Zhao

Dr Emmanuel Ibarboure

Prof. Dr Elisabeth Garanger
email: garanger@enscbp.fr

Sébastien Lecommandoux
email: sebastien.lecommandoux@u-bordeaux.fr

Thermosensitive vesicles from chemically encoded lipid-grafted elastin-like polypeptides

Keywords: Biomimetic membrane, elastin-like polypeptides, lipoproteins, self-assembly, vesicles

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

Introduction

The design of functional membranes mimicking biological materials represents a critical challenge for the study of the origin of life, for artificial cell/protocell construction, biotechnology or pharmaceutical industry. 1,2 Biomimetic models of cellular membranes that can provide an optimal environment to perform multiple complex biochemical reactions (e.g., selective catalytic transformations, complex enzymatic reactions, protein expression, RNA replication) may offer outstanding opportunities for the understanding of cell membrane properties (e.g., endo/exocytosis, cell adhesion, membrane permeation). 1,[3][4][5] Considering the average 60% 6 lipid composition of natural membranes (i.e., cholesterol, phospholipids, glycerophospholipids, sphingolipids, etc.), the first basic synthetic analogues of biological cells reported are 'liposomes' prepared from naturally-occurring phospholipids and composed of a 3-5 nm thick bilayer membrane. 7,8 Later developed 'polymersomes', built from amphiphilic synthetic polymers/polypeptides, provided more mechanically stable vesicle structures owing to macromolecules of higher molar mass and thicker membranes (5-50 nm). [8][9][10][11] Recent advances in cell-mimicking systems have drawn the attention to genetically encoded protein/peptide-based membrane forming bioamphiphiles for potential design of artificial living systems with self-replication, growth and division properties. 6,[12][13][14] Among proteins of interest, elastin-like polypeptides (ELPs) have been proposed as a relevant class of intrinsically disordered proteins (IDPs). ELPs consist in repetitive (-VPGXG-) pentapeptide sequences (X is a guest residue) and are ideal biomaterials for artificial cells design. 12,13,15,16 ELPs exhibit unique lower critical solution temperature (LCST) phase transition behavior, which drives their transition from a soluble state below their cloud point temperature (Tcp) to a dehydrated coacervate state above Tcp. 17 In this context, recent synthetic approaches have been proposed to synthesize protein-based amphiphiles as membrane forming materials. Schiller's group introduced genetically encoded amphiphilic proteins built from recombinantly produced and fluorescently labelled diblock elastin-like polypeptides (dbELPs) which formed complex architectures varying from vesicles to twisted fibers upon solvent (n-butanol or 1-octanol) assisted self-assembly. 18 Kiick's lab also reported conjugates of elastin-like and collagen-like polypeptides (ELP-CLP) and the production of thermoresponsive, collagenbinding drug delivery vehicles. 19,20 Champion's lab developed another approach to access protein vesicles from ELPs, especially using charge interactions with peptide zippers. 21,22 Francis' lab also reported series of fusion proteins that have been developed to self-assemble spontaneously into stable micelles after enzymatic cleavage of a solubilizing protein tag. 23 Natural lipidated proteins are reference models for the development of relevant artificial lipid-protein conjugates due to their diverse properties, such as bioactivity, self-assembly and cell membrane anchoring properties that can regulate different cellular functions. 24 In this context, Chilkoti's group has firstly produced post-translationally modified ELPs conjugates with cholesterol 25 and aliphatic myristoyl group 26 , exhibiting temperature-triggered self-assembly into a diverse array of structures including fibers, beads-on-a-string coacervates, and networks of fibers. In addition, the production of different derivatives of linear lipid-ELPs via post-translational modification, self-assembly into micelles, 27,28 elongated fibers and bottle-brush structures, 29 have also been reported recently by Mozhdehi et al. Nevertheless, none of these previous studies using linear lipid-ELP bioamphiphiles have shown or investigated their membrane forming property. In this work, we have explored a simple and versatile synthetic strategy to chemoselectively graft aliphatic mono-cis-unsaturated lipid tails to recombinant ELP 30 backbones to access, for the first time, brush-like biohybrid lipoproteins (Figure 1), that are able to self-assemble into vesicles, also referred to as "lipoproteinosomes" with a simple solvent free thin film hydration technique. We hypothesized that this brush-like architecture, where each lipophilic alkyl tail is mimicking the phospholipids' acyl chain, should facilitate vesicle formation stabilized by the peptide backbone which connect 11 lipid tails distributed every 20 probiotic amino acid residues. This novel class of biohybrid amphiphiles combine the best features of the ELP (e.g. bioactivity, non-immunogenicity, chemical diversity, multifunctionality and thermal responsiveness) together with the membrane-forming properties of lipids, enabling the growth of vesicles with temperature responsive on/off release profiles.

Results and Discussion

Synthesis of lipid-graft-ELP[M1V3-40]

The lipoprotein designed in this work is based on a recombinant ELP with the exact protein sequence MW[(VPGVG)(VPGMG)(VPGVG)2]10, noted as ELP[M1V3-40], produced in Escherichia coli bacteria. 31,32 The latter was chemoselectively alkylated using glycidyl propargyl ether to introduce an aliphatic alkyne group on the side chain of the eleven methionine (Met) residues of the ELP as previously reported by our group. 30,31 In parallel, the chlorine group at the chain end of oleyl chloride was converted into an azide group (Scheme S1) for subsequent coupling to ELP[M(Alkyne)1V3-40] via Huisgen's copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction. (Scheme S2) Full substitution of the halogen atom by the azide group was confirmed by 1 H NMR spectroscopy by the characteristic 0.2 ppm downshifting of α-methylene group's resonance peak from 3.56 to 3.27 ppm, (Figure S1) and by the emerging large -N3 stretching vibration band at 2,092 cm -1 in the ATR FT-IR spectrum. (Figure S2 S5) The 1 H NMR spectrum of ELP[M(Oleyl)1V3-40] showed a welldefined and separated resonance peak at 8.02 ppm corresponding to the newly formed triazole proton which was used as reference to quantify the reaction conversion. (Figure S3) Setting triazole proton peak integral to 11, the expected number of protons for all the well identified and separated peaks were found as indicated in the experimental part. (Table S1) Comparative SEC chromatograms of the native recombinant ELP[M1V3-40] and of ELP[M(Alkyne)1V3-40] indicated a retarded elution of the latter despite a higher molar mass. This can be attributed to the branching introduced on the polypeptide backbone which significantly affects the hydrodynamic volume of the macromolecule, 33 but also to the positively charged sulfonium groups and associated counterions that may interact with the columns. 30 (Figure S5-A) After grafting of the monounsaturated lipid tail, ELP[M(Oleyl)1V3-40] displayed a slightly shorter retention time consistent with an increased molar mass. SEC analysis also evidenced a significant peak shoulder attributed to dimer species. The band associated with these dimers was found much weaker in SDS-PAGE analysis which confirmed their noncovalent nature, hydrophobic inter-chain interactions likely occurring between the brush-like lipopeptide chains. (Figure S5-B)

The conjugation was further confirmed by ATR FT-IR spectroscopy following the disappearance of the stretching vibration band of the azide group of the oleyl azide chains and the emergence of new stretching vibration bands at 2,962 and 2,927 cm -1 attributed to the asymmetric and symmetric stretching bands of -CH2- 34 indicating a successful lipidation of the ELP. (Figure S2) The structural characteristics of ELP[M(Oleyl)1V3-40] are provided in Table 1 and compared to those of synthetic intermediates. Considering the temperature responsive property of ELP[M1V3-40], 32 a monounsaturated aliphatic tail 35 with a low melting transition temperature (Tm = -25 °C, Figure S6) was selected in order to not interfere with the phase transition temperature (Tt) of the ELP. The olefin bond present in natural lipids/fatty acids has a significant impact on the membrane packing and on its fluidity owing to the conformational (cis/trans) difference. 36 The planar structure, torsion angle and faster rotational motions of the first neighboring single C-C bonds on the cis alkyl chains have a significant impact on the formation of well-packed lipid bilayers in natural cell membranes. 36 Whereas the trans conformation behaves similar to saturated fatty acids. 36 ATR FT-IR spectroscopy was used to confirm the presence of the characteristic =C-H stretching vibration band of the cis conformation at 3,005 cm -1 and the absence of the trans conformation band at 3,034 cm -1 . 34 (Figure S2) S9-A) The average hydrodynamic diameter of the nanoparticles formed expectedly decreased above 30 °C while the derived count rate increased, as a consequence of the ELPs dehydration resulting in a conformational change from coils to β-spiral structures above Tcp and an increased density of the dispersed particles. 17,37 Additionally, heating-cooling cycles (up to 37 °C and down to 20 °C) confirmed the reversibility of the temperature response, while the size of particles decreased and increased of approximately 45 nm in response to temperature change. (Figure S9-B) Considering the physico-chemical parameters and phospholipid like structure of Met residues' side chains, we hypothesized that such hybrid brush-like lipoprotein may self-assemble in the aqueous milieu into membrane systems via lateral packing of alkyl tails, 38 in a similar manner to natural phospholipids above Tm and be stabilized by the ELP backbone below its Tcp. A dense packing of lipid tails may induce the formation of entropically constrained ELP (VPGVG)3 loops in the internal and external surface of the vesicles shell, lipid tails being separated by 20 amino acid residues. To induce the formation of large vesicles, we have chosen the simplest, organic solvent free, conventional vesicle growth technique namely 'thin film hydration' 39 consisting in the formation of a thin lipopolypeptide film on a solid surface that is subsequently swollen by the addition of an aqueous solution to facilitate budding and vesicles growth. (Figure 2-A, experimental section of supporting information (SI)) To increase the surface area of the lipopolypeptide film per volume, provide extra curvatures and have better control on the vesicles growth process, 0.5 and 5 mm size glass beads were used. [START_REF] Vogele | [END_REF]41 Film hydration on glass beads yields narrow size distribution and allows to reduce the volume of the swelling solvent to microliters thus known as a robust and rapid method. 12,41 Therefore, a thin layer of ELP[M(Oleyl)1V3-40] lipopolypeptide was formed directly on the surface of the beads by evaporating the solvent via nitrogen flow (CHCl3: MeOH, 8:2). Considering the thermal characteristics of ELP[M(Oleyl)1V3-40], (Table S2) the film hydration process was conducted in pure deionized water by incubation over 48 h at a temperature below 20 °C. As expected, 5 mm size beads yielded giant lipoproteinosomes (GLPs), (Figure 2-B, 3 and Figure S9) with micrometer sizes. In contrast, the smaller 0.5 mm beads yielded nanosized lipo-proteinosomes (NLPs) with an average hydrodynamic radius of 372 nm and an estimated membrane thickness of 7.8 nm, consistent with a bilayer packing model. (Figure 2-D, andFigures S11-13) In order to visualize the asformed vesicles, 5 v% of rhodamine labelled Rhd-ELP[M(Oleyl)1V3-40] (Scheme S3 and Figure S14) was mixed with the unlabeled lipopolypeptide solution before the film was formed. Images captured by epifluorescence and confocal microscopy confirmed the formation of GLPs with homogeneous membranes as can be seen in Figure 3 and Figure S9 without any defect or domain formation. The use of an excess swelling solvent to cover the large beads yielded a dilute vesicles solution with a broad size distribution varying between 6-23 μm (obtained from confocal images, Figure 2-C). In contrast, the small beads yielded NLPs with an average hydrodynamic radius of 372 nm in good yield and with rather narrow size distributions (PDI=0.25, Figure 2-E) which was in a good agreement with the size obtained from TEM images (295±62 nm, Figure 2-D).

Thermo-responsive permeability of lipo-proteinosomes

The vesicles (NLPs) were then subjected to DLS analysis to evaluate the temperature-responsive hydration-dehydration of closely packed ELP chains on the lipoprotein membrane as illustrated in Figure 4-C.

Conclusion

We reported here a straightforward and versatile design of brushlike ELP[M(Oleyl)1V3-40] lipopolypeptides allowing the formation of model thermo-responsive membranes with great potential applications in protocell design, nano/micro reactors, drug delivery vehicles with biomimetic character and controlled burst release properties. Such combination of genetically engineered elastin-like polypeptides with chemical post-modification reactions to introduce alkyl chains offers promising opportunities for biohybrid materials design, featuring compartmentalization properties from natural phospholipids combined with temperature responsiveness, bioactivity, sequence-defined structure, reproducibility, easy scalability and narrow molar mass distribution of the genetically engineered and recombinantly produced ELPs.

Entry for the Table of Contents

Temperature-sensitive lipo-proteinosomes were engineered by grafting lipids onto temperature-responsive elastin-like polypeptides (ELPs). Maintained at 37°C, folded ELP loops on the internal/external surface of the biohybrid membrane prevents the leaching of encapsulated molecules, while cooling of the dispersion induces a burst release, in correlation with the decompaction of ELP chains.

Institute and/or researcher Twitter usernames: @LCPO_Bordeaux, @LCPO_Poly4Life, @VUSALAIBRAHIMO2, @zhaohang1989, @ElisGaranger, @Biomac_ACS_Bord

Table of Contents

Figure 1 .

 1 Figure 1. (A) Chemical structure and (B) schematic representation of ELP[M(Oleyl)1V3-40].

  ) The quantitative grafting of oleyl azide onto ELP[M(Alkyne)1V3-40] and successful purification of ELP[M(Oleyl)1V3-40] were confirmed by different techniques namely by 1 H NMR spectroscopies, size exclusion chromatography (SEC), SDS-PAGE and ATR FT-IR. (Figures S2-

Formation

  of lipo-proteinosomes The Tcp of the final conjugate, ELP[M(Oleyl)1V3-40], was determined in water (10 mg•mL -1 , 417 μM colloidal dispersion) by differential scanning calorimetry (DSC) analysis. (Figure S7) The onset temperature was measured at 25.4 °C on the second heating ramp. This value is very close to the Tcp of the unmodified recombinant ELP[M1V3-40] (i.e., 22.2 °C, 100 mg•mL -1 in water, 5,900 μM), and only slightly lower than the Tcp of ELP[M1V3-40] at identical molar concentration (417 μM, 30 °C) as expected by turbidimetry assays. (Figure S8) This confirmed that the ELP backbone preserved its thermo-responsiveness after the lipidation. The ELP[M(Oleyl)1V3-40] dispersion in water was further investigated by dynamic light scattering (DLS) measurements through the scanning of lipopolypeptide solutions in the range of 15-75 °C at 90° angle. (Figure

Figure 2 .

 2 Figure 2. (A) Schematic representation of the glass beads thin film hydration process in water. (B) Confocal laser scanning microscopy image (red channel) of giant lipo-proteinosomes (GLPs obtained using 5 mm beads) at room temperature, and (C) size distribution histogram plotted from the counting of 25 vesicles. (D) Uranyl acetate-stained TEM image of nanometer sized lipoproteinosomes (NLPs obtained using 0.5 mm beads) with an average size of 295±62 nm (n=20) and (E) 90° angle DLS intensity-averaged distribution of

Figure 3 .

 3 Figure 3. Optical microscopy images of giant lipo-proteinosomes (GLPs) at room temperature. (A) Bright field and (B) red channel images taken with a confocal laser scanning microscope. (C) Fluorescence intensity profile along the white solid line in (B).

Figure 4 .Figure 5 .

 45 Figure 4. (A) Derived count rate (DCR) versus temperature (20-50 °C) during repetitive heating (red trace) and cooling (blue trace) cycles of NPLs (n=3). (B) Hydrodynamic diameter of NPLs at 20 and 37 °C (average of Z-average (d, nm) size has been used in the figure for each data point, n=3 cycles). (C) Schematic illustration of the thermal responsiveness of NPLs. Repetitive heating-cooling cycles of NLPs showed an increase in derived count rate as temperature increased above Tcp due to the dehydration and collapse of ELP chains, forming a compact vesicle shell. (Figure 4-A) Repetitive heating (n=3) to 37 °C and cooling 20 °C cycles further confirmed the reversibility of the hydration and dehydration process at physiological and room temperatures. (Figure 4-B) After confirming thermoresponsiveness of the ELP[M(Oleyl)1V3-40] membrane, the effect of temperature on membrane permeability was subsequently evaluated. Self-quenched fluorescein was loaded in NLPs via film hydration in 10 mM fluorescein solution over 48 h. Excess dye was removed by Sephadex G-100 size-exclusion chromatography and the presence of vesicles was confirmed by DLS and TEM measurements. (Figure 5-A)

Experimental

  Procedures .......................................................................................................................................................... 1. Materials and methods. ............................................................................................................................................................ 2. Synthetic procedures ................................................................................................................................................................ 2.1 Synthesis of oleyl azide .......................................................................................................................................................... 2.2 Synthesis of ELP[M(Oleyl)1V3-40] .......................................................................................................................................... 3. Characterization of the temperature responsive behaver of the ELP[M(Oleyl)1V3-40] bioconjugate ....................................... 3.1 Differential scanning calorimetry (DSC) measurement .......................................................................................................... 3.2 Dynamic light scattering (DLS) measurement ...................................................................................................................... 4. Thin film hydration .................................................................................................................................................................. 4.1. General procedures for thin film hydration on glass beads. ................................................................................................ 4.2. Fluorescein loading into NPLs ............................................................................................................................................. 5. Fluorescent labeling ............................................................................................................................................................... References ................................................................................................................................................................................ Author Contributions ...............................................................................................................................................................

Table 1 .

 1 Characteristics of ELP derivatives. Number average molar mass (Mn, g.mol -1 ) and polydispersity index (Đ) were determined by SEC. Dextran used as the standard and dimethysulfoxide (DMSO + lithium bromide LiBr 1 g.L -1 ) was used as an eluent.[b] Theoretical molar mass (MW, g.mol -1 ) [c] Grafting yield determined by 1 H NMR (%) [d] Product yield of reaction (%)

	Compound	Mn [a]	Đ [a]	MW [b]	Yield [c]	Yield [d]
	ELP[M1V3-40]	14,930	1.09	17,035	-	-
	ELP[M(Alkyne)1V3-40]	10,270	1.23	18,917	100	91
	ELP[M(Oleyl)1V3-40]	12,520	1.44	23,957	100	64
	[a]

Acknowledgements

For financial support EuroNanoMed3 TEMPEAT (ANR-17-ENM3-0009) and SIRIC BRIO (INCA, COMMUCAN project) are gratefully acknowledged. Authors wish to thank Bertrand Garbay and Guillaume Goudounet for gene design, cloning and production of ELP[M1V3-40]. We gratefully acknowledge Amelie Vax and Anne-Laure Wirotius from LCPO for SEC and NMR analyses, respectively. Continuous support from Univ. Bordeaux, CNRS and Bordeaux-INP is acknowledged.

SUPPORTING INFORMATION

Supporting Information

Thermosensitive vesicles from chemically encoded lipidgrafted elastin-like polypeptides

Experimental Procedures

Materials and methods.

The following reagents were used as received. Copper(II) sulfate pentahydrate (CuSO4•5H2O, 99%) and anhydrous sodium sulfate (Na2SO4, 99.5%), sodium azide (NaN3, 99.5%), sodium sulfate (Na2SO4, anhydrous, 99.0%) and 5-Carboxy-Xrhodamine N-succinimidyl ester were purchased from Sigma-Aldrich. (+)-sodium L-ascorbate (C6H7NaO6, 99%) was obtained from Alfa Aesar. Cis-1-chloro-9-octadecene (oleyl chloride, C18H35Cl, 70%) and N,N-Diisopropylethylamine (DIPEA, 99.0%) were purchased from TCI. Fluorescein disodium salt dehydrate was purchased from Fluka. Cuprisorb® was purchased from Seachem.

The following solvents were used without additional purification: Methanol (MeOH, analytical grade, 100%, VWR chemicals), ethanol (EtOH, absolute, 99.9%, VWR chemicals), N-N-Dimethylformamide (DMF 99.9%, HPLC grade, VWR chemicals), diethyl ether (Et2O, 97%, VWR chemicals), dimethyl sulfoxide (DMSO, 99.9%, Sigma-Aldrich) and chloroform (CHCl3, HPLC grade, 99.8%, Sigma-Aldrich). The following deuterated solvents were purchased from Sigma-Aldrich and used without additional purification: Trifluoroacetic acid-d (TFA-d, 99.5 atom % D) and chloroform-d (CDCl3-d, 99.8 atom % D). Ultrapure water (18 MΩ•cm) was obtained by using a Millipore Milli-Q Biocel A10 purification unit. Spectra/Por Regenerated Cellulose (RC) and Float-A-Lyzer G2 dialysis tubes were used for the purification of ELP-conjugate. Glass beads (0.5 and 5 mm) were purchased from Sigma-Aldrich. ELP production: ELP[M1V3-40] was produced recombinantly in Escherichia coli bacteria and purified by inverse transition cycling following established procedures. [1][2] Nuclear Magnetic Resonance (NMR): 1 H NMR spectra were recorded with a Bruker AVANCE III HD (Liquid-state 400 MHz NMR spectrometer with 5 mm BBFO probe). Deuterated chloroform (CDCl3, Euriso-top, 99.8%), and deuterated water (D2O, Euriso-top, 99.8%) were used as solvents and references for the lock. Bruker Topspin Software was used for data treatment.

Size Exclusion Chromatography (SEC):

Analyses were performed on an Ultimate 3000 system from Thermoscientific equipped with a diode array detector (DAD). The system from Wyatt technology also includes a multi-angle light scattering detector (MALS) and a differential refractive index detector (dRI). Polypeptide conjugates were separated on two TOSOH successive columns (one G4000PWXL (7.8*300) column with exclusion limits from 2,000 Da to 300,000 Da and one G3000PWXL (7.8*300) column with exclusion limit below 40,000 Da). Analyses were performed at a flow rate of 0.5 mL•min -1 and columns temperature was held at 80 °C. Dextran (PSS) was used as the standard. Dimethysulfoxide (DMSO, lithium bromide LiBr 1 g•L -1 ) was used as eluent and ethylene glycol was used as a flow marker.

Differential scanning calorimetry (DSC):

In a dry state, measurement of 10 mg sample was performed using a Q 100-RCS apparatus from TA Instruments over temperature range from -80 to 120 °C, in a heating cooling mode at 5 °C•min -1 . The analysis was carried out in a nitrogen atmosphere with aluminum pans. In a liquid state, measurements were carried out using high pressure crucibles filled with 30 µL of solution sample at a concentration of 10 or 100 mg•mL -1 in water. The temperature range was 5 to 50 °C with a heating rate of 2 °C•min -1 . Sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS-PAGE) analysis: Precision Plus Protein TM Standards (BIO-RAD, unstained) was used as size marker. 20 μL of sample were loaded in gel wells (BIO-RAD, 4-20% Mini-PROTEAN® TGX™ stain-free gel). Tris-Glycine-SDS Buffer (BIO-RAD, TGS 1x) was used as loading buffer. Laemmli Sample Buffer (BIO-RAD, 2x) was used as a running buffer.

Dynamic light scattering measurements (DLS):

The measurements were performed on a NanoZS 90 instrument (Malvern, UK) at a 90° angle and at a constant position in the cuvette (constant scattering volume) at different temperatures. The derived count rate (DCR) was defined as the mean scattered intensity normalized by the attenuation factor. Imaging of giant lipo-proteinosomes: Giant lipo-proteinosomes (GLPs) were collected and imaged in an Ibidi chamber slide (µ-Slide VI 0.4, Ibidi) by using confocal laser scanning microscopy and epifluorescence microscopy. Confocal images were acquired by a confocal laser scanning microscopy (Leica, SP5 AOBS) through an HCX PL APO 63x oil immersion objective. A diode laser (561 nm) was used to excite rhodamine that is labelled on the ELP backbone. Bright field and epifluorescence microscopy images of GLPs were taken on a Zeiss Axiovert 40 CFL inverted microscope with a 100x oil immersion objective captured with a digital Gigabit Ethernet CCD camera (Viewvorks VG-2M). For epifluorescence microscopy, a mercury lamp was used as source with an excitation and emission filter of narrow band pass window in the red for the rhodamine fluorophore (532 to 544 nm for excitation and 573 to 637 nm for emission). Fluorescence spectrometry: Fluorescence spectra were measured on a JASCO Spectrofluorometer FP-8500 equipped with mini circulation bath (JACOB MCB-100). Light source is 150 W xenon lamp with 1.0 nm wavelength accuracy. The device is equipped with Silicon photodiode (Ex. monochromator) and Photomultiplier (Em. monochromator) detectors. The measurements were taken three times in a 0.5 mL volume quartz cell (2x10 mm light path) and averaged data were used for the final plot. @ABOH?P/Q&'*C!P"==>Q! *",2*#, !?qT!%+<!F:

SUPPORTING INFORMATION %"

SUPPORTING INFORMATION

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Author Contributions

The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. Funding Sources: Euronanomed III TEMPEAT projet (ANR-17-ENM3-0009) and SIRIC BRIO (INCA, COMMUCAN project) are gratefully acknowledged for financial support. Notes: The authors declare no competing financial interest.


J.-Y Wang
email: j.wang3@exeter.ac.uk

J C Mansfield

Sophie Brasselet

Claudio Vergari

Judith R Meakin

Peter C Winlove

J C Mansield

C P Winlove

Micro-mechanical damage of needle puncture on bovine annulus ibrosus ibrils studied using polarization-resolved Second Harmonic Generation (P-SHG) microscopy

Keywords:

Introduction

Intervertebral discs (IVD), which lie between vertebral bodies, play a key role in maintaining the lexibility of the spine. Macroscopically, IVD consist of an inner gel-like nucleus pulposus (NP) and a tough outer annulus ibrosus (AF) which has a concentric layer structure with 7-15 lamellae. AF and NP are mostly composed of collagen, proteoglycan, and 60-80% of water. Any structural damage or mechanical failure can lead to internal disc disruption and result in discogenic pain which has been recognised as one of the main causes of low back pain [START_REF] Izzo | Spinal pain[END_REF][START_REF] Ehrlich | Low back pain[END_REF]. Mechanical injuries can cause strength reduction and delamination of the annulus ibrosus and depressurisation of the nucleus pulposus [START_REF] Iatridis | Localized intervertebral disc injury leads to organ level changes in structure, cellularity, and biosynthesis[END_REF]. Localised mechanical injuries can lead to organ-wide disc damage which results from a disruption in the biochemical balance between cells, extracellular matrix, and disc mechanical stress [START_REF] Iatridis | Localized intervertebral disc injury leads to organ level changes in structure, cellularity, and biosynthesis[END_REF][START_REF] Vergroesen | Mechanics and biology in intervertebral disc degeneration: a vicious circle[END_REF]. Furthermore, the accumulation of mechanical injuries leads to long-term progressive dysfunction and alteration of biosynthesis in IVD at the macroscopic level [START_REF] Iatridis | Localized intervertebral disc injury leads to organ level changes in structure, cellularity, and biosynthesis[END_REF]. One of the suspected causes of acute injuries is needle puncture which can potentially lead to mild or moderate disc degeneration and low back pain in the long term [START_REF] Iatridis | Does needle injection cause disc degeneration? News in the continuing debate regarding pathophysiology associated with intradiscal injections[END_REF][START_REF] Cuellar | Does provocative discography cause clinically important injury to the lumbar intervertebral disc? A 10-year matched cohort study[END_REF][START_REF] Manchikanti | Systematic Review of lumbar discography as a diagnostic test for chronic low back pain[END_REF][START_REF] Carragee | A gold standard evaluation of the "discogenic pain" diagnosis as determined by provocative discography[END_REF]. Needles are used in spinal diagnostic procedures, such as discography [START_REF] Bernard | Lumbar discography followed by computed tomography[END_REF]Guyer et al., 2003), spinal disc repair, and in potential treatments such as electrothermal therapy [START_REF] Kleinstueck | Acute biomechanical and histological intradiscal electrothermal therapy on human lumbar discs[END_REF][START_REF] Cohen | Nucleoplasty with or without intradiscal electrothermal therapy (IDET) as a treatment for lumbar herniated disc[END_REF][START_REF] Pollintine | Intradiscal electrothermal therapy can alter compressive stress distributions inside degenerated intervertebral discs[END_REF][START_REF] Tsou | Intradiscal electrothermal therapy in the treatment of chronic low back pain: experience with 93 patients[END_REF]. The use of needles has been found to accelerate disc degeneration and create progressive degeneration with an increasing number of cell deaths surrounding the puncture site [START_REF] Masuda | A novel rabbit model of mild, reproducible disc degeneration by an annulus needle puncture: correlation between the degree of disc injury and radiological and histological appearances of disc degeneration[END_REF][START_REF] Sobajima | A slowly progressive and reproducible animal model of intervertebral disc degeneration characterized by MRI, X-ray, and histology[END_REF][START_REF] Korecki | Needle puncture injury affects intervertebral disc mechanics and biology in an organ culture model[END_REF]. Using different sizes of needles can result in different internal damage [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]Keorochana et al., 2010;[START_REF] Michalet | The effects of needle puncture injury on microscale shear strain in the intervertebral disc anulus ibrosus[END_REF]. For example, the use of small needles can cause the reduction of the disc height and nucleus pressurization while using larger needles can disrupt annular function and lower disc stiffness [START_REF] Michalet | The effects of needle puncture injury on microscale shear strain in the intervertebral disc anulus ibrosus[END_REF][START_REF] Korecki | Needle puncture injury affects intervertebral disc mechanics and biology in an organ culture model[END_REF].

Due to the fact that the damage caused by annular puncture can lead to alterations of biochemical compositions in NP such as water content and collagen, many studies have been done to understand the biomechanics, function, and composition of AF at the microscopic level [START_REF] Iatridis | Localized intervertebral disc injury leads to organ level changes in structure, cellularity, and biosynthesis[END_REF]. Many microscopic techniques have been used, such as light microscopy [START_REF] Michalet | The effects of needle puncture injury on microscale shear strain in the intervertebral disc anulus ibrosus[END_REF][START_REF] Kobielarz | Qualitative and quantitative assessment of collagen and elastin in annulus ibrosus of the physiologic and scoliotic intervertebral discs[END_REF], X-ray diffraction [START_REF] Scott | Proteoglycan-ibrillar collagen interactions[END_REF][START_REF] Klein | X-ray diffraction demonstrates reorientation of collagen ibres in the annulus ibrosus during compression of the intervertebral disc[END_REF][START_REF] Hickey | Aging changes in the macromolecular organization of the intervertebral disc: an X-ray diffraction and electron microscopic study[END_REF], electron microscopy (Hickey andHukins, 1981, 1982;[START_REF] Hayes | Collagen ibrillogenesis in the development of the annulus ibrosus of the intervertebral disc[END_REF][START_REF] Iatridis | Mechanisms for mechanical damage in the intervertebral disc annulus ibrosus[END_REF][START_REF] Tavakoli | The ultra-structure organization of the elastic network in the intra-and inter-lammellar matric of the intervertebral disc[END_REF][START_REF] Nosikova | Characterization of the annulus ibrosus-vertebral body interface: identiication of new structural features[END_REF], differential interference contrast optical microscopy [START_REF] Pezowicz | Intralamellar relationships within the collagenous architecture of the annulus ibrosus imaged in its fully hydrated state[END_REF][START_REF] Schollum | A microstructural investigation of intervertebral disc lamellar connectivity: detailed analysis of the translamellar bridges[END_REF][START_REF] Schollum | A microstructural investigation of intervertebral disc lamellar connectivity: detailed analysis of the translamellar bridges[END_REF][START_REF] Brown | Staying connected: structural integration at the intervertebral disc-vertebra interface of human lumbar spines[END_REF], and second harmonic generation microscopy (SHG) for collagen [START_REF] Vergari | Lamellar and ibre bundle mechanics of the annulus ibrosus in bovine intervertebral disc[END_REF](Vergari et al., , 2017a(Vergari et al., , 2017b;;[START_REF] Reiser | Quantitative analysis of structural disorder in intervertebral disks using second harmonic generation imaging: comparison with morphometric analysis[END_REF]. While many successes have been made using those techniques, SHG has the advantages of imaging collagen without any staining process [START_REF] Bueno | Second harmonic generation microscopy: a tool for quantitative analysis of tissues[END_REF] and enabling the study of collagen ibre orientation [START_REF] Brasselet | Polarization-resolved nonlinear microscopy: application to structural molecular and biological imaging[END_REF][START_REF] Ávila | Polarization dependence of aligned collagen tissues imaged with second harmonic generation microscopy[END_REF]. The working principle of SHG is based on the simultaneous excitation of the sample by two photons of the same frequency, with instantaneous emission of a single photon at twice the frequency [START_REF] Cox | Second-harmonic imaging of collagen[END_REF]. Collagen is a strong source of SHG signal, and collagen ibres can be imaged in tissue without the need of any staining process [START_REF] Chen | Second harmonic generation microscopy for quantitative analysis of collagen ibrillar structure[END_REF]. Moreover, the SHG emission from the sample depends on the alignment of the collagen molecules relative to the incident light polarization, where the strongest signal occurs when the molecules are aligned with the incident polarization [START_REF] Brasselet | Polarization-resolved nonlinear microscopy: application to structural molecular and biological imaging[END_REF][START_REF] Anceau | Local orientational distribution of molecular monolayers probed by nonlinear microscopy[END_REF]. This polarization-resolved (or polarization-dependent) SHG has further enabled the analysis on the orientation of collagen and used to study the proportion of disorganised collagen in the tissue [START_REF] Brasselet | Polarization-resolved nonlinear microscopy: application to structural molecular and biological imaging[END_REF][START_REF] Ávila | Polarization dependence of aligned collagen tissues imaged with second harmonic generation microscopy[END_REF]. Furthermore, two-photon luorescent (TPF) signals are often used to image cells while simultaneously acquiring the SHG signal from collagen [START_REF] Mansield | Collagen iber arrangement in normal and diseased cartilage studied by polarization sensitive nonlinear microscopy[END_REF][START_REF] Mansield | The elastin network: its relationship with collagen and cells in articular cartilage as visualized by multiphoton microscopy[END_REF][START_REF] Matcher | What can biophotonics tell us about the 3D microstructure of articular cartilage?[END_REF][START_REF] Reed | Two-photon luorescence and second harmonic generation characterization of extracellular matrix remodeling in post-injury murine temporomandibular joint osteoarthritis[END_REF][START_REF] Bowles | Self-assembly of aligned tissue-engineered annulus ibrosus and intervertebral disc composite via collagen gel contraction[END_REF][START_REF] Dittmar | Intervertebral Disc Visualization by Combined Two-Photon Excitation Fluorescence and Second-Harmonic Generation Microscopy[END_REF]. TPF is also a multiphoton imaging technique and is based on the absorption of two photons followed by the emission of a single photon at a shifted frequency with respect to SHG. As a result of the combined system, SHG-TPF microscopy can provide comprehensive tissue information on the extracellular matrix and cells, and therefore stands out as a very powerful investigative tool [START_REF] Lacomb | Quantitative second harmonic generation imaging of the diseased state osteogenesis imperfecta: experiment and simulation[END_REF][START_REF] Campagnola | Second harmonic generation imaging microscopy: applications to diseases diagnostics[END_REF].

This work aimed to investigate the short-term progressive damage caused by needle puncture to the AF, and quantify how this damage is affected by needle size. It is still unclear how a localised needle puncture can induce organ-wide disc degeneration clinically. Based on our previous study, punctures produced no signiicant changes in elastic properties of AF strips at a relatively large distance from the puncture site (4-6 times the needle diameter) even using different size of needles (Vergari et al., 2017a). In the current study, polarization-resolved SHG combined with TPF microscopy was exploited to study collagen ibres and cell nuclei in the outer AF surrounding the punctures to investigate local microscopic damage. The progressive mechanical damage after needle puncture was also investigated by applying cyclic loadings axially to imitate the effects of spinal motion.

Materials and methods

Sample preparation, cell staining

Twenty-nine spinal motion segments (an intervertebral disc and two vertebral bodies) from ten cow tails were obtained from a local abattoir. Tails were frozen at -20 • C on the day of death and thawed a few hours before testing at room temperature, with maximal frozen time of less than two months. Each motion segment was carefully dissected to remove muscles and ligaments. Intervertebral disc height and diameter were measured using callipers. A thin layer of petroleum jelly was applied on the surface of the outer annulus ibrosus and endplates except for an area of less than 5 × 5 mm 2 on outer AF, which was left exposed for imaging.

Prior to imaging, a few drops of propidium iodide solution with concentration 1 mg/mL in phosphate-buffered saline (Propidium Iodide 10 mg, Sigma-Aldrich, UK) was applied on the 5 × 5 mm 2 imaging area to stain the cell nuclei in the outer annulus ibrosus. Samples were then covered with cling ilm and aluminium foil and left in a 37 • C incubator for 40 min. After the staining, disc samples were rinsed with PBS to remove excess propidium iodide on the sample surface.

Tensile loading and needle puncture

A custom-built micro-straining rig was utilized to test the samples as described in our previous study (Vergari et al., 2017a). In brief, the rig is motorized with a drive and the driving distance is set at 17 μm per step.

The sample was mounted in the rig (Fig. 1(a)), and it was loaded with the protocol shown in Fig. 1(b). Each sample was strained between -5% and +5% in 1% steps at 5 mm/min (corresponding to 0.65% strain/s). Each cycle lasted between 18.4 s and 30.8 s (with frequency range between 0.033 and 0.054 Hz) corresponding to the disc thickness 7.65 mm-12.85 mm. Polarized SHG images were acquired after each 1% strain step, with the method described below, while the strain was kept constant. The sample was then unloaded, punctured with the needle radially into the nucleus pulposus using the protocol described below, and then loaded again between -5% and +5% with an image acquired at each 1% loading step. Finally, 70 cyclic loadings were performed, with one SHG image acquired every 10 cycles. Images from 1% loading steps were not included in this study and will leave for future work in comparison with our previous studies in [START_REF] Vergari | Lamellar and ibre bundle mechanics of the annulus ibrosus in bovine intervertebral disc[END_REF]2017).

Four different needles (Fig. 1(c)) were used to puncture the discs: two hypodermic needles with diameters of 0.5 mm (25G) and 0.8 mm (21G) (Birmingham Gauge) [START_REF] Yentis | Anaesthesia and Intensive Care A-Z E-Book: an Encyclopedia of Principles and Practice[END_REF], and two spinal needles with diameters of 0.7 mm (22G) and 0.9 mm (20G) (BD™ Quinke Spinal Needles, UK). The tips of hypodermic needles have a smaller bevel angle (30 • ) than the tips of the spinal needles (45 • ). Punctures were approximately located in the centre of each discs. The exact circumferential position of the puncture sites were not recorded in this study and the locations could vary. The openings of needle tips were randomly aligned. The puncture scheme was shown in Fig. 1(d) and (e) where the needle punctured radially and applied loading axially. Each needle type was applied to ive to six discs, and an additional eight discs without puncture formed a control group. Each disc sample was rinsed once using a few drops of PBS solution on the surface of the 5 × 5 mm 2 imaging area prior to the puncture. The acquisition time for each images was approximately 2 min and 34 s. For control group, the whole protocol lasted within 2.5 h. For needle groups, the whole protocol lasted less than 3.5 h with less than 2.5 h just before the puncture and after a few drops of PBS being applied on the imaging area.

The effect of dehydration could affect the collagen behaviour. Although the outer annulus and endplates were covered with petroleum jelly except an 5 × 5 m 2 area, while two vertevrate were also exposed to the air. Force-distance curves were recorded (as force-distance) and compared the changes between the initial and last cyclic loadings within total time period of 2.5 h using the sample protocols mentioned above.

Multiphoton imaging

The second-harmonic generation imaging system was set up as previously described [START_REF] Vergari | Lamellar and ibre bundle mechanics of the annulus ibrosus in bovine intervertebral disc[END_REF]. Both SHG and TPF signals were collected to visualise collagen and cell nuclei, respectively. An 810 nm mode-locked femto-second Ti:Sapphire laser (Mira 900-D, Coherent Inc.) with a repetition rate of 76 MHz and a pulse width of 100 fs pumped by a 532 nm solid-state laser (Verdi V10, Coherent Inc.) was used as an illumination source. A polarization half-wave plate (WPH05M-488, Thorlabs, Newton, NJ) was mounted on a motorized rotation stage (PR50CC, Newport, Irvine, CA) and the polarization angle was rotated from 0 • to 162 • relative to the laser polarization at 18 • steps which generated a set of ten SHG and TPF images. A confocal microscope (Olympus Fluoview BX51, OLYMPUS Corp., USA) itted with a 10x/0.4NA air objective (UPlanSApo, OLYMPUS Corp.le, USA) was used to collect both SHG and TPF signal in the backwards direction. The air objectives was used due to it can give the largest imaging areas or ield of view, compared to other water or oil objectives designed for magniication higher than 20x. The TPF and SHG signals were separated using a dichroic beam splitter (Semrock Di02-R405), and detected using PMTs (R3896 Hamamatsu Japan). At the SHG PMT the following ilters were used (blue green glass ilter CG-BG-39 CVI laser and 405 nm band pass ilter, FF01-405/10-25 Semrock) and at the TPF PMT a 655 nm band pass ilter (FF01-655/40-25 Semrock, IDEX Health & Science, LLC, USA) was used. Disc samples were itted on the rig and placed under the objective, as shown in Fig. 1(a). Images were mainly collected at two different sizes: 800 × 600 pixels (1600 μm × 1200 μm, for needle group 22G, 21G, 20G) with each pixel 2 μm × 2 μm or 512 × 512 pixels (1413 μm × 1413 μm, control group and needle group 25G, 21G) with each pixel size 2.76 μm × 2.76 μm due to puncture shape being more rounded for the 25G needle group and more elongated for three samples in the 21G needle group. The damage on one sample in 20G (0.9 mm diameter) created a much larger size of puncture and thus the image was taken as 1024 × 1024 pixels (1.86 μm × 1.86 μm).

Polarization-resolved SHG and image analysis

The SHG signal results from how the incident light interacts with non-centrosymmetric assemblies of molecules, in which each molecule can be seen as a dipole radiating at the exactly twice the frequency of the incident light. The polarization dependence of the SHG intensity can be deduced from a theoretical distribution of nonlinear induced dipoles, supposing a coherent superposition of all dipoles in the focal spot of the excitation objective [START_REF] Brasselet | Polarization-resolved nonlinear microscopy: application to structural molecular and biological imaging[END_REF]. Under the dipolar approximation that holds within the size of the diffraction limit size, each nonlinear contribution can be summed up coherently [START_REF] Brasselet | Polarization-resolved nonlinear microscopy: application to structural molecular and biological imaging[END_REF]. SHG from a single molecule (or peptide bond in the case of collagen) is the result of the radiation from the induced individual dipole being linearly polarized along an angle α with respect to X in the sample plane (X, Y) [START_REF] Brasselet | Polarization-resolved nonlinear microscopy: application to structural molecular and biological imaging[END_REF]. At the ensemble level, these dipoles radiate coherently within the focal volume, which results in a macroscopic nonlinear induced dipole P SHG [START_REF] Mansield | Collagen reorganization in cartilage under strain probed by polarization sensitive second harmonic generation microscopy[END_REF]. The measured SHG intensity I SHG can be deduced from the resulting radiated ield which leads to a paraxial approximation

I SHG ∝|P SHG | 2 ∝|E (α)| 4
, where E(α) is the incident radiation ield. SHG intensity can thus be decomposed in contributions of different harmonics [START_REF] Mansield | Collagen reorganization in cartilage under strain probed by polarization sensitive second harmonic generation microscopy[END_REF])

I SHG (α) ∝ a 0 + a 2 cos 2 α + b 2 sin 2 α + a 4 cos 4 α + b 4 sin 4 α (1)
The coeficients can be grouped into amplitude (I 2 , I 4 ) and phase coeficients (ϕ 2 , ϕ 4 ) of different orders (2nd or 4th) of symmetry:

I SHG (α) ∝ a 0 + I 2 cos 2(α -ϕ 2 ) + I 4 cos 4(α -ϕ 4 ) (2)
With: These parameters are related to how the nonlinear induced dipoles from collagen molecules distribute in the sample plane, which is directly related to the isotropic organization of collagen ibre [START_REF] Duboisset | Generic model of the molecular orientational distribution probed by polarization-resolved second-harmonic generation[END_REF][START_REF] Mansield | Collagen reorganization in cartilage under strain probed by polarization sensitive second harmonic generation microscopy[END_REF][START_REF] Tilbury | Differentiation of Col I and Col III isoforms in stromal models of ovarian cancer by analysis of second harmonic generation polarization and emission directionality[END_REF][START_REF] Aït-Belkacem | Microscopic structural study of collagen aging in isolated ibrils using polarized second harmonic generation[END_REF][START_REF] Deniset-Besseau | Measurement of the second-order hyperpolarizability of the collagen triple helix and determination of its physical origin[END_REF]. The second-order parameters (I 2 , ϕ 2 ) represent the magnitude and orientation of the anisotropic contribution to the polarization response. i.e. the degree of the order and the overall alignment of the collagen molecules. Higher values of I 2 represent more anisotropic distribution of collagen molecules, i.e. more orientally ordered or more tightly aligned collagen molecules and thus more organised collagen ibrils within one focal spot [START_REF] Duboisset | Generic model of the molecular orientational distribution probed by polarization-resolved second-harmonic generation[END_REF][START_REF] Mansield | Collagen reorganization in cartilage under strain probed by polarization sensitive second harmonic generation microscopy[END_REF][START_REF] Tilbury | Differentiation of Col I and Col III isoforms in stromal models of ovarian cancer by analysis of second harmonic generation polarization and emission directionality[END_REF][START_REF] Aït-Belkacem | Microscopic structural study of collagen aging in isolated ibrils using polarized second harmonic generation[END_REF][START_REF] Deniset-Besseau | Measurement of the second-order hyperpolarizability of the collagen triple helix and determination of its physical origin[END_REF]. The fourth-order parameters (I 4 ,ϕ 4 ), which are not exploited in this work, are the magnitude and orientation signatures of a more reined higher-order dependence which provides information about the shape of the distribution (Gaussian, cone, or cone surface) and identiies the effect of birefringence [START_REF] Duboisset | Generic model of the molecular orientational distribution probed by polarization-resolved second-harmonic generation[END_REF][START_REF] Mansield | Collagen reorganization in cartilage under strain probed by polarization sensitive second harmonic generation microscopy[END_REF].

I 2 = ̅̅̅̅̅̅̅̅̅̅̅̅̅̅ ̅ a 2 2 + b 2 2 √ a 0 , I 4 = ̅̅̅̅̅̅̅̅̅̅̅̅̅̅ ̅ a 2 4 + b 2 4 √ a 0 , ϕ 2 = 0.5 tan -1 ( b 2 a 2 ) , ϕ 4 = 0.25 tan -1 ( b 4 a 4 ) (3)
In this work, we focused on the degree of order of the collagen molecules, to identify how the collagen ibril organization affected by needle puncture. By calculating the coeficients (a 0 , a 2 , b 2 ), I 2 value was derived from this generic model at each pixel. A custom programme was written using commercial software (MATLAB, 2017b; Mathwork, USA) to calculate the parameters and I 2 was normalised by the total number of pixels. I 2 distribution was plotted as histograms and itted by two Gaussian distribution curves to provide two mean values μ 1 and μ 2 . This is due to the apparent non-normal distributions observed, for which a bi-Gaussian function corresponds to the lowest order itting basis [START_REF] Stanciu | Bi-Gaussian itting of skewed peaks[END_REF]. 200 intervals were set to I 2 distribution histograms from 0 to 0.8 with 0.0004 in each interval. 900 iterations were set to obtain the best itting curves using the MATLAB. The mean I 2 , μ 1 and μ 2 values, and their changing rates with cyclic loadings were calculated.

The other two parameters, the standard deviations (σ 1 , σ 2 ) and the mixing proportions (P 1 , P 2 ), were listed in Supported Material SI2 for all the samples at cycle 0, 30 to cycle 70.

Statistics

I 2 , μ 1 and μ 2 values calculated from polarized-SHG results were taken average from whole image, and separated in different needle groups. The mean ± standard deviation were plotted for each needle group. The changing rates versus cyclic loadings of I 2 , μ 1 and μ 2 values were taken as the slopes of the itted linear curve. The mean ± standard deviation of the changing rates are plotted for each needle group. The Kruskal-Wallis tests were performed to compare the I 2 , μ 1 , μ 2 values and their slopes against cycles between needle groups at 5% signiicance level. I 2 , μ 1 , μ 2 values and their slopes were also normalised by their values from cycle 0, in order to eliminate the contribution of damage from dissections. Pearson correlation coeficients weer also calculated to obtain the linear correlations between needle size and the averaged I 2 , μ 1 , μ 2 values. The Kruskal-Wallis tests were also performed to compare two mixing proportions P 1 and P 2 (both non-normalised and normalised values by cycle 0) against needle sizes at 5% signiicance level and Pearson correlation coeficients were calculated to obtain their linear correlations with the cycle number. Percent overlap of I 2 histogram was calculated against cycle number and Kruskal-Wallis tests were also performed between groups. Pearson correlation coeficients were calculated to obtain the linear correlation between percent overlap and cycle number.

Change in puncture length and width were compared after the puncture (cycle 0) and after the cyclic loadings (cycle 70). This is to compare the effect of applied loadings on puncture size changes. The length and width were chosen as along the perpendicular or in parallel to the loading direction, respectively. The change of percentage in length (ΔL) was deined in this study as the maximum length difference between cycle 0 (L0) and cycle 70 ((L7) devided by L0, ie, ΔL=(L7-L0)/ L0. The change of percentage in width (Δw) was deined as the maximum width found in cycle 0 (w0) and the change the width in the same position after cycle 70 (w7) and devided by w7, ie, Δw=(w7-w0)/ w0. The mean and standard deviation for each needle group were calculated. The Kruskal-Wallis tests were performed among and between each needle group, where the change in length and width were compared between each group. Pearson correlation coeficients were calculated to obtain the correlation between needle sizes and percentage changes in puncture sizes (width and length).

Cell length (l) to diameter (r) ratios were calculated and compared between an unpunctured and a punctured sample near the puncture site from the SHG images. l to r ratio of the cells from the whole images were taken average and the standard deviation were calculated.

Results

Needle puncture damage on outer annulus ibrosus

Fig. 2 shows the SHG images from forty samples (with eight samples in each group, including the control group and four needle groups), at the puncture site immediately after the puncture and before cyclic loading. The shape and size of the puncture varied greatly even using the same needle; with puncture dimensions ranging between 200 and μm. Overall, damage from the puncture included both cutting and splitting of collagen bundles, where collagen ibres were cut through or pushed aside, with bundles being torn apart. From the observation, the shape of the puncture can be classiied into elongated cracks along the ibre orientation (25G-2, 21G-3), round holes (25G-4, 22G-2 21G-1), or a combination of both. Furthermore, some puctures can be seen with part of the surrounding ibres being pushed into (22G-1,3:6, 21G-2,4:6, 20G-2,4:5,7:8) or lifted (21G-2:5) from the sample surface.

Comparing the damage before and after cyclic loadings, i.e. cycle 0 and cycle 70, changes in shape and size of the puncture were observed as shown in the representative samples in Fig. 3(a). Those representative samples have apparent puncture damage of both tearing and cutting. Among them, some punctures expanded over time (25G-6, 20G-8) while some appeared to shrink (22G-7, 21G-5). Changes in lengths (L) and widths (w) of the puncture before and after 70 cyclic loadings (cycle 0: L0, w0, cycle 70: L7, w7) from four needle groups were plotted in Fig. (b-c), respectively. A few samples were not taken into account (G25-6, G22-1, G22-4, G21-1, G21-4, G20-3, G20-5) due to part of the tissue were lifted up towards the end of 70 cycles rather than shrinking or expanding in puncture size. The results from the Kruskal-Wallis tests showed that no signiicant difference was found between Δw and ΔL within each needle group where p-values = 0.564 (25G), 0.602 (22G), 0.513 (21G), 0.756 (20G). Between needle groups, there was no signiicant difference in change of length (ΔL) where the p value p(ΔL) = 0.116, and width (Δw) where p(Δw) = 0.402 within 5% conidence. Negative correlation was found between needle size and ΔL with correlation coeficient -0.539 while positive correlation was found between needle size and Δw with correlation coeficient 0.648. For all those 24 samples, the average length change ΔL was -13.62 ± 16.11% (Mean ± Std Dev) and width change Δw was -16.60 ± 15.21% (Mean ± Std Dev). There were 1 samples out of 24 expanding in width, and samples expanding in length. These indicated that the majority of the punctures tend to shrink and the applied cyclic loadings caused 3% less shrinkage in length which is perpendicular to the loading compared to in width. SHG images from all the samples taken at cycle 0, cycle 30, and cycle 70 are provided in supported material SI1.

Dehydrations can affect the mechanical properties of collagen. The force-distance curves (cycle 1, 2-10, 61-70) from a representative sample in control, needle groups (25G and 20G) were plotted in Fig. (d-f). Some intervals appeared in cycle 1 with relaxations due to the imaging at 1% steps. Comparing cycle 2 and cycle 70, forces changed between -10.4% and -16.2% from Fig. 3(d-f). From all samples, on average, forces changed -10.2 ± 3.14% for control group, and changed -14.5 ± 3.5%, -17.2 ± 2.2%, -14.4 ± 2.5%, -18.9 ± 3.4% for needle groups 25G-20G, respectively.

Polarized-SHG image analysis

Fig. 4 shows I 2 mapping from the same representative samples shown in Fig. 3 at cycle 0, 30 and 70. I 2 value ranges from 0 to 0.8 where the higher I 2 represents more ordered collagen molecules present in each pixel, i.e. more collagen molecules are aligned to the same orientation. Comparing the needle group with the control group, the change in I 2 over cyclic loading, indicate that collagen ibril organization decreased, especially in the regions surrounding the puncture, while little change was observed in the control group, even with surface dissected and exposed to the air.

Fig. 5 shows the histogram of I 2 from the images in Fig. 4, after cycle 0, 30 and 70, normalised by the total number of pixels. Shifts in overall I 2 values over cyclic loadings can be observed from the needle groups compared to the control group. For each I 2 distribution, non-normal distributions were observed and two Gaussian curves were itted as shown in the inset of Fig. 5(d ande: Cyc 70) with two mean values μ 1 and μ 2 representing a population of more organised collagen and less organised collagen respectively. The itted parameters of those Gaussian distributions, including mean values (μ 1 , μ 2 ) standard deviations (σ 1 , σ 2 ) and mixing proportions (P 1 , P 2 ) were listed in Supported Material SI2.

26 out of 29 samples where μ 1 decreased with increasing cycle number while no obvious trends for the other two parameters in both distributions. Sample 20G-1 had very high μ 2 from cycle 30-70 (>1) but with very low proportion P 2 (<0.1%) which were assumed as single perfect normal distributions with P 2 , μ 2 , σ 2 being disregarded.

The averaged percent overlap of I 2 distribution compared to cycle 0, as illustrated in wavy area in Fig. 5(c), from ive groups were plotted in Fig. 5(f) against cycle numbers. Decreases in percent overlap were observed in all groups ranging from 15% to 25% on average after cyclic loadings. Kruskal-Wallis tests were performed compared control and four needle groups where p=0.361 (>5%), which indicated no signiicant difference found between groups, ie, different needle sizes. Decreasing trends have been observed in all ive groups against cycles, and the correlation coeficients for different group against cycle number were -0.9073 (Control), -0.8427 (25G), -0.970 (22G), -0.788 (21G) and -0.9165 (20G) which indicated strong correlations between the loss of percentage overlap against increasing cycles.

The mean I 2 , μ 1 and μ 2 values from each group are shown in Fig. (a-c) and their normalised values are shown in Fig. 6(d-f). All the values were taken as averages from all the samples over 70 cycles, i.e., the mean values from all samples with eight data points for each sample. Error bars represent the standard deviation from eight samples. For nonnormalised results, on average larger needles caused more damage compared to the smaller needles, and there is an overall decrease of I values with increase of needle size while variations occurred in needle Comparing these ive needle groups, statistical Kruskal-Wallis tests (5% conidence) showed that there were no signiicant differences between these groups in non-normalised I 2 and μ 2 where the p-values were 0.058and 0.0596, while differences could occur in μ 1 where p-value was where p= 0.215, 0.331, 0.134 for I 2 , μ 1 , μ 2 , respectively. This indicated no signiicant differences could be concluded in normalised values between different needle groups. Decreasing trends were observed in those three values against the needle sizes, the correlation coeficients for nonnormalised results against needle sizes (0.5-0.9 mm) were -0.744 (I 2 ), -0.512 (μ 1 ), and -0.810 (μ 2 ); for normalised values the correlation coeficients were -0.903 (I 2 ), -0.876 (μ 1 ) and -0.655 (μ 2 ). These indicated that normalised I 2 and μ 1 showed strong linear correlations with needle size.

Two mixing proportions P 1 and P 2 of those two populations μ 1 and μ 2 showed less obvious trend with increasing cycle numbers or needle sizes (Supported Material 2). No signiicant differences were found among groups (with 5% conidence) where p-values from Kruskal-Wallis tests were 0.380 (non-normalised P 1 ), 0.423 (non-normalised P 2 ), 0.292 (normalised P 1 ) and 0.431 (normalised P 2 ). Weak correlations were also found where Pearson's correlation coeficients between the needle size and non-normalised P 1 was 0.579, between non-normalised P 2 was -0.593, between normalised P 1 was -0.2879, and between normalised P 2 was -0.416. The correlations between cycle numbers varied among needle groups. For non-normalised P 1 , the correlation coeficients between cycle numbers were -0.299 (Control), 0.858 (25G), 0.1263 (22G), -0.336 (21G) and 0.397 (20G); for non-normalised P 2 were 0.291 (Control), -0.863 (25G), 0.051 (22G), 0.285 (21G), and -0.403 (20G). For normalised P 1 , the correlation coeficients between cycle numbers were -0.659 (Control), 0.903 (25G), 0.499 (22G), -0.705 (21G), and 0.758 (20G); for normalised P 2 were -0.052 (Control), -0.809 (25G), 0.768 (22G), 0.440 (21G) and 0.148 (20G).

In order to investigate any progressive damage due to axial cyclic loading, the results of the mean I 2 values taken from the polarized-SHG images were plotted against cycle number, as shown in Fig. 7(a). A linear approximation was itted where the slope was also plotted from one representative sample in each group. The mean slopes of I 2 , μ 1 , μ 2 from all the samples separated to each group were plotted in Fig. 7(b-d) and their normalised values were plotted in Fig. 7(e-g). The correlation coeficients between non-normalised I 2 and cycle numbers were -0.627 (control), -0.893 (25G), -0.851 (22G), -0.978 (21G), -0.924 (20G); and were -0.562 (control), -0.871 (25G), -0.518 (22G), -0.974 (21G) and -0.924 (20G) for normalised I 2 . The correlation coeficients between non-normalised μ 1 and cycle numbers were -0.588 (control), -0.829 (25G), -0.518 (22G), -0.937 (21G), -0.939 (20G); and were -0.582 (control), -0.835 (25G), -0.644 (22G), -0.936 (21G) and -0.934 (20G) for normalised μ 1 . The correlation coeficients between non-normalised μ 2 and cycle numbers were -0.055 (control), -0.962 (25G), -0.955 (22G), -0.925 (21G), -0.798 (20G); and were -0.039 (control), -0.952 (25G), -0.935 (22G), -0.913 (21G) and -0.934 (20G) for normalised μ 2 . For both normalised and non-normalised results, I 2 , μ 1 , μ 2 were less negatively correlated to cycle numbers for the control group compared to the needle groups, which indicated the cyclic loadings had less damaging effects on the control group compared to the needle groups.

Overall decreases were observed in both normalised and unnormalised mean I 2 slopes with increasing needle size where the correlation coeficient was -0.664 for non-normalised slopes and -0.603 for normalised slopes. Similar trends of decrease also occurred in nonnormalised μ 1 , μ 2 where correlation coeficients were -0.257 and -0.591; however, the decreasing trends were less obvious in normalised slopes where the correlation coeficient was -0.075 for normalised μ 1 slope and -0.005 between normalised μ 2 slope and needle size. For both normalised and un-normliased slopes, the p-values from Kruskal-Wallis tests were also found much higher than 5% conidence, where p = 0.372, 0.228, 0.610 (for un-normliased I 2 , μ 1 , μ 2 slopes, respectively) and p = 0.713, 0.265, 0.662 (for normalised I 2 , μ 1 , μ 2 slopes respectively). These indicated no signiicant difference was found between groups at 5% signiicance level.

Cell nuclei images

AF collagen ibre bundles and cell nuclei were imaged using SHG and TPF microscopy as shown in Fig. 8(a-c) where images were taken from a representative disc sample punctured by a 25G hypodermic needle at the site of the puncture. Images from a control-group sample are shown in Fig. 8(d-e). In Fig. 8(a) the needle puncture is located at the centre of the image where some rounding in cell shape was found surrounding the puncture in Fig. 8(b) compared to the elongated spindle-shaped cells embedded in unpunctured collagen bundle in Fig. 8(d). Higher magniication of the image in Fig. 8(c) was taken at the corner of the puncture within a square in Fig. 8(b).

This shows the cell nuclei are more irregular, rounder and accumulated near the damage site where the collagen ibre was pushed down

C t r l 2 5 G 2 2 G 2 1 G 2 0 G -4 -2 0 2 I 2 slope 10 -3 C t r l 2 5 G 2 2 G 2 1 G 2 0 G -4 -2 0 2 1 slope 10 -3 C t r l 2 5 G 2 2 G 2 1 G 2 0 G -4 -2 0 2 2 slope 10 -3 0 20 40 60 Cycle number 0 0.1 0.2 0.3 0.4 I 2 Ctrl 25G 22G 21G 20G C t r l 2 5 G 2 2 G 2 1 G 2 0 G -10 -5 0 5 I 2 slope -norm C t r l 2 5 G 2 2 G 2 1 G 2 0 G -10 -5 0 5 1 slope -norm C t r l 2 5 G 2 2 G 2 1 G 2 0 G -10 5 0 5 2 slope -norm (c) (d) (e) (b)
(f) x10 -3 x10 -3 x10 -3 (g) (a)

Discussion

This study aimed to investigate the effect of needle puncture on the outer surface of the annulus ibrosus using SHG. It has previously been found, in animal models, that short-term damage can be effectively restrained when small needles are used, due to the laminated and ibrous structure of the annulus ibrosus [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]. Four different sizes of needles were thus included in this study to investigate the damage to the outer annulus ibrosus. Needle punctures have been suspected to cause long term back pain due to structural damage which can lead to biochemical dysfunction [START_REF] Michalet | The effects of needle puncture injury on microscale shear strain in the intervertebral disc anulus ibrosus[END_REF], cyclic loadings were applied axially in this study to investigate progressive damage surrounding the puncture. Low-frequency cyclic loading was used in this study since it has previously been found to affect delamination and wall distortion in the annulus [START_REF] Schollum | A microstructural investigation of intervertebral disc lamellar connectivity: detailed analysis of the translamellar bridges[END_REF].We had previously studied the damage further away from the puncture site and the results showed that the needle puncture did not cause any changes in the elastic modulus of annulus ibrosus (Vergari et al., 2017a). However, the tests were performed on annulus ibrosus samples which had been cut into strips. In this study, whole motion segments were thus used with disc samples remaining attached to two vertebrae.

Whilst the amount of dehydration was minimised in the experiments by applying petroleum jelly around the AF and endplates, leaving a small window exposed to the air at the puncture site and by rinsing the puncture site with PBS just before puncture, some sample dehydration will have taken place. Whole motion segments were used to give an as realistic as possible puncture and loading protocol, however these could not be maintained in a water bath, and therefore some water may have been lost from the cut ends of the bone. Additionally an air objective, instead of water or oil objective, was used for the imaging to allow a large enough ield of view to cover the whole puncture site, however in doing so moisture could also be lost at the image site. Any dehydration taking place may account for the -10.2% of axial strength loss on average within imaging period 2.5 h on unpunctured. For punctured samples, the axial strength loss was found between -14.4% and -18.9% on average within imaging period of 3.5 h while the on AF imaging area was rinsed in between by PBS just before the puncture. Dehydration could also cause minor bone shrinkage and slippage with reduced diameter [START_REF] Lievers | Specimen size effect in the volumetric shrinkage of cancellous bone measured at two levels of dehydration[END_REF][START_REF] Lievers | Effects of dehydrationinduced structural and material changes on the apparent modulus of cancellous bone[END_REF], while the main dehydration in this study was still assumed to come from the direct imaging area exposed to the air as the dehydration from the vertebrae would require the water to diffuse through the endplates. Needle damage would also cause loss of axial strength in the needle groups [START_REF] Michalek | Needle puncture injury of the rat intervertebral disc affects torsional and compressive biomechanics differently[END_REF][START_REF] Michalek | Height and torsional stiffness are most sensitive to annular injury in large animal intervertebral discs[END_REF][START_REF] Zirbel | Intervertebral disc degeneration alters lumbar spine segmental stiffness in all modes of loading under a compressive follower load[END_REF]. In this study, although not being able to differentiate two factors explicitly from the same samples, axial strength loss in the needle group were 4.2-8.7% greater compared to the control group which could come from the needle puncture.

Due to the geometry of the microscope, to carry out images of the tissue deformation whilst the disc was being punctured was not possible which is one of the main limitations of SHG. It was thus not possible to make any direct comparison before and after the puncture on the same sample surface as it was inevitable to puncture at a different spot. This was also due to the small needle diameters (0.5-0.9 mm) compared to the size of 5 × 5 mm 2 area left for imaging. Otherwise to make direct comparision between the collagen ibre orientations at the site before and after puncture would have further enhanced the study and provided valuable information about how the size and shape of the puncture and the amount of peripheral damage were affected. The SHG imaging was from a single focal plane with a depth of approximately 7 μm, and due to the scattering nature of AF tissue we were only able to image close to the sample surface. Changes out of the focal plane caused by the needle puncture and loading therefore could not be monitored. In addition, the circumferential locations of punctures were not recorded in this study. The main challenge also came from the fact that the imaging was limited to the 7 μm focal depth and the imaging windows could not be all located at posterior portion after the initial dissection. The microstructure of AF can vary on the same disc where outer AF were found thinner and the ibres in the adjacent layers were found to be more in parallel in posterior portion compared to anterior or lateral portion [START_REF] Markolf | The structural components of the intervertebral disc. A study of their contributions to the ability of the disc to withstand compressive forces[END_REF]. Clinical lumbar puntures locate at posterior part of the disc which are expected to cause disc damage more easily. To determine long-term damage in terms of puncture locations would require larger sample sizes and precise microdissections while this study presented a preliminary results of prepherial damages on outer AF surface after short period of cyclic loadings.

Two different types of needles, the hypodermic and spinal needle, with two different sizes were used in this study. The damage caused by the needle shape were not compared in this study. Some of the collagen bundles were cut through with part of the collagen ibres being pushed inside or lifted up (Fig. 2). Those collagen ibres which were lifted up were out of the imaging focal depth (7 μm) and had low SHG intensity and thus were not included in this study. In Fig. 3, the puncture size change could indicate the loading might have caused tearing in the puncture in which less shrinkage occurred in the direction perpendicular to the loading and larger needles caused more tearing with negative correlation to needle size. However, the complex combination of mechanical stretching, orientation of needle tip openings, the cutting of the collagen ibres, drying of the sample surface could also take place. Similar to the clinical applications, the openings of the tips were random and not monitored in this study. The effect of dehydration could also contribute to shape changes which is the limitation in this study. Larger sample sizes were required to verify the signiicant changes under hydrated states. Furthermore, all the samples were ixed on the rig, those changes (shrinkage or expansion) have been found occurred only locally, i.e. within adjacent ibre bundles, while this study only focused on the imaging site of around 1.5 mm × 1.5 mm area with pixel size 2 μm × 2 μm to cover the whole puncture site and compare the progressive damages.

From the polarized-SHG imaging results, the overall I 2 value decreases with increasing needle size, as shown in Fig. 6. Non-normal distributions were found in I 2 and two Gaussian curves were itted with two mean values (μ 1 , μ 2 ) being compared. Those two values represent the populations of more organised (μ 1 ) and less organised (μ 2 ) collagen molecules across the whole images. However, how those two populations relate to damages at molecular level such as damages on different molecules and thus collagen network will require further invests with larger sample sizes and higher magniication objectives imaging the damaged ibrils. Each itted Gassian distribution curve was described in three parameters, the mean (μ), the mixing proportion (P) and the standard deviation (σ). 26 out of 29 samples having decreasing μ 1 with increasing cycle number indicated an overall shift of average values in more organised collagen population. However, no obvious trends were observed in P and σ which suggested more complex mechanism were taken places where two polulations did not simply shift rather than changing in both proportions and distributions.

A progressive decrease in overall I 2 over short-term cyclic loading was observed, with a larger needle size having more decrease as shown in Fig. 7. Strong correlations were found between cycle numbers and I 2 , μ 1 and μ 2 in needle groups with correlation coeficients higher than 0.79, while weaker correlations were found in control group with correlation coeficients less than 0.59. These indicated the needle damage could cause more progressive damage. It can be further explored and applied in clinical practice, since this result suggests that using larger needle can cause more long-term damage. However, the statistical results in this study, although the percent overlap I 2 distribution decreased with increasing cyclic loadings for all samples, no statistical differences could be concluded between needle groups. Similarly, decreasing trends were found in normalised and non-normalised I 2 , μ 1 , μ 2 and their slopes, while no signiicant difference could be concluded within 5% conidence. These also verify the results from other research groups [START_REF] Iatridis | Does needle injection cause disc degeneration? News in the continuing debate regarding pathophysiology associated with intradiscal injections[END_REF][START_REF] Cuellar | Does provocative discography cause clinically important injury to the lumbar intervertebral disc? A 10-year matched cohort study[END_REF] in which mild or moderate damage have been observed. While it does not come as a surprise that using a larger needle can increase damage, this study showed that the reason for this increase is not the larger hole produced by the larger needle, but rather the microscopic local disruption around it.

Finally, the outer AF cell nuclei at the puncture site showed rounded shape (Fig. 8). Clustered and round cells have also been found surrounding puncture and between bundles, which has been little studied. Round cells are often observed in aging and degenerative discs, instead of elongated ibroblast-like in healthy young discs [START_REF] Johnson | The effect of needle size inducing degeneration in the rat caudal disc: evaluation using radiograph, magnetic resonance imaging, histology, and immunohistochemistry[END_REF][START_REF] Pattappa | Diversity of intervertebral disc cells: phenotype and function[END_REF][START_REF] Gruber | Human disc cells in monolayer vs 3D culture: cell shape, division and matrix formation[END_REF][START_REF] Gruber | Cell shape and gene expression in human intervertebral disc cells: in vitro tissue engineering studies[END_REF]. Cell shape can inluence cell function and mitogenesis capacity, and in turn the interactions between cells and extracellular matrix can regulate cell shape, cell gene expression and cell functions [START_REF] Grad | Cells and biomaterials for intervertebral disc regeneration[END_REF][START_REF] Bruehlmann | Regional variations in the cellular matrix of the annulus ibrosus of the intervertebral disc[END_REF]. In this study, disc samples had undergone the freezing and thawing processes, cell nuclei rounding could be due to membrance lysis during the process, or the needle damages of the extracellular matrix causing the AF cells return to their lower mechanical energy state which tend to be round in nature. To have clearer understanding of the cause would require cell membrane of cytoskeletal imaging which will be left in future work.

In this study, the number of samples was small with only 5 to 6 samples in each group and some results have not shown statistical differences in this study, further improvement including the effect of cyclic loadings with longer period would require larger sample sizes with samples kept in hydrated state for statistical studies.

Conclusion

In this study the progressive damages on collagen ibres in outer annulus ibrosus of bovine intervertebral discs caused by needle puncture were investigated using polarization-resolved SHG at the site of puncture. By using a generic model to analyse the SHG response, the degree of the collagen ibril organization was presented pixel by pixel. There were two main categories of the puncture shape which agree with the results from other research groups, corresponding to more or less damage induced on the collagen organization. Larger needles have been found to cause more moderate damages over short-term cyclic loadings while statistically no signiicant differences can be concluded with such small sample sizes. There are a few limitations in this study, including the dehydration which caused 10-19% of axial strength loss, the SHG limited to prepheral images, and small sample size.

In conclusion, although with small sample sizes, mechanical effects of needle puncture found in this study on disc microstructure were not signiicantly affected by needle type, and only marginally by needle size. This conirms that puncturing the disc for diagnostic or therapeutic aims should be avoided when possible, because all types and size of needles induced similar damage, and it should be done with smaller needle size when necessary.

Fig. 1 .

 1 Fig. 1. (a) A bovine tail motion segment was itted onto a custom-built rig and placed under a microscope. (b) Protocol for puncture and cyclic loadings axially.Images (marked by *) were taken every 1% step of axial strain during loading and then every 10 cycles during cyclic loadings. A few drops of PBS were applied for rinsing the imaging area on AF surface (marked by blue drops) after initial loading and prior to the puncture. (c) Four different needles used in this study: two hypodermic needles with diameter 0.5 mm (25G) and 0.8 mm (21G); two Quinke spinal needles with diameter 0.7 mm (22G) and 0.9 mm (20G). (d) Illustration of needle puncture along radial axis (z) while loading was applied axially along x. (e) Imaging area (puncture site) on x-y plane.

Fig. 2 .

 2 Fig. 2. The SHG images for 29 samples, including the control group and four needle groups (needle size 0.5mm-0.9 mm) with eight samples in each group. Samples are numbered 1 to 8 in the order of increasing progressive damage. Scalebars represent 200 μm. The arrow represents the direction of loading.

Fig. 3 .

 3 Fig. 3. (a) SHG collagen ibre images in outer annulus ibrosus from representative samples in each group with obvious damage of cutting and tearing (specimen number referred to Fig. 2). Images taken from before and after 70 cyclic loadings (marked as cycle 0 on top row and cycle 70 on bottom row) are shown here to compare the effect from cyclic loading. All scale bars represent 200 μm. The arrow represents the direction of loading. The maximum width (w) and length (L) of the puncture before and after the cyclic loading (w0, L0, w7, L7) were marked in each needle groups, respectively, (b) change of maximum puncture length ΔL and (c) change of maximum puncture width Δw. (d-f) The force (F) versus strain (D) cuves for three representative samples from control, 25G and 20 G needle group on cycle 1 (Cyc1) with intervals for imaging every 1% step, cycles 2-10 (Cyc 2-10), and cycles 61-70 (Cyc 60-70). Force readings from pressure transducers changes between 10.4 and 16.2% compared cycle 2 and cycle 70.

Fig. 4 .

 4 Fig.4. I 2 mapping of the collagen ibre from the polarization SHG images. One representative sample was taken from each group (the same samples shown in Fig.3). In each sample images, the top row (cycle 0) images were taken right after the puncture before the cyclic loadings (cycle 0), the middle row were images taken after 30 cyclic loadings (cycle 30), and the bottom row were taken after 70 cyclic loadings (cycle 70). All scale bars represent 200 μm.

Fig. 5 .

 5 Fig. 5. (a-e) Histogram of normalised I 2 distributions from the images shown in Fig. 4. Two Gaussian distribution curves were itted to each I 2 distribution as shown in the inset in (d) and (e) to provide two mean values μ 1 and μ 2 due to non-normal distributions have been observed. (f) The percent overlap against cycle number from control and needle groups. An example of overlap was shown in the wavy patterns in (c). Error bars represent standard deviation from those samples in each group.

Fig. 6 .

 6 Fig.6. The mean value of I 2 , μ 1 , μ 2 for whole images from 5 groups in (a-c), their normalised values in (d-f). Each value was taken as the averaged value from all those samples over 70 cycles. Error bars represent the standard deviation from eight samples in each group.

Fig. 7 .

 7 Fig. 7. I 2 values of representative samples taken from each group versus cycle number in (a). The slopes of decreasing I 2 , μ 1 , μ 2 values over 70 cycles from 5 groups in (b-d). The slopes of decreasing normalised I 2 , μ 1 , μ 2 values over 70 cycles from 5 groups in (e-g). Error bars represent the variation of I 2 values from each group.

Fig. 8 .

 8 Fig. 8. AF Collagen ibre (red) and cell nuclei (green) images using SHG and TPF microscopy respectively. (a) Image was taken using an 10× air objective at the puncture site where the puncture was located at the centre of the image highlighted by a smaller square. (b) A higher ampliication re-scanned image from the same puncture site in the centre of (a). (c) A higher ampliication of image taken at the edge corner of the puncture from (b). Length (l) to diameter (r) ratios were calculated across the image. (d) AF image taken from an unpunctured disc sample: cells were elongated within collagen ibre. Length (l) to diameter (r) ratios were calculated across the image. (e) AF image between two collagen bundles. All images were taken at 512 × 512 pixels with pixel size 2.76 μm × 2.76 μm. Error bars represent (a) 200 μm, (b) 100 μm, (c) 50 μm, (d) 50 μm and (e) 20 μm.

Acknowledgements

The authors thank the Henry Smith Charity for funding this study [grant number 20141464].

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Declaration of competing interest

The authors declare that they have no known competing inancial interests or personal relationships that could have appeared to inluence the work reported in this paper.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi. org/10.1016/j.jmbbm.2021.104458.


Anna Rufà

Véronique Laroulandie

Les oiseaux charognards prédatés. L'exemple du parc « les loups du Gévaudan » The predated scavenging birds. Example of "les loups du Gévaudan" park

INTRODUCTION

Birds are among the most frequent scavengers 1,2 . This study aims to illustrate how wolves can prey on carrion birds, and describe the modifications they could produce.

MATERIAL AND METHOD

DISCUSSION AND CONCLUSION

There may be situations in which scavenging birds and wolfs interact during the consumption of carcasses. This interaction leads to conflicts between both agents looking for the same resources. These circumstances could be lethal for the carrion bird [3][4][5][6][7] . Wolves could take advantage of the bird carcasses, consuming them and producing modifications on bones. Despite the small sample size, the present study provides a starting point for better understanding and identifying interactions between wolf and scavenging birds in the archaeological record.

  Origin of the assemblage: park "les Loups du Gévaudan" Period/reason of bone recollection: March 2018 • Hand picked in one of the enclosures of the scientific park • Wolfs are the only carnivores in the park that are able to hunt large-sized birds Methodology applied: Taphonomy RESULTS NISP: 15 (Fig. 1) MNI: 5 Taxa: Griffon vulture (Gyps fulvus) Kite (Milvus sp.) Common raven (Corvus corax) Completeness: 40% • Long bones present fresh fractures with jagged and/or oblique edges Carnivore modifications: 80% (Fig. 2) • 53% with pits, punctures and notches (multiple and bilateral) • Presence of furrowing and crenulated edges • Two vulture bones show scores • No digestive alterations

Fig. 1

 1 Fig.1 Skeletal representation of the assemblage. Drawings by M. Coutureau (Inrap, ArcheoZoo.org). Fig. 2 Mechanical modifications observed on a raven (upper left) and a kite (upper right) humeri, and griffon vulture sternum (lower left) and humerus (lower right).

ACKNOWLEDGEMENTS: Research funded by the IdEx University of Bordeaux Investments for the Future Programme (ANR No.-10-IDEX-03-02). A.R. participates in the projects HAR2016-76760-C3-1-P, PID2019-103987GB-C31 (MICINN); CLT009/18/00053, CLT009/18/00054, CLT009/18/00055 and 2017 SGR 836 (Generalitat de Catalunya-AGAUR), and PCR « paléoécologie du Lazaret » (DRAC PACA). We would like to thank Jean-Baptiste Mallye and Myriam Boudadi-Maligne who, during their experiments at the park "les Loups du Gévaudan", collected the bird remains and entrusted them to us. Forthcoming publication in Arkeogazte. REFERENCES: 1 SELVA, N. & FORTUNA, M.A. (2007). The nested structure of a scavenger community. Proc. Royal Soc. B-Biol. Sci., 274: 1101-1108. / 2 SELVA, N. et al. (2005). Factors affecting carcass use by a guild of scavengers in European temperate woodland. Can. J. Zool., 83: 1590-1601. / 3 ALLEN, D. (1979). Wolves of Minong: Their vital role in a wild community. Houghton Mifflin, Boston. / 4 HUNTER, J.S. et al. (2007). Patterns of scavenger arrival at cheetah kills in Serengeti National Park Tanzania. Afr. J. of Ecol., 45: 275-281. / 5 PAQUET, P.C. (1992). Prey use strategies of sympatric wolves and coyotes in Riding Mountain National Park, Manitoba. J. Mammal., 73: 337-343. / 6 PETERSON, R.O. (1977). Wolf Ecology and Prey Relationships on Isle Royale. Government Printing Office, Washington. / 7 STAHLER, D. et al. (2002). Common ravens, Corvus corax, preferentially associate with grey wolves, Canis lupus, as a foraging strategy in winter. Anim. Behav., 64: 283-290.