Alejandro Garcia Garcia

Jean-Baptiste Perot

Megane Beldjilali-Labro

Quentin Dermigny

Marie Naudot

Sophie Le Ricousse

C Legallais

Fahmi Bedoui
email: fahmi.bedoui@utc.fr

Le Ricousse

Monitoring mechanical stimulation

Keywords: Electrospinning, Mechanical stimulation, Stem cells, Tissue engineering, tendon Electrospinning, Mechanical stimulation, Stem cells, Tissue engineering, tendon 31 32

come

Introduction

33

The main objective of tendon tissue engineering is to design and produce a cell-construct 34 that will help to regenerate damaged tissue or mimic it for comprehensive in vitro studies. [START_REF] Kubo | Effect of stretching training on the viscoelastic 589 properties of human tendon structures in vivo[END_REF] To achieve this goal, different groups have searched in recent years for the most efficient Bone marrow stromal cells (BMSCs) were isolated from rat bone marrow thanks to their 123 short-time adherence to plastic, in accordance with previously described protocols [START_REF] Li | Evaluation of isolation methods and culture conditions for rat bone 528 marrow mesenchymal stem cells[END_REF]. ST2, disinfected with ethanol 70% (Sigma-Aldrich, USA) for 45 min, 141 and then washed three times with PBS for 10 min. Disinfected scaffolds were soaked in 142 fresh α-MEM for 48h before seeding the cells. After that, the media was discarded and each 143 scaffold was plated with a density of 6x10 4 cells cm -2 . 144  Mechanical stimulation 145 After two days of static culture, each construct was placed in a bioreactor for mechanical 146 stimulation or in well dishes for static culture. Mechanically-stimulated cell constructs were 147 stretched twice a day at 5% strain for 1h at 1Hz with 11h of rest between each cycle for 5 or Proteins were isolated and hydrolysed in 6N HCl (Sigma-Aldrich, USA), and total 175 hydroxyproline content was determined using hydroxyproline assay (Hydroxyproline Assay 176 Kit, Sigma-Aldrich, USA) [START_REF] Neidert | Enhanced fibrin remodeling in vitro 530 with TGF-beta1, insulin and plasmin for improved tissue-equivalents[END_REF]. Hydroxyproline content was related to the collagen 177 content [START_REF] Marturano | Characterization of 533 mechanical and biochemical properties of developing embryonic tendon[END_REF]. between the curves in each sinus. Because sinusoidal stress was applied, we also 209 determined the storage modulus (E'), loss modulus (E''), complex modulus (E*) and tan δ. Although not statistically significant, some trends are present in each of the mechanical 332 properties analysed. In the absence of cells, all mechanical parameters presented lower 333 percentages than those obtained in cell-constructs (Figure 8). Tan(δ) presented a slight 334 increase for static controls (4 ± 7%) and decreased for dynamic ones (-3 ± 6%). For 335 dissipated energy, controls presented a decrease of -4 ± 17% and -10 ± 2% for static and 336 dynamic conditions respectively. E'' presented the same trend as dissipated energy, with the 337 highest decrease obtained for static conditions (-25 ± 4) compared to dynamic ones (-9 ± 338 6%). The elastic parameter (E') appeared to decrease particularly for the static control (-20 ± 339 9%), compared to a slight reduction for the dynamic control (-6 ± 0.1%). E* followed the 340 same trends as E', with the highest decrease found with the static control with -20 ± 9% vs -341 6 ± 0.2% for the dynamic control.

342

In the presence of cells, Tan δ seemed to increase in both static and dynamic cultures, with 343 an enhancement of 22 ± 16% and 16 ± 5% respectively. Dissipated energy presented a 344 similar trend with enhancement of 25 ± 7% and 14 ± 9% for static and dynamic culture of 345 cell-constructs, respectively. In terms of E'', cell-constructs presented an increase of 45 ± 5% sine strain simulating physiological activities for tendons [START_REF] Ikoma | Effects of stress-shielding 544 on the dynamic viscoelasticity and ordering of the collagen fibers in rabbit Achilles 545 tendon[END_REF].

367

Our first concern was thus to develop a biomaterial suitable for this approach. with BMSCs, where more cells were found after 2 weeks of culture compared to shorter 387 culture times under 5% of stretching [START_REF] Choi | Effects of mechanical 556 stimulation on the proliferation of bone marrow-derived human mesenchymal stem 557 cells[END_REF]. Other studies have shown similar results with 388 fibroblasts [START_REF] Kuang | Influence of mechanical stimulation on human 559 dermal fibroblasts derived from different body sites[END_REF] and TDSCs [START_REF] Xu | The effect of mechanical stimulation 562 on the maturation of TDSCs-poly(L-lactide-co-e-caprolactone)/collagen scaffold 563 constructs for tendon tissue engineering[END_REF]. While an increase in cell proliferation is found as a response 389 to mechanical stimulation, the mechanisms involved in this mechano-response still need to 390 be clarified [START_REF] Wang | Cell adhesion and mechanical stimulation in the regulation of 565 mesenchymal stem cell differentiation[END_REF].

405

Collagen I is the most abundant collagen in tendons, and tenomodulin is a late tendon 406 differentiation marker, and a key glycoprotein for the mature state of tendons [START_REF] Dex | Tenomodulin is Required 581 for Tendon Endurance Running and Collagen I Fibril Adaptation to Mechanical Load[END_REF][START_REF] Nagasawa | Static and dynamic biomechanical 584 properties of the regenerating rabbit Achilles tendon[END_REF]. Both 407 were more abundant under mechanical stimulation when compared to static culture 408 samples. Furthermore, they appeared to be arranged in the stretching direction. Taken 409 together, these data with cell alignment achieved in dynamic conditions, revealed a tendon- (2) Mechanical stimulation; impact of the culturing conditions.

413

Concerning the evolution of the biomechanical behaviour and to avoid the noise, we 414 decided to analyse and represent the V% of the targeted parameters variations as 415 described before with the equations, from the 1200 to 3600 sinus where a neat loop was 416 observed, similar to the mechanical behaviour observed in tendons [START_REF] Kubo | Effect of stretching training on the viscoelastic 589 properties of human tendon structures in vivo[END_REF]. For both static and 417 dynamic controls, E', E'' and Tan δ seem to present a reduction while for cell constructs The elastic modulus E´ considers the elastic properties both from the material and for cells.

421

The increase over time could be explained as the result of newly deposited type I collagen The cell-constructs showed an increase of E" for both static and dynamic conditions. 435 However, this increase seemed to be higher for static conditions with 45% increase, Representative stress vs strain curve (B) of the first sinus from the first cycle and the last sinus from the last cycle for cell-constructs subjected to dynamic culture conditions. After 2 days of static culture, the cell-construct or the control scaffolds were fixed inside the or the control scaffolds were fixed inside the bioreactor chamber and the mechanical test was launched. Both signal force and displacement were recorded over time and found to be smooth, without any background noise, making it possible to monitor mechanical (elastic and viscoelastic) properties.



  3200, TA, USA) and elongation at 0.1 mm s -1 was performed with a 119 working load of 22N. The applied force was measured each second and the modulus 120 obtained from the slope of the linear region.121 Cell harvesting and culture 122

124Briefly, 6 -

 6 week-old male Sprague Dawley rats (n=4) were sacrificed, and both right and left 125 femurs were aseptically removed and washed 3 times with 1x PBS 7.4 (phosphate buffered 126 saline, Gibco Invitrogen, USA). Next, bone marrow was flushed out using α-MEM culture 127 medium (PAN BIOTECH, Germany) supplemented with 10% foetal bovine serum (FBS, Gibco 128 Invitrogen, USA), 1% penicillin-streptomycin (Gibco Invitrogen, USA) and 1% amphotericin B 129 (PAN BIOTECH, Germany). The released cells were then collected into 6-well dishes (BD 130 Falcon™, USA). After 24h, non-adherent cells were carefully discarded and adherent cells 131 were cultured with fresh α-MEM for 6-7 days, the time needed for BMSC colonies to reach 132 confluence. The cell culture media were replaced every 3 days. When the culture dishes 133 started to approach confluence, the cells were detached and serially subcultured. The cells 134 at the third passage (P3) were used for the cell seeding experiments. Experimental 135 procedures involving animals were carried out on euthanised animals within the approved 136 structure (E60 159 01). Both the procedures and the treatment of the animals complied 137 with the principles and guidelines of the French legislation on animal welfare (No. 2013-118) 138 and the Directive if the European Communities (2010/63/EU). 139 Electrospun mats were cut into strips measuring 40 x 12.5 mm or 35 x 9 mm as shown in 140 Supplementary Table

  considered as 1 or 2 weeks of culture time respectively). For this purpose, two 149 different bioreactors were used: (1) the MechanoCulture T6 Mechanical Stimulation System 150 (CellScale Biomaterials Testing, Waterloo, ON, Canada), consisting of an actuator and screw-151 driven clamp grips mounted inside a cell culture chamber capable of applying uniaxial 152 stretching to 6 parallel samples, was chosen for the biological assays. Cell culture media 153 were replaced every 5 days, and (2) the Bose Biodynamic 5100 (TA Electroforce®, USA), 154 consisting of a cell culture chamber connected to a flow pump in which one sample was 155 attached thanks to a system of rods and clamps. One rod was attached to a step motor 156 making it possible to apply uniaxial displacement. The other rod was connected to a force 157 transducer of 22N making it possible to constantly monitor the force applied to each 158 displacement. Cell culture media circulated continuously through the flow pump connected 159to a reservoir of 500 mL, making it possible to maintain the culture for up to two weeks. This 160 system was chosen for biomechanical evaluation. For static culture, each construct was 161 cultured for 5 or 12 days without tension and the cell culture media were replaced every effects of dynamic culture on cell activity, seeded PCL 10-wt % scaffolds 165 were secured in the grips of the T6 CellScale bioreactor after two days of static culture and 166 cultured for 5 or 12 days under dynamic culture conditions (5% strain for 1h at 1Hz with 11h 167 of rest). After this time, the cell-constructs were removed for biological analyses. 168 Total deoxyribonucleic acid (DNA) and collagen from each sample were extracted at 5 or 12 169 days of static or dynamic culture with the reagent, Trizol (TRI Reagent®, Sigma-Aldrich, USA) 170 according to the manufacturer´s protocol. Briefly, once lysed by the action of 1ml of Trizol, 171 chloroform (Sigma-Aldrich, USA) was added to obtain a colourless upper aqueous phase 172 with RNA, an interphase with DNA, and a lower red phenol-chloroform phase with proteins. 173 DNA was then isolated and quantified using NanoDrop ND-1000 (Thermo Scientific, USA).

  174

  12 days of static or dynamic culture, cells-constructs were fixed in a solution of 4% 179 (w/v) paraformaldehyde solution (PAF, Agar Scientific, United Kingdom) in PBS for 15 min 180 then rinsed three times with PBS 7.4. After 10 min of permeabilization in a solution of PBS-181 Triton X-100 0.5% (v/v) (VWR, United Kingdom), cell-constructs were blocked at 4ºC 182 overnight with a solution of 1% (w/v) BSA (Sigma-Aldrich, USA). The morphology of the 183 rBMSCs under static or dynamic culture was assessed using rhodamine phalloidin 184 (Invitrogen, USA) to selectively stain the F-actin. For immunofluorescence staining, cell-185 constructs were treated with mouse primary antibodies anti-rat collagen type I (COL1, 186 1:100, Abcam, United Kingdom) or to anti-rat tenomodulin (TNMD, 1:200, Abcam, United 187 Kingdom) overnight at 4ºC. After incubating overnight at 4ºC with secondary fluorescent 188 antibodies donkey anti-mouse 488 (Invitrogen, USA). Hoechst 33342 (Sigma-Aldrich, USA) 189 was added as counterstaining for cell nuclei. Z-stacks were then acquired on an Inverted 190 ZEISS 710 confocal microscope (Zeiss, Germany). 191 To investigate the effects of dynamic culture on the evolution of the mechanical properties 192 of the cell-constructs, seeded scaffolds or scaffolds without cells were mounted in the Bose 193 Biodynamic 5100 for 12 days under mechanical stimulation. Bose Biodynamic 5100 consists 194 of one culture chamber with two rods, one connected to a motor and the other to a force 195 transducer of 22N. The entire system is placed in an incubator making it possible to control 196 the temperature and C0 2 at 37ºC and 5% respectively. Cell culture media circulated 197 continuously thanks to a system of peristaltic pumps connected to the cell chamber. The 198 bioreactor makes it possible to apply defined cyclic (sinusoidal) strain to the cell-seeded or 199 empty scaffolds, and to concomitantly record the resulting stress. Either stress ( ) or strain ( 200 ) could be set up as the driving parameter. We decided to set deformation, 5% cyclic 201 sinusoidal strain as the control parameter. We recorded the resulting force at 20 points per 202 second, corresponding to 20 points per sinus, during the 12 days of dynamic culture. 203 𝑆𝑡𝑟𝑎𝑖𝑛 𝜀 = 𝜀 0 sin 𝑤𝑡 (1) 204 𝑆𝑡𝑟𝑒𝑠𝑠 𝜎 = 𝜎 0 sin (𝑤𝑡 -𝛿)(2) 205 Where and are the initial strain and stress respectively, and and are the frequency 206 and dephasing angle between stress and strain respectively. Plotting together stress vs 207 strain, we were able to calculate the relative dissipation energy by calculating the surface

  sinus and the results are given as an arithmetic average. After the first analysis, we decided 217 to represent the results from 1200 to 3600 sinus of each cycle, where a closed loop region 218 was found. For statically-cultured cell-constructs and controls, each sample was placed on 219 the bioreactor and, once secured between the rods, one cycle of 3600 sinus was set at a 220 given time (7 and 14 days) to compare in the same way as the dynamic conditions. For the 221 control without cells, scaffolds were subjected to the same parameters as the cell-222 constructs, including disinfestations and incubation with cell culture media. 223 Statistical analysis 224 At least 6 independent experiments, except for the biomechanical analysis (n=3) carried out 225 on the Bose Biodynamic 5100, were performed. Each result (mechanical properties as 226 damping factor, loss and storage modulus) represents an arithmetic average the different 227 analyzed tests. Data are therefore presented as an average along with the appropriate mean 228 standard deviation. The significance of the results was tested by analysing the variance 229 (ANOVA) with Turkey´s post hoc test in the case of multiple comparisons. 230 On behalf of the authors, we declare, that all the methods were carried out in accordance 231 with relevant guidelines and regulations. In addition all experimental protocols were 232 approved by our institution (Sorbonne Université Alliance, Université de Technologie de 233 Compiègne) commitees.

235  252  291 Fig 7 ,

 2352522917 Fig 7, to follow the tissue response continuously instantaneously and after two weeks of

368( 1 )

 1 Electrospinning has been widely used in the development of materials mimicking the fibrous 369 nature of the tendon extracellular matrix[START_REF] Bölgen | 10 -Electrospun materials for bone and tendon/ligament tissue engineering[END_REF]. While our PCL scaffold presented an elastic 370 modulus of 8.05 ± 0.82MPa (Fig3), which is still a long way from native tendon[START_REF] Martin | Mechanical Properties of Ligament and 552 Tendon[END_REF], the 371 stress-strain curve revealed three regions similar to those found in tendons[START_REF] Beldjilali-Labro | Biomaterials in Tendon and Skeletal Muscle Tissue Engineering: Current Trends and 500 Challenges[END_REF]. Taking into 372 account this stress-strain response, and comparing them with the mechanical behaviour of 373 tendon, we decided to apply a cyclic sinusoidal strain of 5% (0-5%) corresponding to the 374 heel region of both tendon and our electrospun scaffold. This was intended to calculate the 375 viscoelastic behaviour of our cell-constructs when submitted to cyclic stretching with 376 repeated stretching cycles and strain rates within a low range. The effect of this mechanical 377 protocol was analysed for cell behaviour, mechanical changes over time and the correlation 378 between both variables.379 Cell behaviour over mechanical stimulation behaviour, after one week of culture, there was no difference in terms of 381 proliferation between static and dynamic conditions (Fig5#). After two weeks, the number 382 of cells was found to have tripled for the static culture and even quadrupled for cells 383 submitted to dynamic stretching. We can thus conclude that the mechanical stimulation 384 enhanced the proliferative ability of BMSCs, inducing an increase in cell proliferation in 385 response to mechanical load. This result is consistent with other experiments conducted 386

391

  When stimulated, BMSCs presented an elongated morphology aligned with the stretching 392 direction (Fig 5* B,D), while cells cultured under static conditions were randomly organised 393 on the scaffold (Fig 5* A,C). Similar behaviour was found in another study that analysed the 394 impact of mechanical stimulation on cell alignment when cultured on randomly-oriented 395 scaffolds in the same range of nanofiber size(27).396Regarding collagen synthesis, the cell-constructs showed continuous production over time, 397 with more collagen found after 2 weeks of culture for each condition (Fig5 §). After two 398 weeks of culture, more collagen was found on cell-constructs subjected to mechanical 399 stretching, in agreement with several other studies[START_REF] Screen | Cyclic tensile strain upregulates collagen 570 synthesis in isolated tendon fascicles[END_REF][START_REF] Huisman | Enhanced collagen type I synthesis by human 573 tenocytes subjected to periodic in vitro mechanical stimulation[END_REF][START_REF] Wu | Living nanofiber yarn-based woven biotextiles for 576 tendon tissue engineering using cell tri-culture and mechanical stimulation[END_REF]. This effect has been shown 400 consistently in in vivo studies carried out on tendons where an increase in collagen synthesis 401 was observed as a part of the tendon adaptation response to continuous mechanical 402 loading[START_REF] Heinemeier | In vivo investigation of tendon responses to mechanical 579 loading[END_REF]. 403 Analysis of the ECM produced by cells in both static and dynamic cultures was also 404 performed by immunofluorescence staining of type I collagen and tenomodulin (Fig 5*).

  BMSCs under dynamic culture conditions, similar to native tendon 411 arrangement[START_REF] Doroski | Techniques for biological characterization of tissue-587 engineered tendon and ligament[END_REF].

  412

  418 those parameters seem to increase over time (Fig 8), which maybe result of cellular 419 behaviour. 420

  422fibres. Type I collagen is effectively an important stress-bearing component of connective 423 tissues. It is secreted by cells and hierarchically assembled into fibrils from a packaging of 424 collagen molecules, embedded in a soft matter formed of water and proteoglycans. This 425 organisation gives the collagen structure enough rigidity to be considered an elastic 426 beam[START_REF] Licup | Stress controls 592 the mechanics of collagen networks[END_REF]. In addition, the aligned orientation adopted by collagen fibres (Fig5*) through 427 the stretching direction could explain the higher E' percentage (16%) for dynamically-428 cultured cell-constructs compared to static culture (7%), as aligned collagen fibres are better 429 suited up to supporting tensile stress[START_REF] Martin | The relative effects of collagen fiber orientation, porosity, density, 595 and mineralization on bone strength[END_REF]. 430 The loss modulus (E'') takes into account the viscous behaviour, therefore its energy 431 dissipation behaviour and confirmed our previous results. As seen before, when initial and 432 last values were compared (Fig 8), the controls without cells presented a reduction in E'' 433 with -25% for static and -9% for dynamic controls.

  434

  436compared with 31% for dynamic cultures. Taken together, these results consistently 437 demonstrated that cell-constructs, presented increased viscosity over time, compared to 438 nude biomaterials. Finally, dynamic cultures exhibited a greater increase in the elastic 439 properties of the cell-constructs, with a lesser gain in viscous properties, while the static greater influence on the viscous properties, with a lower increase in 441 elasticity.442This effect may be explained by the effect of the alignment of the extracellular matrix, 443 particularly the collagen fibres, which align through the tensile axis, resulting in an increase 444 in the elastic properties of the cell-constructs exposed to dynamic culture conditions (parameters as tan δ seem to be in accordance with these results. The 447 value of tan δ is an indicator of how efficiently the material absorbs and dissipates energy 448 due to fiber-cells rearrangements and internal friction. When we compared its evolution 449 over time (Fig 8), both controls presented the lowest values. 450 Cell-constructs, in both static and dynamic cultures, presented an enhanced tan δ of 22% 451 and 16% respectively, compared to cell-free control scaffolds (Fig 8). Because tan δ 452 represents the ratio of Loss over storage modulus, the highest values of tan δ were related 453 to a higher damping factor. In a rabbit Achilles tendon regeneration model, Nagasawa et al. 454 found higher values of tan δ for regenerated tendons after surgery compared to 455 controls[START_REF] Nagasawa | Static and dynamic biomechanical 584 properties of the regenerating rabbit Achilles tendon[END_REF]. This increase was explained as the result of the neosynthesis of collagen fibres.456As this "new" fibre presented a lower amount of mature cross-linking, collagen mobility was 457 increased within the tissue, resulting in more energy dissipation. This may explain the 458 increase in tan δ in our cell-constructs, where cells produced collagen, compared to controls 459 without cells. In addition, the random organisation of collagen fibres for static conditions 460 may explain the greater tan δ percentages. These results are in accordance with others 461 mechanical parameters studied here (Supplementary data) as energy dissipation and E*. 462 Correlating both the biological and mechanical results (Fig 9), it seems that mechanical 463 stimulation has a positive effect on cell proliferation and collagen synthesis, which was 464 greater than static culture conditions. Cell and collagen alignment, together with an increase 465 in tenomodulin under dynamic culture conditions, provides a better environment for BMSCs 466 to differentiate towards a tendon-like phenotype. Furthermore, the mechanical properties 467 of the cell-constructs were enhanced over time compared to the control scaffolds without 468 cells. This could be explained as a result of the cellular activity, translated into the 469 production of an ECM.

Figure 1 .Figure2.Figure3.scaffolds (B). 615 Figure 4 .Figure 5 .Figure 6 .Figure 7 .Figure 8 .Figure 9 .

 1615456789 Figure 1. Electrospinning device.

Figure 1 .

 1 Figure 1. Electrospinning device.

Figure 4 .Figure 5 .

 45 Figure 4. Stress (MPa) vs strain (%) curve for 10-wt % PCL scaffolds in a dry (dark grey) or wet (light grey) state, obtained using the Electroforce 3200 system.

FFigure 6 .

 6 Figure 6. Design of the dynamic culture process during 12 days of stimulation (A).Representative stress vs strain curve (B) of the first sinus from the first cycle and the last sinus from the last cycle for cell-constructs subjected to dynamic culture conditions. After 2 days of static culture, the cell-construct or the control scaffolds were fixed inside the or the control scaffolds were fixed inside the bioreactor chamber and the mechanical test was launched. Both signal force and displacement were recorded over time and found to be smooth, without any background noise, making it possible to monitor mechanical (elastic and viscoelastic) properties.

John Wiley & Sons, Inc. Journal of Biomedical Materials Research: Part A

For analysis, each 1h cycle, consisting of 3600 sinuses, was divided into six intervals of 600

John Wiley & Sons, Inc. Journal of Biomedical Materials Research: Part A F o r P e e r R e v i e w

Data availability

485

The data supporting the findings of this study are available from the authors upon 486 reasonable request. Correlation between cellular activity (Cell Scale T6) and biomechanical performance (Bose Biodynamic 5100). Briefly, after 2 days of static culture, scaffolds were transferred to the different bioreactors (Electro Force and Bose Biodynamic). Once attached, the dynamic culture was launched for 2