Clément Rieu

Nicolas Rose

Anissa Taleb

Gervaise Mosser

Bernard Haye

Thibaud Coradin

Fabien Le Grand

Lea Trichet

Léa Trichet

Differential myoblast and tenoblast affinity to collagen, fibrin and mixed threads in the prospect of muscle-tendon junction modelisation

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Differential myoblast and tenoblast affinity to collagen, fibrin and mixed threads in the prospect of muscle-tendon junction modelisation

Introduction

Tendons are connective tissues that anchor skeletal muscles to the bones, transmitting the force generated by the muscles to the skeletal structure. As such, tendons play an obvious role in everyday movement and posture control. In case of injury, partly due to their low vascularization, they exhibit poor regeneration capacity resulting in loss of mobility, pain, etc. Injury of the muscle-tendon unit often occurs in the junction area as reported in many articles [START_REF] Armfield | Sports-Related Muscle Injury in the Lower Extremity[END_REF][START_REF] Palmer | MR imaging of myotendinous strain[END_REF][START_REF] Tavernier | Lésion isolée de l'infraépineux à la jonction myotendineuse : une nouvelle lésion[END_REF][START_REF] Hertel | Supraspinatus rupture at the musculotendinous junction[END_REF][START_REF] Taneja | MRI Findings of Rotator Cuff Myotendinous Junction Injury[END_REF] . In particular, muscle injuries are often located close to the junction [START_REF] Garrett | The effect of muscle architecture on the biomechanical failure properties of skeletal muscle under passive extension[END_REF][START_REF] Woo | Injury and repair of the musculoskeletal soft tissues[END_REF] , leading to the rupture of the myofibres rather than the tearing of the collagen fibres from the sarcolemma. Tensile tests on whole muscle-tendon units from rabbits also highlighted the weakness of the myo-tendinous junction (MTJ) area [START_REF] Garrett | The effect of muscle architecture on the biomechanical failure properties of skeletal muscle under passive extension[END_REF] . As muscles often taper in contact with the tendon, stress concentration increases with decreasing cross-section [START_REF] Sharafi | Strains at the myotendinous junction predicted by a micromechanical model[END_REF] .

During muscle regeneration process, quiescent muscle stem cells called satellite cells activate, and give rise to proliferating myoblasts destined to differentiate into myocytes able to fuse with one another or with injured myofibres [START_REF] Zammit | The Muscle Satellite Cell: The Stem Cell That Came in From the Cold[END_REF] . Up to date both the intrinsic transcription factors and the extrinsic cell-to-cell signaling pathways controlling muscle stem cells and myoblasts functions are well characterized. However, an emerging body of evidence supports the notion that myogenic cells integrate biochemical factors together with biomechanical cues that may control their cell fate [START_REF] Li | Cellular Biomechanics in Skeletal Muscle Regeneration[END_REF] .

Muscles and tendons have completely different composition and structure, resulting in radically different mechanical properties: muscles are soft materials with Young moduli between 0.005 and 3 MPa while tendons are much stiffer, with Young moduli ranging from 500 to 1850 MPa [START_REF] Ladd | Co-electrospun dual scaffolding system with potential for muscle-tendon junction tissue engineering[END_REF] . Junctions between materials with such a difference in mechanical properties may result in high interfacial stress and are likely to fail first during mechanical solicitation all the more since the stress borne by the MTJ reaches several tens of kPa [START_REF] Charvet | The development of the myotendinous junction. A review[END_REF] . The increase of the contact surface between tendon and muscle by a factor 10 to 20, with finger-like structures, enables to decrease this stress [START_REF] Knudsen | The human myotendinous junction: An ultrastructural and 3D analysis study: The human myotendinous junction[END_REF] . Along the myofibers the repeating sarcomeric units consist of an overlap of myosin and actin filaments, with Z-lines acting as anchoring points of the actin filaments and delineating the sarcomeric segments. In the peripheral finger-like structures, the last Z line is linked to the interdigitated sarcolemma through actin filament bundles. These actin filaments bind to transmembrane protein complexes containing integrins, and interact with the collagen IV and laminin-rich basal membrane and the extra-cellular matrix of tendons, rich in collagen I. Rebuilding the MTJ following muscle injury may thus subject progenitor cells to highly different environmental stiffnesses that promote divergent cellular responses.

For both muscular and tendon sides, reconstruction of the MTJ is relevant to help reconstruction in case of damaged MTJ or for better integration of the neo-tissue. For instance, in the hypothetical case of the grafting of a tendon de novo, integration at the muscular level through a new MTJ may be more effective and rapid as muscle is highly vascularized and is rich in satellite cells. MTJ structure is much less documented than muscle or tendon ones, and attempts to reproduce the MTJ are also scarce. We can cite the work reported by Ladd et al. 11 in which two different polymers are electrospun with collagen the poly(L lactic acid), being stiffer, for the tendon part and poly(ε -caprolactone) for the muscle part. Merceron et al. [START_REF] Merceron | A 3D bioprinted complex structure for engineering the muscle-tendon unit[END_REF] used co-printing of two synthetic polymers polyurethane (PU) and poly(ε-caprolactone) (PCL) together with a bioink based on hyaluronic acid, fibrinogen and gelatin. The bio-ink contained C2C12 myoblasts on the PU side while the bio-ink on the stiffer PCL side contained NIH/3T3 cells. Both works used two materials with different mechanical properties to mimic the tendon and the muscle sides.

Mechanical properties of the substrate have been highlighted as key factors to control cell migration [START_REF] Vincent | Mesenchymal stem cell durotaxis depends on substrate stiffness gradient strength[END_REF][START_REF] Isenberg | Vascular Smooth Muscle Cell Durotaxis Depends on Substrate Stiffness Gradient Strength[END_REF] and differentiation [START_REF] Engler | Matrix Elasticity Directs Stem Cell Lineage Specification[END_REF][START_REF] Trumbull | Mechanoresponsive musculoskeletal tissue differentiation of adipose-derived stem cells[END_REF] . In particular, substrate stiffness has been shown to play a role in muscle stem cells self-renewal [START_REF] Gilbert | Substrate Elasticity Regulates Skeletal Muscle Stem Cell Self-Renewal in Culture[END_REF] , as well as in myotubes differentiation [START_REF] Engler | Myotubes differentiate optimally on substrates with tissue-like stiffness: pathological implications for soft or stiff microenvironments[END_REF] . Substrate stiffness has also been shown to promote tendon progenitor cells (here called tenoblasts) differentiation [START_REF] Liu | Matrix stiffness regulates the differentiation of tendon-derived stem cells through FAK-ERK1/2 activation[END_REF][START_REF] Patel | Effects of cell adhesion motif, fiber stiffness, and cyclic strain on tenocyte gene expression in a tendon mimetic fiber composite hydrogel[END_REF] . Besides cell interactions with structural proteins have been shown to influence cell phenotype [START_REF] Keselowsky | Integrin binding specificity regulates biomaterial surface chemistry effects on cell differentiation[END_REF][START_REF] Vicente-Manzanares | Non-muscle myosin II takes centre stage in cell adhesion and migration[END_REF] . Among them, collagen and fibrin are among the preferential proteins used in tissue engineering.

Type I collagen is an ubiquitous fibrillar protein found in most of the connective tissues in vivo [START_REF] Portier | Stabilization of Collagen Fibrils by Gelatin Addition: A Study of Collagen/Gelatin Dense Phases[END_REF] . It is widely used in biomaterials [START_REF] Rieu | From Tendon Injury to Collagen-based Tendon Regeneration: Overview and Recent Advances[END_REF] as it is a relatively stiff body protein [START_REF] Wegst | Bioinspired structural materials[END_REF] that bears multiple adhesion sites 28,[START_REF] Jokinen | Integrin-mediated cell adhesion to type I collagen fibrils[END_REF] for integrin receptors [START_REF] Kleinman | Role of collagenous matrices in the adhesion and growth of cells[END_REF] . In particular, it is the major component of tendons, reaching 55 to 80 % of tendon's dry weight [START_REF] Screen | Tendon Functional Extracellular Matrix: tendon functional extracellular matrix[END_REF] . On the other hand, fibrin is a major player in haemostasis, and polymerizes to form a clot after activation of its precursor, the blood-born fibrinogen, by thrombin [START_REF] Semple | Platelets and the immune continuum[END_REF] . This fibrillar protein exhibits high deformability [START_REF] Litvinov | Fibrin mechanical properties and their structural origins[END_REF] and direct integrin adhesion sites [START_REF] Bayless | RGD-Dependent Vacuolation and Lumen Formation Observed during Endothelial Cell Morphogenesis in Three-Dimensional Fibrin Matrices Involves the αvβ3 and α5β1 Integrins[END_REF][START_REF] Lishko | Multiple Binding Sites in Fibrinogen for Integrin α M β 2 (Mac-1)[END_REF][START_REF] Flick | Leukocyte engagement of fibrin(ogen) via the integrin receptor αMβ2/Mac-1 is critical for host inflammatory response in vivo[END_REF] , making it a material of choice for various applications such as for cardiovascular remediation [START_REF] Jockenhoevel | Fibrin gel -advantages of a new scaffold in cardiovascular tissue engineering[END_REF][START_REF] Long | Elastic fiber production in cardiovascular tissue-equivalents[END_REF][START_REF] Ross | ECM gene expression correlates with in vitro tissue growth and development in fibrin gel remodeled by neonatal smooth muscle cells[END_REF] or cartilage repair [START_REF] Fussenegger | Stabilized Autologous Fibrin-Chondrocyte Constructs for Cartilage Repair in Vivo[END_REF][START_REF] Hunter | Dynamic compression of chondrocyte-seeded fibrin gels: effects on matrix accumulation and mechanical stiffness[END_REF][START_REF] Sims | Tissue engineered neocartilage using plasma derived polymer substrates and chondrocytes[END_REF] . It is particularly favored for muscular reconstruction [START_REF] Page | Restoration of Skeletal Muscle Defects with Adult Human Cells Delivered on Fibrin Microthreads[END_REF][START_REF] Kang | A 3D bioprinting system to produce human-scale tissue constructs with structural integrity[END_REF][START_REF] Huang | Rapid formation of functional muscle in vitro using fibrin gels[END_REF] .

In this work, we investigate the effect of collagen I, fibrin and of mixed collagen:fibrin materials on mouse primary tenoblasts and myoblasts. Given the differing mechanical properties of the constitutive proteins, such scaffolds, composed only of biopolymers, might represent new types of tools to screen the respective influence of compositional and mechanical features on cell behavior. As a first step and in the prospect of musculotendinous junction modelisation, the capacity of highly concentrated collagen to offer a proper environment for tenoblasts, and of softer fibrin to favour muscular development, will be tested, as well as the influence of a mixed material. For this purpose, collagen, fibrin and "mix" threads are extruded. Mechanical properties and structure of threads are characterized and primary myoblasts and tenoblasts extracted from mice hind limbs are seeded on the threads.

Material & Methods

Extrusion

Threads were produced based on protocols developed in [Rieu tb free]. Briefly, collagen solutions (extracted from rat tail tendons) were concentrated at 60 mg/mL in 3 mM HCl 0.8 mM citrate. Fibrinogen solutions were made from fibrinogen stock solutions (Merck) at 40 mg/mL by addition of HCl to reach 60 mM HCl. To obtain fibrin solutions, the acidic fibrinogen solution was supplemented with CaCl2 (10 mM final) and thrombin (Sigma, 20 U/mL final). Such solution was either directly mixed with collagen at a ratio 1:1 (volume:volume) to prepare mix solution, or incubated 10 mn to reach suitable viscosity for pure fibrin extrusion.

Solutions were then transferred to a 1 mL syringe and centrifugated for 8 mn at 3000 x g to remove bubbles. They were then extruded in 100 mM HEPES 2.5 w.% PEG solutions, pH 7.4, through a 370 µm needle. The resulting threads (Fig. 1.A) were left for at least 10 days in the HEPES/PEG solution and then rinsed at least three times with cell culture medium and left in it for 24 hours to remove PEG.

Tensile Testing

Tensile testing was done on an Instron 3343 machine equipped with a 10N load-cell and a watertight column to perform the tests in hydrated conditions. Tests were performed in PBS 1X, to mimic physiological conditions and thus give an idea of the possible mechanical behavior in vivo. Threads were immobilized into clamps with Velcro tape to prevent sliding of the threads. Strain was calculated with the displacement of the clamps.

Transmission Electron Microcopy

Hydrated samples, crosslinked with PFA, glutaraldehyde and osmium tetroxide 4 wt. %, were subsequently dehydrated using baths with increasing concentrations in ethanol. They were then progressively transferred to propylene oxide and to araldite resin prior to full embedding in pure araldite. After inclusion, 70 nm ultrathin sections (Leica microtome) were made and contrasted with uranyl acetate and observed on a transmission electron microscope FEI Tecnai Spirit G2 operating at 120 kV. Images were recorded on a Gatan Orius CCD camera.

Cell isolation

Satellite cells were extracted from the hindlimb muscles (hamstring muscle group, quadriceps, tibialis, extensor digitorum longus, gastrocnemius, soleus, gluteus) of 2 to 3 months-old mice while tenoblasts were extracted from the mice Achilles tendons.

Muscles were dissected and minced to a pulp using forceps then digested with collagenase B solution (0.75 U/mL final, from Roche) with dispase II (1.2 U/mL final, from Roche) and CaCl2 (2.5 mM) at 37 °C for 40 minutes. Preparations were washed twice in culture media, filtered through a 70 µm cell strainer and subjected to immunomagnetic cell sorting using a "satellite cell isolation" kit and a MACS "midi" column (Miltenyi Biotech).

Tendons were either digested in 600 U/mL of collagenase II (Worthington) or in 1.5 U/mL of collagenase B (Roche) at 37°C for 18 hours, mechanically destroyed by up and down pipetting and filtered through a 100 µm cell strainer.

Primary myoblasts and tenoblasts were then amplified and cultured on collagen-coated plates for several passages.

Thread seeding

Threads were cut in approximately 1-cm-long pieces. They were seeded with myoblasts or tenoblasts at 10 5 cell/cm² in 24-well plates. Cells used were between P5 and P10 for both types. For tenoblasts, 3 threads of each condition were seeded with a first cell line and 1 thread with a different one. For myoblasts, two threads were seeded with a first line and two others with a second one.

Growth medium (high-serum medium -79 % Hams F-10 from Gibco, 20 % fetal bovine serum, 1 % antibiotic-antimycotic Gibco, with 2.5 ng/mL recombinant human fibroblast growth factor R&D Systems) and fusion medium (low-serum medium -94 % Dulbecco's Modified Eagle's Medium, Gibco, 5 % horse serum, 1 % antibiotic/ antimycotic Gibco) were changed every other day. Unless specified, the differentiation process involves 7 days of proliferation in the growth medium, followed by differentiation in the fusion medium. Aminocaproic acid (6-aminohexanoic acid, Sigma) at a final concentration of 2 mg/mL was added to both media. Recombinant human Wnt7a (Chinese Hamster Ovary cell line-derived, R&D Systems) at a concentration of 50 ng/mL was used for the culture displayed on Supp.Fig. 1.

2D culture

Culture of myoblasts in 2D was performed in 24 well plates coated with the different materials. Plates were coated with collagen, mix and fibrin with the same solutions as the ones used for extrusion. A thin layer of the solution was deposited, with further addition of growth medium to trigger fibrillation. Matrigel-coated plates, used to promote fusion, were seeded as a reference (BD Biosciences). They were prepared by incubation at 37°C for 30 mn of a thin layer of Matrigel at 1 mg/mL and subsequent drying under the hood. They were then rehydrated using growth medium. Each well was seeded with a density of 2x10 4 cells/cm², with triplicates per material, and left 1 day in proliferation medium for the myoblasts to attach and then cultured in fusion medium for 6 days. Aminocaproic acid at a final concentration of 2 mg/mL was added to both media.

Fluorescence microscopy

To be observed the seeded threads were rinsed with PBS 1X and then fixed with 4 % PFA for 15 minutes at room temperature. After a blocking step with BSA 4% for 45 minutes, cells were stained using primary (Myosin heavy chain: primary hybridoma mouse IgG2b; Myogenin: myogenin antibody (5FD) Santa Cruz biology;) and secondary antibodies (Red: Alexa Fluor 546 goat anti-mouse IgG2b, In vitrogen; Magenta: A647 anti-rabbit, Life Technology). Nuclei were stained with Hoechst (Life Technology). Observations were made on a calibrated EvosR fluorescence inverted microscope.

To assess homogeneity of mix threads, fibrinogen-FITC was used. To label fibrinogen, 20 mg of FITC CeliteR was dissolved in 3 mL of 0.13 M carbonate buffer pH 9.2. Fibrinogen stock solution at 40 mg/mL was then mixed to the FITC solution at a ratio 1:3. The resulting solution was then dialysed 48 hours against 2 L of a solution of 20 mM citrate pH 7.4, with regular changes of the dialysing bath. Final solution was aliquoted and stored at -80°C. For extrusion, 10% in volume of FITC-labelled fibrinogen [Fbg-FITC] was added to fibrinogen stock solution, before addition of HCl, and following the usual protocol afterwards.

Statistical analysis

For statistical analysis, imaging of every part of the threads was carried out at 4x magnification to minimize deviation due to image sampling. ImageJ software was used to measure cell density. An area taking only the central part of the thread was outlined and measured using ImageJ. The central area was chosen to be approximately two thirds of the thread width. This was done for two reasons. First, the projected area measured on the picture in 2D was smaller than the actual area in 3D, due to the curvature of the threads. For a ratio r = 2/3, the relative error made is around 8% which is reasonable. Second, due to the curvature of the thread, the cells outside the central part tend to appear as cell aggregates and are more difficult to distinguish and count. Cells inside the outlined area were manually counted using the cell counter plug-in from ImageJ. To count cells positive to MF20, a composite picture was used with both the blue channel (Hoechst, nuclei) and the red channel (MF20, heavy chain myosin).

Prism software was used to draw the whisker boxes as well as to perform Mann Whitney tests to compare two distributions.

Results

Mechanical properties

Results of mechanical tests are displayed on Fig. 1.B. First, we noticed that the three types of threads exhibit a strain stiffening behavior. Two Young moduli were then measured: at low strain, noted E0, and during strain-stiffening, written E1 (Fig. 1.B.1). For both moduli, the difference between collagen and fibrin stands out: collagen is much stiffer (E0 = 0.57 ± 0.05 MPa, E1 = 1.11 ± 0.13 MPa) than fibrin (E0 = 0.10 ± 0.02 MPa, E1 = 0.21 ± 0.06 MPa). Mix threads exhibit intermediate stiffness (E0 = 0.33 ± 0.05 MPa, E1 = 0.83 ± 0.19 MPa) between collagen and fibrin.

Fibrin threads exhibit higher strain at break than collagen (251 ± 63 % vs. 75 ± 15 %) (Fig. 1.B.2) but lower ultimate tensile strength (0.33 ± 0.04 MPa vs. 0.70 ± 0.11 MPa) (Fig. 1.B.3). Interestingly, mix threads seem to break at even lower strain than collagen (57 ± 21 %) with an ultimate tensile strength slightly higher than for fibrin (0.42± 0.2 MPa).

Structure

Investigation of the microstructure of threads was performed on ultrathin transverse sections of threads using Transmission Electron Microscopy (Fig.

Tenoblasts culture on threads

Tenoblasts were seeded on the three types of threads and observed after 4 days in high-serum medium (Fig. 2.A) and after 7 days in high-serum medium plus 3 days in low-serum medium (Fig. 2.B). After 4 days, we observed that tenoblasts on collagen threads would form cell "crusts" that detached from the thread (Fig. 2.A.1). On mix and fibrin threads, tenoblasts cover threads with high cell density, without cell crust detaching from them (Fig. 2.A.2 & A.3 resp.). The detachment of cells causes the cell density to drop on collagen (median ≈ 200 cell/mm²) compared to mix (med. ≈ 1100 cell/mm², p=0.012) and fibrin (med. ≈ 1000 cell/mm², p=0.006) threads, with no significant difference between mix and fibrin (Fig. 2.A.4).

After 7 days in high-serum medium plus 3 days in low-serum medium, few cells remain on collagen threads (Fig. 2.B.1) while mix and fibrin threads still appear covered with cells (Fig. 2.B.2 & B.3 resp.). On both types of threads, cells seem to align along the main axis of the threads. Cell density is extremely low on collagen threads (med. < 100 cell/mm²), much below the ones on mix threads (med. ≈ 700 cell/mm², p<0.0001) and fibrin threads (med. ≈ 350 cell/mm², p=0.020) (Fig. 2.B.4). Compared to cell density after 4 days in high-serum medium, a decrease is observed on mix threads (p=0.0014) and even more on fibrin threads (p=0.0016), resulting in a lower density on fibrin than on mix threads (p=0.0059). The decrease occurs during the low-serum culture, as no significant change in cell density could be observed for both types of threads at day 7 (data not shown).

Myoblasts culture on threads

Seeding of myoblasts on the three types of threads resulted in a poor adhesion of the cells on collagen threads after 4 days in high-serum medium (Fig. 3 ). Most myoblasts on mix and fibrin threads were positive to MF20 staining exhibiting production of myosin heavy chains, a late-differentiation marker. Quantitatively, after 4 days, cell density was dramatically lower on collagen (med. = 3 cell/mm²) than on mix (med. ≈70 cell/mm², p<0.001) and fibrin threads (med. ≈ 90 cell/mm², p<0.001) (Fig. 3.A.4). After 3 days of differentiation, cellular densities exhibited higher median value on both mix (med. ≈200 cell/mm² p=0.035) and fibrin (med. ≈ 300 cell/mm², ns) compared to 4 days of proliferation. Myoblasts on collagen were too scarce to enable statistically relevant count. No significant difference in cell density between mix and fibrin could be observed either after 4 days of proliferation or 3 days in differentiation. A slight difference in MF20 positive myoblast ratio was however noticed with 84 % of MF20+ cells on mix compared to the 80% on threads (p=0.015) (day 7+3). Interestingly, myoblasts remained round-shaped on both types of threads and no trace of spindle-shaped or fused cells could be observed. Prolonged culture in low-serum medium (5 days) and in presence of Wnt7a at 50 ng/mL fostered faint spindle shape but no fusion as seen on Supp.

Myoblast culture in 2D

To investigate the role played by the threads substrates on myoblasts fusion, compared to 2D substrates, the same solutions as the ones used for thread production were coated on well-plates. Myoblasts were also seeded on Matrigel-coated wells as a control. As seen on Fig. 4, several features stand out. Myoblasts seeded on collagen exhibited scarce spindle shapes and extremely low numbers of fused cells (Fig. 4.A). On the contrary, myoblasts cultured on mix (Fig. 4.B) and fibrin wells (Fig. 4.C) could elongate and formed myotubes, with an apparent number of myotubes being higher on mix. Myoblasts on Matrigel formed more and much larger myotubes (Fig. 4.C).

Discussion

We have produced in this work three types of threads, one made of pure collagen, one of pure fibrin and the last one of a mix of collagen and fibrin at a ratio 3:2 in mass. These threads differing in terms of present also different mechanical properties.

Mechanical properties

In tensile testing, we noticed that the three types of threads exhibit a strain stiffening behavior characteristic of fibrin and collagen materials [START_REF] Munster | Strain history dependence of the nonlinear stress response of fibrin and collagen networks[END_REF] . As expected, collagen threads exhibited stiffer behavior, with higher strength, and lower strain at break compared to fibrin, which could be stretched up to approximately 250%. In accordance with first intuitions, mix threads had intermediate stiffness and strength between fibrin and collagen. Mix threads had however lower strain at break than collagen.

Interestingly, our results are consistent with the ones obtained by Lai et al. [START_REF] Lai | Mechanical behavior of collagenfibrin co-gels reflects transition from series to parallel interactions with increasing collagen content[END_REF] who studied mechanical properties of lower concentration (2 to 3 mg/mL in proteins) collagen/fibrin gels with different proportions from 100 % collagen to 100 % fibrin to understand the nature of the networks and their relationships in fibrin/collagen composites. The reported values of modulus and strain at failure of composites at 60 % collagen are closer to those for pure collagen than for pure fibrin. They demonstrated that such behavior could not be explained by a simple parallel (i.e. non interacting networks) or series (i.e. co-dependent networks) model of collagen and fibrin materials. They partially explained mechanical properties obtained in this study in a second article using a model of non-percolating network in which islands of the diluted protein are embedded in the concentrated one [START_REF] Nedrelow | Mechanics of a two-fiber model with one nested fiber network, as applied to the collagen-fibrin system[END_REF] . Such conclusion is in total agreement with the structure observed in mix threads, with islands of fibrin inside the collagen matrix (Fig. 1.C.2). While mix threads are macroscopically homogeneous as observed on threads made from FITClabelled fibrinogen, it seems that a phase separation occurs microscopically, resulting in islands of fibrin in a collagen matrix, for the ratio collagen:fibrin used here.

Structure of collagen threads with small fibrils with local orientation is typical of highly concentrated collagen scaffolds [START_REF] Gobeaux | Fibrillogenesis in Dense Collagen Solutions: A Physicochemical Study[END_REF] and was already observed in threads extruded in high osmotic pressure buffer [START_REF] Picaut | Pure dense collagen threads from extrusion to fibrillogenesis stability[END_REF] .

Fibrin's structure is similar to the structure we observed in fibrin threads extruded in Dulbecco's Modified Medium with a fibrillar structure on the outside of the aggregates instead of the thin bridging fibers, probably due to high osmotic pressure exerted by the high PEG concentration in the extrusion buffer. Same structure is observed for fibrin islands in mix threads. The small size of islands and the low distance between them (<1µm) ensure that cells can access both collagen and fibrin materials. It may be interesting to think about the "clustering" effect of these islands on cellular response, compared to a situation where collagen and fibrin would be thouroughly mixed, as clustering effect has shown to have paramount effect on cellular response.

Tenoblasts

Several features stood out from the culture of tenoblasts on collagen, mix and fibrin threads. First, tenoblasts rapidly proliferated rapidly on collagen threads, due to high affinity, and rapidly formed a crust around the threads, made of interconnected tenoblasts and secreted ECM [START_REF] Stoll | Extracellular matrix expression of human tenocytes in three-dimensional air-liquid and PLGA cultures compared with tendon tissue: Implications for tendon tissue engineering[END_REF][START_REF] Subramanian | Tendon development and musculoskeletal assembly: emerging roles for the extracellular matrix[END_REF] . After only 4 days in culture the crust detached from collagen threads suggesting that collagen is not a suitable substrate to enable long-term culture of tenoblasts. The difference in mechanical properties as well as the tendency of cells to contract their surrounding matrix may cause the observed crust-detaching effect.

Second, the cell density drops on mix and especially on fibrin threads between day 7 and day 7+3. This drop is likely to be caused by the switch from high-serum to low-serum medium. While mix and fibrin had similar densities at day 4 and 7, cell density is lower at day 7+3 on fibrin than on mix. The combination of collagen and fibrin thus seems beneficial to maintain tenoblasts survival in low serum.

On fibrin threads, cell density seems rather stable during the 7 days of proliferation. After 7 days (data not shown), some small cell plaques seem to detach but without significantly affecting cell density when compared to density at day 4. After 3 days in differentiation medium, cell density decreased. This may be due to the change to low serum-content medium, or to the detachment due to confluence in a similar fashion as for collagen.

Third, seem to spontaneously align along the direction of the thread on both mix and fibrin threads. This may be explained by a minimization of the stress on internal structure due to bending as already pointed out [START_REF] Rovensky | Morphogenetic response of cultured normal and transformed fibroblasts, and epitheliocytes, to a cylindrical substratum surface[END_REF] .

Myoblasts

The observed low adherence of myoblasts on collagen threads may appear surprising as primary myoblasts are routinely cultured on collagen-coated plates for proliferation [START_REF] Grand | Six1 regulates stem cell repair potential and self-renewal during skeletal muscle regeneration[END_REF] . Actually, on collagencoated dishes myoblasts adopt a round shape demonstrating low affinity to collagen whereas they strongly adhere on coating containing laminin and/or fibronectin such as Matrigel® and adopt tapered shape [START_REF] Lukjanenko | Loss of fibronectin from the aged stem cell niche affects the regenerative capacity of skeletal muscle in mice[END_REF] . In fact, myoblasts do not bind directly to collagen but through fibronectin [START_REF] Kleinman | Role of collagenous matrices in the adhesion and growth of cells[END_REF] , and the one provided by the addition of serum is supposed to be sufficient to permit adhesion of myoblasts [START_REF] Chiquet | Fibronectin mediates attachment of chicken myoblasts to a gelatin-coated substratum[END_REF] .

Given the higher densities on mix and fibrin threads, myoblasts seemed to adhere more on fibrin. Myoblasts can bind directly to fibrin through integrin receptors [START_REF] Chiron | Complex Interactions between Human Myoblasts and the Surrounding 3D Fibrin-Based Matrix[END_REF] . The cell density on mix threads was each time comparable to the one on fibrin. It is surprising that mix threads induced similar response as pure fibrin, given the relatively low content in fibrin compared to collagen. Fibrin islands contained in mix threads were thus sufficient to provide similar adhesion of cells to threads as on pure fibrin. Given the absence of adhesion of myoblasts on collagen threads, we can hypothesize that myoblasts attach to fibrin islands instead of collagen matrix, and thus "feel" a fibrin composition. However, given the size of islands embedded in the collagen matrix, it is probable that cells, when pulling on focal adhesion, rather "feel" the stiffness of the collagen matrix. Mix materials may then appear as a way to decouple the composition and mechanical properties of the substrate and thus show that the lack of adherence of myoblasts to collagen threads is rather due to the poor binding to collagen compared to fibrin, rather than a too high stiffness of the substrate.

Concentration of proteins in threads may also play a role. Gerard et al. [START_REF] Gerard | Fibrin Gel Improves the Survival of Transplanted Myoblasts , Fibrin Gel Improves the Survival of Transplanted Myoblasts[END_REF] that concentration of fibrin had an effect on myoblasts entrapped in a fibrin gel. They demonstrated that loose gels (3mg/mL) promoted higher proliferation and metabolic activity compared to 50 mg/mL gels.

The absence of fusion of myoblasts into myotubes was unexpected as fibrin has been widely used for muscle constructs [START_REF] Page | Restoration of Skeletal Muscle Defects with Adult Human Cells Delivered on Fibrin Microthreads[END_REF][START_REF] Kang | A 3D bioprinting system to produce human-scale tissue constructs with structural integrity[END_REF][START_REF] Huang | Rapid formation of functional muscle in vitro using fibrin gels[END_REF][START_REF] Bian | Engineered skeletal muscle tissue networks with controllable architecture[END_REF] and is believed to be the gold-standard material [START_REF] Koning | Current opportunities and challenges in skeletal muscle tissue engineering[END_REF] . However, myoblasts on threads expressed myogenin and heavy myosin chains, two markers muscle cell terminal differentiation. The high density of multinucleated myotubes on 2D Matrigel control proves that the absence of fusion on the threads is not due to low cellular density or to an inability of the primary myoblasts we used to fuse. We tested on differentiated cells on threads the stimulation by Wnt7a that, as previously reported, is an inducer of muscle cell fusion [START_REF] Lacour | R-spondin1 Controls Muscle Cell Fusion through Dual Regulation of Antagonistic Wnt Signaling Pathways[END_REF] . We only observed a modest effect on cell sprawling and polarization [START_REF] Grand | Wnt7a Activates the Planar Cell Polarity Pathway to Drive the Symmetric Expansion of Satellite Stem Cells[END_REF] , still too low to enable cell fusion. Given the fact that most cells expressed were terminally differentiated, and kept a round "myoblastic" shape on threads, it seemed that the absence of fusion is due to a problem of adhesion of cells to the matrix.

The comparison between culture on threads and 2D culture on coated wells enables to point out two distinct reasons that seem to play a role in the absence of fusion of myoblasts. The first is due to the composition of the thread. Collagen alone seems to be particularly non-permissive to myocyte fusion. Differentiation of myoblasts without fusion has already been observed on collagen-coated plates [START_REF] Heino | The collagen family members as cell adhesion proteins[END_REF] . On the contrary to collagen, fibrin-coated plates comports multiple myotubes. The combination of collagen and fibrin mix plates may have a synergetic effect and as they seem to promote even more myotube fusion. Number and size of myotubes are however much lower than on Matrigel, which contains multiple ECM components. The lack fusion may thus result from the lack of certain components of the ECM such as laminin, shown to be paramount for cell fusion [START_REF] Goody | Hanging on for the ride: Adhesion to the extracellular matrix mediates cellular responses in skeletal muscle morphogenesis and disease[END_REF] and often used to promote myoblasts adhesion and fusion [START_REF] Larkin | Structure and functional evaluation of tendon-skeletal muscle constructs engineered in vitro[END_REF][START_REF] Lam | Microfeature guided skeletal muscle tissue engineering for highly organized 3-dimensional free-standing constructs[END_REF] .

A clear difference could however be observed between myoblasts seeded on 2D fibrin or mix, compared to myoblasts seeded on threads. Multiple myotubes and spindle-shaped cells could be observed in 2D while no fusion was witnessed on threads, and cell polarization and elongation was tediously and limitedly obtained using Wnt7a. As the materials used in 2D and threads are identical, the absence of fusion is likely to be due to the topology of the threads, ie. the curvature. Curvature has already been reported to influence cell migration and differentiation 67 but to our knowledge, no study has shown that substrate curvature inhibited myoblast fusion. In 1994, Rovensky et al. reported that cells with straight actin filament such as fibroblasts tend to align in the direction of lower curvature to minimize stress [START_REF] Rovensky | Morphogenetic response of cultured normal and transformed fibroblasts, and epitheliocytes, to a cylindrical substratum surface[END_REF] . We can hypothesize that the myoblasts do not fuse and do not even elongate because they are highly sensitive to internal stress.

It is known that muscle satellite cells do not fuse on an isolated myofibre while they fuse once plated on a Matrigel®-coated plate [START_REF] Zammit | Muscle satellite cells adopt divergent fates: a mechanism for self-renewal?[END_REF] . The shape and curvature of the substrate may then inhibit fusion. Myoblasts on our threads seemed to behave in the same fashion as satellite cells on isolated myofibres, adopting the same shape and unable to fuse. The topological inhibition is surprising given the difference of dimension compared to myofibers with diameters of a few tens of micrometers [START_REF] Harber | Aerobic exercise training improves whole muscle and single myofiber size and function in older women[END_REF][START_REF] Collins | Stem Cell Function, Self-Renewal, and Behavioral Heterogeneity of Cells from the Adult Muscle Satellite Cell Niche[END_REF] . The curvature inhibition may prevent differentiation of satellite cells at the surface of undamaged myofibers.

The threads developed may then represent an interesting model of isolated myofibers. This is also encouraging for muscular reconstruction as satellite cells adopt such a behavior when anchored to an isolated fiber but behave differently in a 3D environment, in the niche comprised between multiple fibers. Use of multiple braided threads for instance, possibly held together by a loose gel, may then form a niche model for myoblasts and effectively promote myotube formation and muscle reconstruction.

Conclusion

We developed here collagen, fibrin and mixed threads and used them as substrates of different mechanical properties, collagen threads being stiffer than fibrin ones. Mix threads demonstrated properties similar to collagen due to their non percolating fibrin island network in the collagen matrix. Such substrates enable to decouple mechanical properties from chemical interaction by providing fibrin islands for the cells to adhere with the stiffness of the embedding collagen network. Both primary tenoblasts and myoblasts on mix threads behave similarly to fibrin threads, rather than collagen, highlighting the importance of the chemical nature of the cell/matrix interaction.

Even myoblasts differentiated on mix and fibrin threads, they did not fuse. Investigations carried out seem to point out a possible inhibition of fusion due to substrate curvature. Further characterization of such phenomenon should be undertaken as it may provide a better understanding of the satellite cells differentiation commitment all the more since prevention of satellite cell differentiation is a challenge for muscular reconstruction [START_REF] Quarta | An artificial niche preserves the quiescence of muscle stem cells and enhances their therapeutic efficacy[END_REF] . Indeed, surface curvature seems to be a parameter of choice to foster or not the differentiation of satellite cells and ensure differentiation only upon fiber damage.

The work reported here is a first step toward myo-tendinous junction modelisation. The next step would be to co-culture both tenoblasts and myoblasts on our substrates, as we demonstrated their viability in the same medium, to understand the cooperative role they may play. As fibrin, collagen and mix threads are produced in the same conditions, collagen:fibrin ratio of mix threads can be tuned to investigate cell response to substrate and find the optimal composition for tenoblasts and myoblasts. Even further, pluripotent stem cells could be used and composition of mix threads adapted to favor tenogenic or myogenic commitment.

  1.C). Collagen threads are made of thin fibers with different orientations, among which a significant part is pointing toward the direction of observation, i.e. along the thread axis (blue arrow, Fig.1.C.1). Fibrin threads exhibit dense aggregate-like structures with some fibrous-like structures on the outside (red arrow, Fig.1.C.3). Mix threads structure is composed of small islands of fibrin materials (noted F on Fig.1.C.2), embedded into a collagen matrix (C Fig.1.C.2), made of thicker fibers than the ones observed in collagen alone.

Figure 1 :

 1 Figure 1 : Threads structural & mechanical properties. (A) Macroscopic photos of the three types of threads: collagen, mix and fibrin. (B) Mechanical properties of the three types of threads: (B.1) low-strain E0 (full histogram) and high-strain E1 (empty histogram) moduli, (B.2) strain at break, (B.3) ultimate tensile strength (UTS). Cross sections of the (C.1) collagen, (C.2) mix and (C.3) fibrin threads as observed by transmission electron microscopy. Blue arrow points at collagen fibers aligned along the thread. Red arrow points at the fibrous-like structure at the surface of the fibrin aggregates.

Figure 2 :

 2 Figure 2: Tenoblast colonization of the three types of threads after day 4 (A) and 7+3 (B). Fluorescence microscope images of a collagen, mix and fibrin threads after 4 days in high-serum medium (A.1, A.2 and A.3 resp) and after 7 days in high-serum medium and 3 days in low serum medium (B.1, B.2 and B.3 resp.), and the resulting tenoblasts densities at day 4 (A.4) and day 7+3 (B.4). Blue staining: Hoechst; red: F-actin.

Figure 3 :

 3 Figure 3: Myoblast colonization of the three types of threads after day 4 (A) and 7+3 (B). Fluorescence microscope images of a collagen, mix and fibrin threads after 4 days in high-serum medium (A.1, A.2 and A.3 resp.) and after 7 days in high-serum medium plus 3 days in low serum medium (B.1, B.2 and B.3 resp.), and the resulting myoblasts densities at day 4 (A.4) and day 7+3 (B.4). (B.5) Proportion of MF20 positive myoblasts over the whole number of myoblasts on mix and collagen threads. Blue staining: Hoechst; red: F-actin. Staining Blue: Hoechst, Red: myosin heavy chains MF20.

  .A.1) while mix and fibrin threads were well colonized (Fig.A.2 & A.3 resp.). Same trend was observed after 3 days of differentiation, with rare cells found on collagen (Fig.3.B.1) and well colonized mix and fibrin threads (Fig.3.B.2 & B.3 resp.

Fig. 1 .Fig. 1 :

 11 A &B. As a reference, same line cultured on a 2D substrate coated with Matrigel exhibited high number of myotubes (Supp. Fig. 1.C), in spite of the same cellular density (Supp. Fig. 1.D). Supp. Primary myoblasts cultured for 7 days in proliferation and 5 days in differentiation with Wnt7a at 50 ng/mL. Immunofluorescence staining images of myoblasts on mix (A) and fibrin (B) threads and on Matrigel®-coated plates (C) (blue: Hoechst, red: myosin heavy chains, magenta: myogenin, green: fibrin marked FITC). (D) Resulting cell densities on plates and threads.

Figure 4 :

 4 Figure 4: Primary myoblasts cultured for 1 day in proliferation and 6 days in differentiation . Immunofluorescence staining images of myoblasts on collagen (A), mix (B), Matrigel® (C) and fibrin (D) coated plates (blue: Hoechst, red: myosin heavy chains


Clément Rieu
email: clement.rieu@upmc.fr

Gervaise Mosser
email: gervaise.mosser@upmc.fr

Bernard Haye
email: bernard.haye@upmc.fr

Nicolas Sanson
email: nicolas.sanson@espci.fr

Thibaud Coradin
email: thibaud.coradin@upmc.fr

Lea Trichet
email: lea.trichet@upmc.fr

Léa Trichet

Thrombin-free polymerization leads to pure fibrin(ogen) materials with extended processing capacity

come

Introduction

Fibrin is a gold standard material in tissue engineering 1,[START_REF] Janmey | Fibrin gels and their clinical and bioengineering applications[END_REF] . It exhibits high bioactivity thanks to the direct adhesion sites for integrins [START_REF] Flick | Leukocyte engagement of fibrin(ogen) via the integrin receptor αMβ2/Mac-1 is critical for host inflammatory response in vivo[END_REF][START_REF] Lishko | Multiple Binding Sites in Fibrinogen for Integrin α M β 2 (Mac-1)[END_REF] , which impact cell migration, proliferation and fate [START_REF] Catelas | Human Mesenchymal Stem Cell Proliferation and Osteogenic Differentiation in Fibrin Gels in Vitro[END_REF][START_REF] Willerth | Optimization of fibrin scaffolds for differentiation of murine embryonic stem cells into neural lineage cells[END_REF] . Fibrin also indirectly interacts with cells through binding to extracellular matrix proteins [START_REF] Makogonenko | Interaction of Fibrin(ogen) with Fibronectin: Further Characterization and Localization of the Fibronectin-Binding Site[END_REF] , polysaccharides [START_REF] Weigel | The specific interaction between fibrin(ogen) and hyaluronan: possible consequences in haemostasis, inflammation and wound healing[END_REF] and growth factors [START_REF] Sahni | Binding of Basic Fibroblast Growth Factor to Fibrinogen and Fibrin[END_REF] . It is widely used clinically, mainly as a hemostat, sealant and adhesive. In vivo, fibrin is a major actor of haemostasis [START_REF] Semple | Platelets and the immune continuum[END_REF] , which is induced by a complex reaction cascade initiated by the adhesion of platelets at the site of injury [START_REF] Ham | Thrombin use in surgery: an evidence-based review of its clinical use[END_REF][START_REF] Davie | The coagulation cascade: initiation, maintenance, and regulation[END_REF] . A key step is the thrombin mediated conversion of circulating fibrinogen into fibrin, which subsequently polymerizes to form a tight plug [START_REF] Wolberg | Thrombin generation and fibrin clot structure[END_REF] .

Fibrinogen is a 340 kDa protein made of three pairs of chains, called α, β and γ that assemble in a specific symmetric shape, forming 3 aligned globules linked together by coiled coil connectors constituted by the three different chains [START_REF] Medved | on behalf of fibrinogen and factor XIII subcomittee of the scientific standardization comittee of the international society on thrombosis and haemostasis. Recommendations for nomenclature on fibrinogen and fibrin[END_REF] (Fig. 1.A.1). The central domain, called E, gathers the amino-terminal parts of the 6 chains, linked together by di-sulfide bonds. The distal domains, called D, are made of the carboxy-terminal ends of the β and γ chains. The carboxy-terminal ends of α chains, called αCs, further extend in a random-coiled manner, with globular domains at their ends. Those domains interact together and with the E domain, and hence do not float freely. During clotting, thrombin cleaves two pairs of fibrinopeptides FpA and FpB from the central E nodule, (Fig. 1.A.2 (0), where only two out of four Fps are drawn for clarity purpose) [START_REF] Doolittle | Fibrinogen and Fibrin[END_REF] . This cleavage reveals two sites A and B which bind to sites a and b, respectively located on the γ and β chains in the D nodule of an adjacent molecule. Such interactions promote the polymerization of fibrin monomers in a half staggered manner (Fig. 1.A.2 (0')), resulting in gels (FbN) (Fig. 1.A.3) with a highly fibrillar network (Fig. 1.A.4). A certain delay, called clotting time, exists before the formation of a macroscopic clot [START_REF] Wolberg | Thrombin generation and fibrin clot structure[END_REF] . Lateral assembly of protofibrils only occurs at the end of the clotting time, which can last from seconds to hours depending on pH and ionic strength [START_REF] Scheraga | The thrombin-fibrinogen interaction[END_REF] .

Difficulty to shape natural materials into 3D structures remains a major obstacle to mimic cell environment [START_REF] Tibbitt | Hydrogels as extracellular matrix mimics for 3D cell culture[END_REF] . The necessity to mix fibrinogen and thrombin to trigger self-assembly and the involved delay may account for the scarcity of work on fibrin shaping into 3D structures. Fibrin is mostly used in the form of gels that can be molded [START_REF] Jockenhoevel | Fibrin geladvantages of a new scaffold in cardiovascular tissue engineering[END_REF] , or in addition to other materials [START_REF] Kang | A 3D bioprinting system to produce human-scale tissue constructs with structural integrity[END_REF] . Some attempts have been carried out to produce fibrin microbeads [START_REF] Gorodetsky | The use of fibrin based matrices and fibrin microbeads (FMB) for cell based tissue regeneration[END_REF] or nanostructures [START_REF] Praveen | Fibrin nanoconstructs: a novel processing method and their use as controlled delivery agents[END_REF][START_REF] Rejinold | Development of novel fibrinogen nanoparticles by two-step co-acervation method[END_REF] . So far, only one process has been reported for pure fibrin extrusion [START_REF] Cornwell | Discrete crosslinked fibrin microthread scaffolds for tissue regeneration[END_REF] , where fibrinogen and thrombin kept in different solutions are mixed together with a Y connector before extrusion. In sharp contrast, collagen, another gold standard material in tissue engineering, has been extensively studied [START_REF] Picaut | Pure dense collagen threads from extrusion to fibrillogenesis stability[END_REF] . The simplicity of collagen self-assembly, induced by neutralization or 37°C heat activation, makes it favorable for multiple shaping techniques, such as molding or extrusion [START_REF] Rieu | From Tendon Injury to Collagen-based Tendon Regeneration: Overview and Recent Advances[END_REF] . To investigate if similar processes could be used to trigger fibrin polymerization and thus widen the possibilities in fibrin shaping, we thus focused on fibrinogen and fibrin solutions at acidic pH.

Acidic fibrinogen gel formation

Stock solutions of fibrinogen (FbgSt) at 40 mg/mL were acidified to pH 3.6, the highest pH below the isoelectric point (~5.5) at which the solution remained stable at ambient temperature (20°C) without precipitating. Without any addition of thrombin these acidic solutions (FbgA) incubated at 37°C formed within an hour a transparent brittle gel (FbgA(G)) (Fig. 1.B.1), while solutions left at ambient temperature set to a gel only after several weeks. Gel formation kinetics was increased at lower pH, with instantaneous gelation at pH 2.5. Formation of fibrinogen transparent gels at acidic pH had already been briefly evoked by Fay & Hendrix 26 and Mihalyi [START_REF] Mihalyi | Physicochemical studies of bovine fibrinogen : III. Optical rotation of the native and denatured molecule[END_REF] . However no study of this phenomenon has been carried out so far.

Gel formation from acidic solutions at pH 3.6 was dynamically investigated by switching the temperature of geometries from 20°C to 37°C after 5 minutes of rheological properties measurement (Fig. 1.B.2). For approximately 15 minutes G' remained constant, and then rose to reach approximately 1 kPa after three hours, overtaking G" (factor > 10). During frequency sweep on the newly formed gels FbgA(G), G' increased with frequency, with an inflection point at 1.6 rad/s, while G" exhibited a reverse bell shape (SI Appendix, Fig. S1). During amplitude sweep, gels exhibited first a plateau with G' around 1 kPa till around 20 % amplitude, then a 3 to 4-fold strain stiffening until 130% strain (Fig. 1.B.3). As a comparison Wufsus et al. reported a G' value one order of magnitude higher for "classical" FbN clots made from 30 mg/mL and 50 mg/mL initial fibrinogen solutions [START_REF] Wufsus | Elastic Behavior and Platelet Retraction in Low-and High-Density Fibrin Gels[END_REF] . Structure-mechanics relationship in "classical" fibrin clots have been widely investigated but no general picture has been proposed yet. Non-linear elasticity of fibrin clots FbN originates from the multi-scale hierarchical structure [START_REF] Piechocka | Multi-scale strain-stiffening of semiflexible bundle networks[END_REF][START_REF] Litvinov | Fibrin mechanical properties and their structural origins[END_REF] . The observed behavior in frequency sweep of FbgA(G) differs from that of classical clots and exhibits a signature reminiscent of rubber plateau in polymer melts with high-molecular-weight entangled chains [START_REF] Liu | Evaluation of different methods for the determination of the plateau modulus and the entanglement molecular weight[END_REF] . However, the strain-stiffening behavior is characteristic of biological gels and in particular of fibrin [START_REF] Piechocka | Multi-scale strain-stiffening of semiflexible bundle networks[END_REF] . AFM force-unfolding experiments on fibrin proteins suggest that clot elasticity may arise from coiledcoil and γ-nodule unfolding, and from αCs interactions [START_REF] Lim | Molecular Basis of Fibrin Clot Elasticity[END_REF][START_REF] Zhmurov | Mechanism of Fibrin(ogen) Forced Unfolding[END_REF] . Coiled-coil and/or γ nodule unfolding might then account for elasticity and strain stiffening behavior of the acidic gels, while it is unlikely that αCs are involved as they do not interact at acidic pH [START_REF] Veklich | Carboxyl-terminal Portions of the Alpha Chains of Fibrinogen and Fibrin[END_REF] .

Transmission Electron Microscopy (TEM) observations of sections of fibrinogen gels FbgA(G) included in araldite display a network of fine entities without any specific pattern (Fig. 1.B.4).

After chopping a gel in water and depositing the supernatant on a carbon coated grid, some large fiber-like structures can be observed (Fig. 1.B.5). In comparison, a "classical" fibrin clot made by mixing fibrinogen and thrombin at neutral pH exhibits a highly fibrillar network with fibers of various sizes (Fig. 1.A.4).

Underlying Mechanism of acidic gel formation

Acidic fibrinogen gels FbgA(G) were surprisingly stable to most conditions known to disperse protein-based hydrogels (SI Appendix, Table S1). Change to neutral or basic pH did not dissolve the gels. They were resistant to low and high ionic strength, as well as to high temperature and salt-in solutions. Gels were however soluble in urea. As expected they could also be dissolved in DTT, since the constituting fibrinogen chains are linked together by multiple disulfide bonds. SDS electrophoretic gels of acidic fibrinogen gels dissolved in urea confirmed that no major cleavage was provoked by the acidic conditions and by the thermal treatment at 37°C (SI Appendix, Fig. S2). MALDI-TOF performed on fibrinogen acidic gels FbgA(G) showed that no fibrinopeptide could be detected after acidic gel formation, while both Fps A and B were visible for fibrin clots FbN (SI Appendix, Fig. S3). Thus, the mechanism involved in gel formation at acidic pH is different from the "classical" one (0+0', Fig. 1.A.2).

Circular dichroism measurements on fibrinogen stock FbgSt and acidified solutions FbgA demonstrate a change in the secondary structure upon acidification, with a decrease in intensity of the 225 nm peak (Fig. 2.A). No further change in acidic solution signature could be observed upon incubation at 37°C, even after formation of a gel FbgA(G). The change appears to be reversible for the solutions FbgA when raising the pH above the pI (Fig. 2.A). CD spectra of Fbgst have already been reported and a consensus of approximately a third of α-helix content and a third of random structure exists, while the content of the remaining structures fluctuates according to studies [START_REF] Chen | Thermal conformational changes of bovine fibrinogen by differential scanning calorimetry and circular dichroism[END_REF][START_REF] Upchurch | Prothrombotic consequences of the oxidation of fibrinogen and their inhibition by aspirin[END_REF][START_REF] Budzynski | Difference in conformation of fibrinogen degradation products as revealed by hydrogen exchange and spectropolarimetry[END_REF] .

In our case the change in structure upon acidification could not be reliably attributed. Similar spectra were however observed in different fibrinogen fragments [START_REF] Budzynski | Difference in conformation of fibrinogen degradation products as revealed by hydrogen exchange and spectropolarimetry[END_REF][START_REF] Medved | Isolation of thermostable structure from the fibrinogen D fragment[END_REF][START_REF] Fan | Effect of pH on the Conformation and Backbone Dynamics of a 27-Residue Peptide in Trifluoroethanol : an NMR and CD study[END_REF] and partially heat-denatured fibrinogen [START_REF] Chen | Thermal conformational changes of bovine fibrinogen by differential scanning calorimetry and circular dichroism[END_REF] , which tends to point out a modification of the helical structure and/or the partial denaturation of the D domain. Differential Scanning Calorimetry of fibrinogen stock FbgSt exhibits a major peak around 63°C (Fig. 2.B). This low temperature peak of fibrinogen at neutral pH is attributed to the D domain [START_REF] Privalov | Domains in the Fibrinogen Molecule[END_REF] .

When acidified, three peaks appear: a major peak around 52°C and a shoulder around 65°C, corresponding to the thermolabile part of the D domain and to the αCs, respectively [START_REF] Privalov | Domains in the Fibrinogen Molecule[END_REF] , followed by a third peak close to 85°C, attributed to the E domain. The thermolabile D domain is thus destabilized by the acidic condition with a low temperature peak shift of -10°C, in accordance with previous results [START_REF] Privalov | Domains in the Fibrinogen Molecule[END_REF] . When the acidic solution FbgA is first kept at 37°C for 1 hour before the scan, only the high temperature peak around 85°C remains (Fig. 2.B). The incubation of acidic solutions at 37°C for 1 hour thus leads to vanishing of the low temperature peak attributed to the thermolabile D domain [START_REF] Privalov | Domains in the Fibrinogen Molecule[END_REF] . After incubation, the absence of the shoulder at 65°C, corresponding to the αCs and reported to be reversible upon cooling [START_REF] Medved | Structural organization of C-terminal parts of fibrinogen Aα-chains[END_REF] , could be due to a too low sensitivity of our setup.

Observation of dilute acidic fibrinogen solutions FbgA by TEM shows that fibrinogen proteins tend to aggregate preferentially by their extremities (Fig. 2.C), on the opposite to fibrinogen proteins from stock solution FbgSt, which appear dispersed (Fig.

Effect of neutralization on fibrinogen and fibrin acidic gels

Fibrinogen

The acidic fibrinogen gels FbgA(G) are not soluble when dipped in a neutral solution and no major change in aspect such as opacification or gel contraction can be observed. The change in rheological properties upon neutralization of acidic gels FbgA(G) was studied by forming a gel at 37°C in acidic condition and then adding a neutral solution of 100 mM Hepes around the geometries (Fig. 3.A, blue). This resulted in a gel FbgN(G) with a two-fold increase in storage modulus as well as a different signature in frequency sweep which now increases linearly with the frequency (SI Appendix, Fig. S5). These modified mechanical properties may be related to a change in structure. The increase in G' and G" with frequency is closer to fibrin clot FbN signature [START_REF] Wufsus | Elastic Behavior and Platelet Retraction in Low-and High-Density Fibrin Gels[END_REF] . This modification is not surprising as protein global charge is modified secondary to pI crossing. In addition to this, the αCs may form intermolecular interactions. Veklich et al. reported that αCs interact together and also with the E domains at neutral pH, while they float freely in acidic conditions. They also demonstrated that αCs cleaved from fibrinogen were dispersed in acidic conditions and polymerized into fibers upon neutralization. It is then likely that the αCs float during gel formation at acidic pH and interact together upon neutralization. This would be facilitated by the forced proximity between proteins in the network. Regarding the amplitude sweep a similar behavior as in the acidic condition was observed (Fig. 3.B), with a G' plateau for low amplitudes, at approximately twice the value, but only a slight increase of peak amplitude between 100 and 200 % strain. Such a phenomenon may be due to a partial initial stretching of the proteins inside the network upon neutralization (Fig. 3.C (4)) because of internal constraints related to electrostatic repulsion and/or αCs interactions in the neutral gels.

Fibrin

Till now, no thrombin was introduced in the solutions, gels being made of fibrinogen proteins (as confirmed by MALDI-TOF results). It was shown in literature that addition of thrombin to fibrinogen in acidic conditions effectively cleaves the Fps, transforming fibrinogen FbgA into fibrin monomer FbA but without inducing clotting [START_REF] Laki | The polymerization of proteins : the action of thrombin on fibrinogen[END_REF] , which becomes instantaneous upon neutralization. The influence of thrombin addition to our system was therefore investigated. FbA(G) gels from acidic fibrin solutions FbA could be formed by incubation at 37°C, in the same fashion as FbgA(G), without any impact on kinetics or gel aspect. Same rheological study of acidic gel formation and subsequent neutralization was carried out from fibrin solution FbA as for fibrinogen FbgA. No significant difference either in formation kinetics or mechanical properties could be observed between FbA(G) and FbgA(G), as well as between FbN(G) and FbgN(G) (Fig. 3.A). MALDI-TOF performed on a FbA(G) gel formed by incubation at 37°C confirmed that FpA and FpB are effectively cleaved upon addition of thrombin to acidic fibrinogen (SI Appendix, Fig. S3).

Thus, the removal of the Fps by thrombin does not seem to affect the acidic gel formation and neutralization.

Effect of neutralization on fibrinogen and fibrin viscous acidic solutions

Fibrinogen

As already mentioned viscosity increases progressively during incubation at 37°C (Fig. 1.B.2), thus giving the possibility to obtain viscous fibrinogen solutions FbgA(VS). When neutralized, these solutions form opaque gels FbgN(VS), stable over time, on the contrary to non-heated solutions FbgA which produce precipitates that dissolve over time. This phenomenon was investigated by examining rheological properties. FbgA(VS) heated prior to measurement were neutralized five minutes after starting the measurement at 25°C by careful addition of a neutral solution of 100 mM Hepes around the geometries, and resulted in gels FbgN(VS) with G' of a few hundred of Pa after 24 hours (SI Appendix, Fig. S6). Solutions FbgA(VS) with different initial viscosities were analyzed and the G' of the final gels FbgN(VS) were plotted as a function of the initial G' of the solutions (Fig. 4.A.1). A sharp increase of final G' as a function of initial G' is observed, followed by a plateau around 350 Pa. Resulting gels FbgN(VS) exhibit dense regions of aggregated proteins, reminiscent of the structure observed in acidic fibrinogen gels FbgA(G) as observed by TEM (Fig.

4.A.2).

It is likely that the mechanism (5) responsible for formation of the gels FbgN(VS) upon neutralization of FbgA(VS) is similar to the mechanism causing acidic gel FbgA(G) strengthening after raising pH (Fig. 3.C (4)), and was thus hypothesized to involve αCs interaction (Fig. 4.A.3).

Fibrin

The same rheological study was carried out by neutralizing acidic fibrin solutions FbA(VS) at different initial viscosities. As seen on Fig. 4.B.1, in spite of measures' dispersity, for non-incubated solutions FbA and shortly-incubated solutions FbA(VS) with low initial storage moduli, the final G' of FbN and FbN(VS) ranged between 500 and 1250 Pa. For longer incubated FbA(VS) solutions, with higher initial G', the final value exceeded 1500 Pa, and even 3000 Pa for certain gels. All the fibrin gels FbN and FbN(VS) reached higher storage moduli than pure fibrinogen gels FbgN(VS), independently of the initial solution viscosity. TEM observation of FbN(VS) gels exhibited a composite structure, with thin fibers connecting aggregate-like islands similar to the ones present in pure fibrinogen gels FbgA(G) and FbgN(Vs) (Fig. 4.B.2). Upon neutralization, the non-denatured fibrin proteins self-assemble following the "classical" scheme, bridging the aggregates islands through thin fibers (Fig. 4.B.3). The formation of aggregates by incubation prior to neutralization has a tremendous effect on final gel properties. Indeed, for non-or shortly-incubated solutions FbA and FbA(VS) with low initial G', the final storage modulus remains below 1.5 kPa, while viscous solutions FbA(VS) incubated for a longer time all reach higher moduli. The high dispersion of the measures may be explained by the fact that the system is more complex than for fibrinogen, with multiple mechanisms involved.

Versatility in acidic solution viscosity and potential applications

The here presented study sheds light on the behavior of fibrinogen and fibrin at acidic pH, and the different steps involved in gelation process are depicted in Fig. 5. Beyond the interest from a biochemical standpoint the investigated phenomena represent a unique opportunity to produce stable materials made of pure fibrin (FbN, FbN(VS), FbN(G) and FbA(G)) and, more significantly, of pure fibrinogen (FbgN(VS), FbgN(G) and FbgA(G)) without any use of chemical or physical crosslinking. Two main processes can be implemented from our work. The first is the formation of stable transparent (FbgA(G) and FbgN(G)) gels by incubation (2+3) of acidic fibrinogen solutions. Such homogeneous materials may be valuable for molding or coating processes since they are much easier to process than those obtained by mixing fibrinogen and thrombin at neutral pH (0+0'). At low ionic strength, those gels do not contract and are transparent, which represents an obvious advantage for such processes. Fibrin materials (FbA(G) and FbN(G)) can also be produced the same way (2'+ 3'), with no obvious benefit on structural and mechanical properties as compared to those made of fibrinogen (Fig. 3.A).

The second process is the instantaneous formation of gels (FbgN(VS), FbN, FbN(VS)) from acidic solutions by a simple change from acidic to neutral pH (5,5'), on the contrary to the delayed gelation (0') initiated by thrombin (0) used in the literature [START_REF] Cornwell | Discrete crosslinked fibrin microthread scaffolds for tissue regeneration[END_REF] . The pH-triggered gelation of stable acidic solutions of fibrin(ogen) enables to transpose multiple technique implemented for collagen to fibrin(ogen). For instance, this makes it possible to produce stable fibrinogen and fibrin threads (Fig. 6.A) by extrusion in neutral buffer, and provides the proof of concept for pure fibrin and fibrinogen printing as shown on Fig 6 .B and SI Appendix, Video S1 where the printing of a honeycomb pattern is displayed.

The ability to tune the viscosity (2 or 2') of the solution is a key asset for processing and allows controlling the mechanical properties of the final gel (Fig. 4.A.1&B.1). Increasing solution viscosity by incubating fibrin(ogen) acidic solution has a paramount effect on thread production by extrusion, as it stabilizes the flow outside the needle. Viscosity of solutions is also a paramount parameter for printing: low viscosity fibrin solutions may be, for instance, interesting for inkjet printing, while viscous solutions are sought for extrusion printing. The acidic pH of the solutions limits the incorporation of cells directly in the inks, on the contrary to classical pathway using thrombin only. Use in bioprinting may then require to print cells and fibrin(ogen) separately.

Fibrin(ogen) inks may be used as a support matrix where cells can be printed in between fibrin(ogen) threads, in the fashion of Atala's group [START_REF] Kang | A 3D bioprinting system to produce human-scale tissue constructs with structural integrity[END_REF] , or by means of co-extrusion [START_REF] Yeo | An Innovative Collagen-Based Cell-Printing Method for Obtaining Human Adipose Stem Cell-Laden Structures Consisting of Core-Sheath Structures for Tissue Engineering[END_REF] .

Additionally, acidic conditions and extrusion are compatible with collagen solutions, enabling production of composite materials. For instance, fibrin(ogen) solution can be mixed with collagen solution (60 mg/mL, 3 mM HCl, 0.8 mM citrate, pH 3.6) at any rate from 100% fibrin(ogen) to 100% collagen and extruded in the same neutralizing buffer (100 mM Hepes, 2.5% PEG). Coextrusion can also be performed, with the possibility to tune dynamically thread composition using a double extrusion device (SI Appendix, Fig. S7) and to produce fibrin(ogen) to collagen gradients (Fig. 6.C).

Test were performed to ensure that modification of fibrin(ogen) caused by acidic incubation did not alter fibrin(ogen) biocompatibility and bioactivity. L929 murine fibroblasts, commonly used to test biocompatibility in the ISO standard, were cultured on wells coated with "classical" fibrin FbN, acidic fibrinogen and fibrin gelled by incubation FbgN(G) and FbN(G), and viscous acidic fibrinogen and fibrin gelled by neutralization, FbgN(VS) and FbN(VS). As observed on Fig. 6.C, all materials induced a similar increase in metabolic activity from day 1 to day 6, due to the high proliferation of L929 on the materials till confluence (SI Appendix, Fig. S8). On all materials, cells adopted typical L929 cells morphology, demonstrating that the various processes do not prevent cell adhesion to matrix. Interestingly, at day 1 and 6, a higher metabolic rate is observed (p<0.0001 & p=0.0019 resp.) on non-self-assembled fibrin(ogen), obtained by total incubation FbgN(G) and FbN(G) or by neutralization of viscous fibrinogen solution FbgN(VS), compared to materials containing self-assembled fibrin, "classical" clot FbN and neutralized viscous fibrin solution FbN(VS).

Further test with primary human fibroblasts seeded on FbgN(VS) fibrinogen and FbN(VS) fibrin, obtained by extrusion of the corresponding solutions in a neutralizing buffer (100 mM Hepes 2.5% PEG, pH 7.4), was performed to ensure that the process and the difference of structure did not alter cell adhesion. Culture of Normal Human Dermal Fibroblasts on threads exhibited high affinity toward both types of threads, with high cellular densities (Fig. 6.E&F). After three days, both types of threads were covered with a monolayer of cells, without colonization inside the threads, as confirmed using confocal microscopy (SI Appendix, Fig. S9). Ability to produce fibrinogen materials represents an obvious interest for biomedical applications. Indeed, it spares the need of thrombin, reducing cost and risks as thrombin used clinically is not autologous whereas fibrinogen can be directly extracted from blood patient [START_REF] Spotnitz | Fibrin Sealant: The Only Approved Hemostat, Sealant, and Adhesive-a Laboratory and Clinical Perspective[END_REF] . On a more academic point of view, ability to produce fibrinogen materials enables the investigation of their effect on cell behavior such as, for instance, the influence on cell phenotype of remaining fibrinopeptides in thrombin-free materials [START_REF] Senior | Effects of fibrinogen derivatives upon the inflammatory response. Studies with human fibrinopeptide B[END_REF][START_REF] Gray | Growth factors for human fibroblasts in the solute remaining after clot formation[END_REF] or the presence of self-assembled fibrin.

In summary, our present results widen the possibilities in terms of fibrin processing, and enable production and shape control of fibrinogen materials. Furthermore, the processes are implemented in similar conditions as those used for collagen, which should enable the development of hybrid scaffolds based on these two proteins. The possibility to tune the biochemical and structural properties of these materials should be an asset in order to induce specific and spatially-controlled interactions with the cells and obtain appropriate cellular response to mimic the complexity of the tissues.

Materials & Methods

Fibrinogen and thrombin solutions

Fibrinogen stock solutions were prepared by dissolving 1 g of human fibrinogen lyophilized from 20 mM citrate pH 7.4 solutions (Merck 341576, >90% clottable proteins) into distilled water as recommended by the manufacturer (final volume of app. 22 mL). The final pH of the stock solutions was 6.6 to 6.8. The concentration (40 ± 2 mg/mL) was measured with the extinction coefficient at 280 nm (1.51 mL/mg) 27. The solutions were aliquoted and kept at -80°C. Lyophilized thrombin from bovine plasma (Sigma) was dissolved in sterile PBS 1X to reach a concentration of 200 U/mL. The solutions were aliquoted and kept at -20°C. FITC-labeled fibrinogen was prepared by dissolution of 20 mg of FITC CeliteR in 3 mL of 0.13 M carbonate buffer pH 9.2 subsequently mixed with fibrinogen stock solution at 40 mg/mL at a 1:3 ratio and dialysed 48 hours against 2 L 20 mM citrate pH 7.4, aliquoted and stored at -80°C. For extrusion, 10% in volume of FITC-labeled fibrinogen [Fbg-FITC] was added to fibrinogen stock solution, following the same protocol as usual afterwards.

Rheology

Rheology was performed on an MCR 302 rheometer (Anton Paar), equipped with a temperature control of both upper and lower geometries (Peltier hood PTD200). A sanded plate-plate geometry (PP25/S) was used for gel formation study and subsequent gel characterization. For acidic gel formation study, significant amount of water was poured around the geometry, and the hood lowered to prevent from drying. Measurements were performed at constant amplitude and frequency (γ=0.1 %, ω=1 rad/s). The temperature was kept at 25°C for 5 mn and then switched to 37°C (zero-gap calibration was performed at 37°C prior to measure). Once gel formed, a frequency sweep (ω = 0.1 to 100 rad/s; γ =1 %) was performed, followed by an amplitude sweep (γ= 0.05 to 1000%; ω = 1rad/s).

Gel neutralization experiments on acidic gels were carried out following the protocols above, except that a solution of 100 mM Hepes pH 7.4 was carefully added around the geometry after 3 hours of measure, once acidic gel formation completed.

For the study of neutralization of viscous solutions a solution of 100mM Hepes, pH 7.4, was carefully added around the geometry, after 5 mn of measure. Temperature was kept constant at 25°C during the whole experiment. Frequency and amplitude sweep were performed as described above.

Transmission electron microscopy

For inclusion in araldite, hydrated samples were crosslinked with paraformaldehyde, glutaraldehyde and osmium tetroxide 4 wt. %. They were subsequently dehydrated using baths with increasing concentrations in ethanol, progressively transferred to propylene oxide and incorporated in araldite resin prior to sectioning.

Observation of solutions was performed by dilution of solutions by a factor 10000 with the corresponding buffer and deposition of a few drops on a charged carbon coated TEM grid. The solution was sucked up after a few seconds with a filter paper on the side of the grid. Gels were chopped on a glass slide and a carbon coated TEM grid was put in contact with the mixture and dried by sucking up the supernatant with a filter paper. A drop of uranyl acetate 0.5% was then added and removed the same way.

The observations were made on a transmission electron microscope FEI Tecnai Spirit G2 operating at 120 kV. Images were recorded on a Gatan CCD camera.

Differential Scanning Calorimetry

Differential Scanning Calorimetry was performed on a DSC Q20 from TA. Small volume (20 μL) sealed pans were used. As a reference, a similar pan with comparable weight of relevant buffer was used. Solution was kept at 20°C for 5 min for equilibration, followed by a ramp at 10°C/min till 100°C.

Circular Dichroism

Circular Dichroism was performed on a J-810 CD from Jasco, with a temperature control Peltier module (PTC-4235). As the intensity was too high for stock solutions, even with a 0.01 mm cuvette, stock solution was diluted by a factor three with 20 mM citrate solution, pH 7.4 to reach a concentration of approx. 13 mg/mL that was further used for all the experiments. Spectra were smoothed with a Savitsky-Golay filter with a polynomial order of 2 and a smoothing window of 10 points.

Dynamic Light Scaterring

Dynamic light scattering was performed on a Malvern Zetameter with solutions 1 mg/mL fibrinogen solutions with or without addition of D or E fragments at a ratio Fbg:fragment = 1:2 in mol. Solutions at pH 7.4 were in 20 mM citrate while acidic solutions were dialysed against 50 mM Glycine buffer and pH adjusted to 3.6.

SDS-PAGE

Acidic fibrinogen gels were made by incubating acidic solutions of fibrinogen at 37°C overnight. They were subsequently dipped into either a 20 mM citrate solution pH 7.4 or a 8 M Urea solution. As for the controls, the same protocol was carried out using a neutral stock solution of fibrinogen and a "classical" clot made by mixing thrombin with the stock solution at neutral pH. After 4 days, the solutions in which the three different forms of fibrin(ogen) had been agitated were sampled and run on 10% SDS-PAGE gels.

Mass spectrometry

Matrix-Assisted Laser Desorption/Ionization (MALDI) coupled with a Time Of Flight (TOF) mass spectrometer was used to study the release of the fibrinopeptides FpA and FpB, respectively 1536.6 and 1552.6 Da in mass [START_REF] Jai-Nhuknan | Negative ion matrix-assisted laser desorption/ionization time-offlight post-source decay calibration by using fibrinopeptide B[END_REF][START_REF] Dashtiev | Positive and negative analyte ion yield in matrix-assisted laser desorption/ionization[END_REF] , after acidic fibrinogen and fibrin gel formation. As the fibrinogen stock solution initially contained some fibrinopeptides A, the stock solution was first dialysed 24 hours against 20 mM citrate pH 7.4 with a 12-14 kDa cut-off membrane. Each gel was rinsed with mechanical agitation using 50 % acetonitrile and 50 % of the relevant buffer for each gel (20 mM citrate pH 7.4 for neutral fibrin gel, 20 mM Citrate 60 mM HCl pH 3.6 for acidic fibrin and fibrinogen gels). The rinsing solutions were measured by MALDI-TOF. As a control, a clot formed by mixing thrombin to fibrinogen stock solution at neutral pH was rinsed and both FpA and FpB could be observed at 1536.6 and 1552.6 Da respectively. The attribution of these peaks to FpA and FpB was further confirmed by MS/MS.

Cell culture

Normal Human Dermal Fibroblasts (PromoCell™) at passage 17 were seeded at 420,000 cells/mL on fibrin and fibrinogen threads in low glucose DMEM with 10% fetal bovine serum (GIBCO). Murine fibroblasts L929 at passage 11 were seeded to a final cell density of 2x10 4 cell/cm² in the wells of 48 well-plates coated with the different fibrin(ogen) materials. Fibrinogen solutions were acidified to reach 60 mM HCl and supplemented with 10 mM CaCl2 final, and 20 U/mL thrombin final for fibrin solutions or PBS 1X for fibrinogen solutions. Classical FbN coating was performed using same stock solution supplemented with same concentrations of CaCl2 and thrombin. Viscous fibrin(ogen) solutions were incubated as for thread production, deposited on wells and neutralized with DMEM, to produce FbgN(VS) and FbN(VS). To produce FbgN(G) and FbN(G), solutions were incubated one hour at 37°C to form a gel and neutralized by adding DMEM. Alamar blue tests were performed on 6 replicates at day 1, 3 and 6 by adding resazurin solution to fresh whole medium to reach a volume of 300 µL at a concentration of 0.01 mg/mL, incubating for 4 hours and measuring fluorescence excitation at 560 nm and emission at 590 nm using a Varioskan Lux reader from Thermo Scientific. Fluorescence imaging was performed by similar labeling as on NHDF cells and observations were done on an EVOS inverted microscope from Thermo Fisher Scientific.

Fig. 1 .

 1 Fig. 1. Two gel formation mechanisms: "classical" neutral thrombin-driven (A) and acidic thrombin-free (B). (A.1) Schematic structure of fibrinogen, made of two distal D domains and one central E domain, linked by coiledcoil connectors. The E domain bears the two pairs of fibrinopeptides (Fps) A and B. The two carboxy-terminal ends of the α-chains further extend and fold into small globular domains, noted αCs, which connect to the central E domain. (A.2) "Classical" mechanism where the fibrinopeptides are cleaved (0) by thrombin (Tb), converting the fibrinogen proteins (FbgSt) into fibrin monomers that self-assemble (0') in a half-staggered manner to form a fibrin clot (FbN). (A.3) Image of a fibrin clot. (A.4) FbN observed by Transmission Electron Microscopy (TEM). (B.1) Image of an acidic gel (FbgA(G)) formed by incubation of a 40 mg/mL fibrinogen solution at pH 3.6 (FbgA) at 37°C. (B.2) Rheological dynamical analysis of gel formation of FbgA(G) initiated by a temperature switch from 20°C to 37°C. (B.3) Mechanical behavior of FbgA(G) under amplitude sweep. (B.4) Structure of an acidic gel FbgA(G) included in araldite and observed by TEM.

Fig. 2 .

 2 Fig. 2. Mechanism of acidic fibrinogen gel formation. (A) Circular dichroism spectra at 20°C of a Fbgst (green) solution, FbgA solution at pH 3.6 with HCl (red), and the same solution brought back to neutral pH by addition of NaOH (blue). (B) Thermograms of Fbgst solution (green), FbgA solution pH 3.6 (red), and FbgA(G) gel pH 3.6. (C & D) FbgA and Fbgst, respectively, dried on a carbon coated grid and observed by TEM. (E) Schematic of the mechanism inferred from the obtained results. Upon acidification to pH 3.6 (1) of FbgSt, the D domains of acidic fibrinogen FbgA are destabilized in a reversible manner (red). Incubation at 37°C provokes the denaturation (2) of the thermolabile D domains of fibrinogen proteins that subsequently interact by their extremities (3) to give an acidic fibrinogen gel FbgA(G).

  2.D).Aggregation of fibrinogen in acidic conditions upon incubation in presence of D and E fragments was investigated by dynamic light scattering. Altered aggregation in presence of D fragments was observed, on the opposite to E fragments (Appendix, Fig.S4), which tends to confirm that D domains are involved in aggregation.As summed up in the sketch (Fig.2.E), stock fibrinogen FbgSt undergoes upon acidification (1) a reversible change in structure that corresponds to a destabilization of its thermolabile D domain (represented by a change of color of the D domain from blue to red) in the acidic fibrinogen FbgA.Upon incubation at 37°C (2), these domains are denatured (represented by the floating carboxyterminal ends of β and γ chains[START_REF] Medved | Domain organization of the terminal parts in the fibrinogen molecule[END_REF] ), promoting interactions between proteins (3) and resulting in a gel FbgA(G).

Fig. 3 .

 3 Fig. 3. Effect of neutralization on acidic fibrin and fibrinogen gels. (A) Formation of acidic gels from acidic fibrinogen FbgA and fibrin FbA solutions by incubation at 37°C followed by gel stiffening upon addition of a neutral solution around the geometry. (B) Comparison of mechanical response in amplitude sweep of a neutralized acidic fibrinogen gel (red) compared to a non-neutralized one (blue). (C) Schematic of the underlying mechanisms inferred from the measures. Fibrinogen proteins FbgA assemble upon incubation (2) forming a gel FbgA(G) (3) as seen on Fig.2. Upon neutralization, creation of additional interactions inside the gel FbgN(G) lead to internal constraints and subsequent partial unfolding of some proteins (4). Similar results are obtained in presence of thrombin (FbA), both during the gel FbA(G) formation in acidic conditions at 37°C (2')(3') and upon neutralization (4') to result in a similar gels FbN(G) as FbgN(G).

Fig. 4 .

 4 Fig. 4. Effect of neutralization on viscous acidic solutions of fibrinogen FbgA(VS) (A) and fibrin FbA(VS) (B) and analysis of the final neutral gels FbgN(VS) and FbN(VS). (A.1 & B.1) Resulting storage moduli (G' final) of respectively FbgN(VS) and FbN(VS), as a function of the initial storage moduli of respectively FbgA(VS) and FbA(VS) fixed by different incubation times at 37°C. (A.2 & B.2) Resulting structures, respectively FbgN(VS) and FbN(VS), as observed by TEM. (A.3 & B.3) Schematic sum up of the neutralization (5 & 5') of respectively FbgA(VS) and FbA(VS), where only parts of the proteins are denatured by short incubation times (2 & 2') and form aggregates. For fibrinogen FbgA(VS), upon neutralization (5) the strengthening of the network is hypothesized to rely on αCs interactions promoted by the proximity of proteins. For fibrin solutions FbA(VS), upon neutralization (5') the non-denatured fibrin proteins selfassemble following the classical scheme, bridging aggregates formed during incubation at acidic pH (2').

Fig. 5 .

 5 Fig. 5. The different mechanisms of fibrin(ogen) gelation. Classically, fibrin FbN is polymerized by the action of thrombin that cleaves Fps from fibrinogen FbgSt (0), causing self-assembly in a half staggered manner (0'). Upon acidification (1), fibrinogen FbgSt undergoes a reversible change in structure which involves the destabilization of the thermolabile D domains and αCs interactions in FbgA. Incubation at 37°C denatures this domain, enabling the interactions of "denatured proteins" by their extremities, which causes an increase in viscosity (2) of the solution FbgA(VS) which eventually sets (3) to a gel FbgA(G). Upon neutralization, the formation (5) of opaque gels FbgN(VS) from viscous solutions FbgA(VS) and the strengthening (4) of the transparent gels FbgA(G) are hypothesized to be partly due to αCs interactions. Addition of thrombin (Tb) to freshly acidified fibrinogen solutions FbgA causes the cleavage of fibrinopeptides but prevents fibrin FbN assembly, which only occurs upon neutralization (N). No effect of the Fps cleavage could be observed on the FbA(VS) viscosity increase (2') and gel FbA(G) formation (3') of acidic solutions FbA upon incubation, compared to (2) and (3), as well as after neutralization of FbA(G) gels (4'), compared to (4). However, neutralization of viscous fibrin solutions FbA(VS) (5') produced composites made of acidic aggregates bridged by "classical" fibers, and resulted in stiffer gels FbN(VS), compared to those obtained (FbN) from non-incubated solutions FbA after neutralization (N). These phenomena are useful to produce transparent fibrinogen FbgN(G) or fibrin FbN(G) gels (4)(4') and enable the production of fibrinogen FbgN(VS) (5) and fibrin FbN(VS) (5') threads, and provide a proof of concept in printing, using incubation of solutions (2)(2') to tune the solution viscosity and/or the threads final mechanical properties.

Fig. 6 .

 6 Fig. 6. Use of acidic fibrin and fibrinogen solutions for production of new materials. (A) Image of the extrusion of a viscous fibrinogen solution in 100 mM Hepes 2.5% PEG solution pH 7.4. (B) Printing of a pure viscous fibrin solution on a Petri dish. (C) Pure fibrin to collagen gradient in a continuous thread produced by alternate extrusion of viscous acidic fibrin and collagen solutions (60 mg/mL, 3mM HCl, 0.8mM citrate pH 3.6) in the same conditions (100mM Hepes & 2.5% PEG) observed with a fluorescent microscope. Green: FITC-labelled fibrin. (D) Reduction rate of L929 murine fibroblasts seeded in wells coated with "classical" fibrin FbN, acidic fibrinogen and fibrin gelled by incubation FbgN(G) and FbN(G), and viscous acidic fibrinogen and fibrin gelled by neutralization, FbgN(VS) and FbN(VS).. (E & F) Normal Human Dermal Fibroblasts 3 days after seeding on respectively a pure fibrinogen FbgN(VS) and a pure fibrin FbN(VS) thread. Blue: nuclei (DAPI); green: actin (phalloidin).

  After 72 hours culture, threads were rinsed with PBS 1X and fixed with PFA 4 %. Cells were permeabilized with Triton X-100. Fluorescent labeling of the nuclei (DAPI, Invitrogen) and the actin filaments (Alexa FluorR 488 phalloidin, Invitrogen) was performed. Observations of the samples were carried out under fluorescence microscope (Axio Imager D.1, Zeiss) and confocal microscope (Leica SP5 upright Confocal).

Fig. S2 :

 S2 Fig. S2: Solubility tests of acidic fibrinogen gels FbgA(G).Samples were left for 4 days in two different solutions: "pH 7.4" (20 mM citrate) and "Urea" (8 M). Supernatants were then 30ipette and run on SDS PAGE gels to investigate the presence of fibrin(ogen) proteins and possible degradation products. The samples were acid fibrinogen gels FbgA(G), neutral fibrin clots FbN and a stock solution thawed 4 days before FbgSt or the day of the SDS PAGE "FbgSt fresh". The three main bands correspond to the three chains α, β and γ of the fibrinogen, of respectively 67, 55 and 48 kDa(1). The band around 31 kDa in FbN sample in urea corresponds to thrombin's B chain.

Fig. S3 :

 S3 Fig. S3: MALDI TOF of fibrinogen and fibrin in various conditions. MALDI TOF spectra of: (A) fibrinogen stock solution FbgSt (negative control), (B) supernatant after rinsing a fibrin clot FbN obtained by mixing thrombin with fibrinogen stock solution (positive control), (C) supernatant after rinsing an acidic fibrinogen gel FbgA(G) obtained after 1 hour incubation at 37°C, (D) supernatant after rinsing an acidic fibrin gel FbA(G) obtained after 1 hour incubation at 37°C.

Fig. S4 :

 S4 Fig. S4: Acidic aggregation of fibrinogen with or without D or E fragments monitored by Dynamic Light Scattering (DLS). DLS counts (number) of fibrinogen (A), fibrinogen + D fragments (B), fibrinogen + E fragments (C), D fragments (D) and E fragments (E) solutions at pH 7.4 25°C (red), pH 3.6 25°C (green) and 37°C (blue).

Fig. S5 :

 S5 Fig. S5: Difference in frequency between an acidic fibrinogen gel FbgA(G) and a neutralized fibrinogen gel FbgN(G). Same rheological study (γ=1%) performed on an acidic fibrinogen gel FbgA(G) (red), polymerized by 37°C-incubation between the geometries prior to measure, and a FbgA(G) gel subsequently neutralized (FbgN(G), blue).

Fig. S6 :

 S6 Fig. S6: Neutralization of a viscous acidic fibrinogen solution FbgA(VS). Measurement of the rheological properties of a pre-incubated solution of acidic fibrinogen FbgA(VS) is performed. Five minutes after the start of the measure, a neutral 100 mM Hepes solution is poured around the geometry.

Figure S7 :

 S7 Figure S7: Schematic of the double extrusion device for gradient production. This lab-made device was designed to dynamically tune fibrin(ogen) and collagen content and produce collagen to fibrin(ogen) gradients thanks to controlled extrusion of both syringes thanks to a controller and to a Y connector to mix both solutions.

Acknowledgements

The authors would like to thank Emmanuelle Sachon, Lucrèce Matheron-Duriez and Gilles Clodic, from the Institut de Biologie de Paris Seine, for performing mass spectrometry experiments. C. R. was supported by a French Ministère de la Recherche fellowship. Other funding source: Emergence program from Sorbonne Université.

Materials & Correspondence

Correspondence and requests for materials should be addressed to L. T. or T. C.

Competing interests

The authors declare no competing interests.

Supplementary Information

Thrombin-free polymerization leads to pure fibrin(ogen) materials with extended processing capacity