Pinpin Wang

Federico Perche

Patrick Midoux

Catia S D Cabral

Virginie Malard

Ilídio J Correia

Hanane Ei-Hafci

Hervé Petite

Delphine Logeart-Avramoglou

Chantal Pichon
email: chantal.pichon@cnrs.fr

In Vivo Bone Tissue Induction by Freeze-dried Collagen-nanohydroxyapatite Matrix Loaded with BMP2/NS1 mRNAs Lipopolyplexes

Keywords: mRNA delivery, BMP2, NS1, collagen-nanohydroxyapatite scaffold, lipopolyplex, de novo bone formation

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Introduction

Bone tissue engineering (BTE) applications and regenerative strategies aim at improving current clinical practices for repairing large bone defects resulting from trauma, congenital malformations, and surgical resections [START_REF] Amini | Bone tissue engineering: recent advances and challenges[END_REF][START_REF] Campana | Bone substitutes in orthopaedic surgery: from basic science to clinical practice[END_REF]. BTE is a multidisciplinary field of research, combining biology, material science, chemistry and engineering, with an ultimate goal to create bone graft substitutes (BTE grafts) for bone repair and regeneration [START_REF] Amini | Bone tissue engineering: recent advances and challenges[END_REF][START_REF] O'keefe | Bone tissue engineering and regeneration: from discovery to the clinic--an overview[END_REF]. A classical BTE graft is composed of a biocompatible and biodegradable scaffold for supportive function, combined with bioactive components, such as osteoinductive growth factors and/or stem cell, for induction of osteogenesis and vascularization [START_REF] Kesireddy | Approaches for building bioactive elements into synthetic scaffolds for bone tissue engineering[END_REF].

Gene therapy is one alternative method of introducing osteoinductive compounds into the body. In this context, use of gene-activated matrices (GAMs) represent a novel and attractive strategy to promote bone repair. This type of BTE graft carries genetic information (i.e. pDNA or mRNA, coding osteogenic proteins) instead of traditional recombinant proteins and cells [START_REF] Mello | Bone Regeneration Using Gene-Activated Matrices[END_REF]. The GAMs are designed for long-term in situ growth factors expression with proper post-translational modifications at physiological tolerated dose. The feasibility of GAMs as a therapeutic for bone defect repair was fist demonstrated by Fang et al.in 1996 [6]. In their study, the GAM contained two-plasmid DNAs encoding bone morphogenetic protein-4 (BMP-4) and parathyroid hormone fragment (PTH1-34: amino acids 1-34). It was implanted into the rat femoral osteotomy gap. The fibroblasts from invaded granulation tissue became transfected and expressed BMP-4 and PTH1-34. Gap bridging was observed as early as 4 weeks post-GAM implantation. In the next decade, more GAMs were developed. They comprised various genes (e.g. vascular endothelial growth factor (VEGF), platelet-derived growth factor-B (PDGF), BMP-2) delivered via either viral vectors (e.g. adenovirus and recombinant adeno-associated virus) or non-viral vectors (e.g. polyethyleneimine, SuperFect TM liposome, and calcium phosphate) [START_REF] Ono | Combination of porous hydroxyapatite and cationic liposomes as a vector for BMP-2 gene therapy[END_REF][START_REF] Ito | Remodeling of cortical bone allografts mediated by adherent rAAV-RANKL and VEGF gene therapy[END_REF][START_REF] Chang | Adenovirus encoding human platelet-derived growth factor-B delivered to alveolar bone defects exhibits safety and biodistribution profiles favorable for clinical use[END_REF][START_REF] Keeney | The ability of a collagen/calcium phosphate scaffold to act as its own vector for gene delivery and to promote bone formation via transfection with VEGF165[END_REF][START_REF] Elangovan | The enhancement of bone regeneration by gene activated matrix encoding for platelet derived growth factor[END_REF].

To avoid the risk of genome integration and low transfection efficiency inherent with pDNA delivery, matrices progressively releasing the osteogenic factors produced from mRNA or colonized by mesenchymal stem cells (MSC) are emerging for neoformation of bone tissue at the implantation site [START_REF] Wang | RNA-based therapy for osteogenesis[END_REF][START_REF] Badieyan | Concise Review: Application of Chemically Modified mRNA in Cell Fate Conversion and Tissue Engineering[END_REF]. Aliasger K. Salem group introduced in vitro transcribed (IVT) mRNA (encoding BMP-2) activated matrices (RAMs) for bone regeneration [START_REF] Elangovan | Chemically modified RNA activated matrices enhance bone regeneration[END_REF]. In a rat calvaria defect model, the RAM induced 2-fold and 4-fold more bone tissue when compared to GAM-pDNA (encoding BMP-2) and empty controls, respectively. These encouraging in vivo outcomes have been followed by other studies based on RAM containing chemically modified IVT mRNAs [START_REF] Khorsand | A Comparative Study of the Bone Regenerative Effect of Chemically Modified RNA Encoding BMP-2 or BMP-9[END_REF][START_REF] Balmayor | Chemically modified RNA induces osteogenesis of stem cells and human tissue explants as well as accelerates bone healing in rats[END_REF][START_REF] Zhang | An Improved, Chemically Modified RNA Encoding BMP-2 Enhances Osteogenesis In Vitro and In Vivo[END_REF]. The chemical modification is known to suppress IVT mRNA induced immune responses [START_REF] Kariko | Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA[END_REF]. Despite the advantage obtained using such chemically modified 4/28 mRNA, the main drawbacks are their high cost and the required tuning of chemical modifications according to gene and cell types [START_REF] Uchida | Screening of mRNA Chemical Modification to Maximize Protein Expression with Reduced Immunogenicity[END_REF].

Instead of using chemically modified mRNA, another strategy is to co-express the mRNA of osteogenic genes with the mRNA coding for an immune evasion protein as the influenza A virus derived NS1, known to inhibit type I interferon (IFNα/β) expression, protein kinase R (PKR) and 2'-5'-oligoadenlate synthase (OAS) activation [START_REF] Hale | The multifunctional NS1 protein of influenza A viruses[END_REF][START_REF] Phua | Non-linear enhancement of mRNA delivery efficiencies by influenza A derived NS1 protein engendering host gene inhibition property[END_REF]. In our previous study, we first demonstrated that co-delivering BMP2 mRNA and NS1 mRNA in murine pluripotent stem cells (C3H10T1/2) was able to promote their osteogenic differentiation in vitro [START_REF] Wang | Co-delivery of NS1 and BMP2 mRNAs to murine pluripotent stem cells leads to enhanced BMP-2 expression and osteogenic differentiation[END_REF]. We have established the optimal weight ratio between BMP2 and NS1 mRNAs (3:1). Dual delivery of BMP2 and NS1 mRNA resulted in the inhibition of immune sensors and inflammatory cytokines production and the improved expression of osteogenic markers in comparison to the delivery of BMP2 mRNA alone.

In this following study, we evaluate the potential of this strategy in vivo, The next step of this work is to assess the potentiality of this strategy in vivo, which requires an effective delivery, the other challenge for mRNA therapy besides the immunogenicity issue [START_REF] Kowalski | Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery[END_REF]. We have developed a lipopolyplex (Lip100/His-lPEI/RNA ternary complex, LPR) platform capable of efficient in vitro and in vivo cell transfection with a negligible toxicity [START_REF] Gonçalves | Lipopolyplexes comprising imidazole/imidazolium lipophosphoramidate, histidinylated polyethyleneimine and siRNA as efficient formulation for siRNA transfection[END_REF][START_REF] Van Der Jeught | Dendritic Cell Targeting mRNA Lipopolyplexes Combine Strong Antitumor T-Cell Immunity with Improved Inflammatory Safety[END_REF]. The imidazole/imidazolium binary Lip100 liposomes are made of cationic O,O-dioleyl-N-(3N-(N-methylimidazolium iodide) propylene) phosphoramidate (KLN25) and neutral O,O-dioleyl-N-histamine phosphoramidate (MM27), at a molar ratio of 1:1 [START_REF] Mevel | Synthesis and transfection activity of new cationic phosphoramidate lipids: high efficiency of an imidazolium derivative[END_REF][START_REF] Mevel | Novel neutral imidazole-lipophosphoramides for transfection assays[END_REF]. KLN25 possesses an N-methylimidazolium polar head conferring a permanent positive charge used for nucleic acids condensation. The imidazole group of MM27 is not alkylated on the nitrogen atoms allowing the acquisition of cationic charges at the environmental pH below 6 and favoring the endosome destabilization and nucleic acids release (endosome escape). The cationic polymer His-lPEI, is a histidylated linear polyethyleneimine [START_REF] Bertrand | Histidinylated linear PEI: a new efficient non-toxic polymer for gene transfer[END_REF]. As in MM27, the protonation of imidazole groups from histidine increases endosome escape of trapped nucleic acids leading to higher transfection efficiency compared to lPEI [START_REF] Midoux | Chemical vectors for gene delivery: a current review on polymers, peptides and lipids containing histidine or imidazole as nucleic acids carriers[END_REF].

In the present work, we evaluated whether i), the LPRs platform is suitable for in vivo mRNA in-situ delivery and ii), the dual mRNA system (BMP2/NS1 mRNAs) is able to promote new bone tissue formation in vivo when formulated as LPRs in suitable matrix.

To this aim, we first prepared LPRs with firefly luciferase mRNA and assessed the luciferase production after intradermal administration. Second, we formulated BMP2/NS1 mRNAs as LPR nanoparticles and incorporated them into collagen-nanohydroxyapatite scaffolds to form mRNA-activated matrices (RAMs).

The release and the translation of the mRNA containing LPRs from RAMs were determined in vitro and 5/28 the efficiency of RAMs to induce de novo bone formation was evaluated by subcutaneously implanting them into mouse back.

Materials and Methods

IVT mRNAs preparation

The pT7-GFP [START_REF] Perche | Neutral Lipopolyplexes for In Vivo Delivery of Conventional and Replicative RNA Vaccine[END_REF], pT7-BMP2 and pT7-NS1 [START_REF] Wang | Co-delivery of NS1 and BMP2 mRNAs to murine pluripotent stem cells leads to enhanced BMP-2 expression and osteogenic differentiation[END_REF] plasmids were linearized with either SpeI or NotI, for mRNAs production with the mMessage mMachine T7 kit (Ambion) according to the manufacturer's instructions. The poly(A) tail was added with PolyA polymerase (Ambion). Synthesized mRNAs were purified with phenol/chloroform and isopropanol precipitation. Purity, size and integrity of the mRNAs were verified by a UV spectrophotometer (NanoDrop One, Thermo Scientific) and denaturing agarose gel electrophoresis, respectively.

Cell culture

C2C12 cells were purchased from American Type Cell Collection (ATCC), and cultured in Dulbecco's modified eagle media (DMEM, Sigma) containing 10% heat-inactivated fetal bovine serum (FBS; Sigma) and 1% antibiotics (Sigma). C2C12-BRE/Luc cells containing BMP-2 response element (BRE), which express luciferase under BMP-2 stimulation, were previously described [START_REF] Logeart-Avramoglou | An assay for the determination of biologically active bone morphogenetic proteins using cells transfected with an inhibitor of differentiation promoter-luciferase construct[END_REF]. Human MSCs were harvested from bone marrow obtained from discarded tissue during routine bone surgery from 3 donors (1 woman and 2 men; 15, 22, and 31 years old, respectively) at the Lariboisiere Hospital, Paris, France.

The tissues were collected with the respective donor's consent in agreement with Lariboisiere Hospital regulations. hMSCs were isolated from each donor's bone marrow using a procedure as previously reported [START_REF] Friedenstein | Osteogenesis in transplants of bone marrow cells[END_REF][START_REF] Moya | Osteogenic potential of adipogenic predifferentiated human bone marrow-derived multipotent stromal cells for bone tissue-engineering[END_REF]. Briefly, hMSCs were harvested by gently flushing the bone marrow using complete growth medium (specifically, alpha-Minimum Essential Medium (α-MEM, PAN Biotech) containing 10% heat-inactivated fetal bovine serum (FBS; Sigma) and 1% antibiotics (Sigma). These cells were characterized by expression of selected CD markers (specifically, positive for CD90, CD73, CD105 and negative for CD45; data not shown). At passage 1, hMSCs from each donor were pooled at an equal ratio, and used for experiments less than passage 8. All cells were cultured at 37°C in a humidified atmosphere containing 5% CO 2 , and mycoplasma-free as evidenced by MycoAlert Mycoplasma Detection Kit (Lonza).

Lipopolyplex (LPR) preparation and characterization

The His-lPEI grafted with 16% histidine residues was synthesized as previously described [START_REF] Bertrand | Histidinylated linear PEI: a new efficient non-toxic polymer for gene transfer[END_REF], and purchased from Polytheragene SAS (Genepole, Evry, France). KLN25 and MM27 were synthesized as 6/28 previously described [START_REF] Mevel | Synthesis and transfection activity of new cationic phosphoramidate lipids: high efficiency of an imidazolium derivative[END_REF][START_REF] Mevel | Novel neutral imidazole-lipophosphoramides for transfection assays[END_REF]. The Lip100 containing an equal molar ratio of KLN25 and MM27 was prepared as previously described [START_REF] Gonçalves | Lipopolyplexes comprising imidazole/imidazolium lipophosphoramidate, histidinylated polyethyleneimine and siRNA as efficient formulation for siRNA transfection[END_REF][START_REF] Perche | Enhancement of dendritic cells transfection in vivo and of vaccination against B16F10 melanoma with mannosylated histidylated lipopolyplexes loaded with tumor antigen messenger RNA[END_REF].

Lipopolyplexes were prepared according to [START_REF] Gonçalves | Lipopolyplexes comprising imidazole/imidazolium lipophosphoramidate, histidinylated polyethyleneimine and siRNA as efficient formulation for siRNA transfection[END_REF] with some modifications (Fig. 3A). In brief, 15 µl HEPES (10mM, pH 7.4) containing 7.5 µg His-lPEI was added into 37.5 µl HEPES (containing 2.5 µg mRNA) under vortex followed by 30 min incubation at RT to allow the polyplexes formation. Then, the polyplexes were added into liposome solution (3 µl 5.4mM Lip100 in 197 µl HEPES), and pipetted up and down for 5 times followed by 15 min incubation at RT, allowing the LPRs formation.

LPR particle size, zeta potential and mRNA encapsulation

LPR particle size and zeta potential were measured in 10 mM Heps buffer, pH 7.4 by dynamic light scattering and electrophoretic mobility using a nanoparticle analyzer SZ-100 (Horiba Scientific). mRNA encapsulation efficiency of the LPR formulation was measured through agarose electrophoresis.

LPRs (5 µg Lipo100/7.5 µg His-lPEI /2.5 µg eGFP mRNA) and 2.5µg free eGFP mRNA were run in 1% agarose gel. The mRNA retardation was observed via a gel documentation system (GeneFlash, Syngene).

LPR morphology

LPR morphology was analyzed by transmission electron microscopy (TEM) using a Philips CM20/STEM electron microscope operating at 50 kV. TEM samples were prepared according to the technique of negative staining using uranyl acetate as reported by Perche et al., [START_REF] Perche | Neutral Lipopolyplexes for In Vivo Delivery of Conventional and Replicative RNA Vaccine[END_REF]. Five mL LPR solution in HEPES buffer was deposited on a carbon-coated copper grid for 5 min; and then adsorbed with filter paper. 5 mL uranyl acetate 2% in RNase-free water was then, deposited on the grid for 10 s. Samples were dried at room temperature for 20 min before TEM observation.

LPR transfection efficiency

One day before in vitro transfection, 1 x 10 5 C2C12 cells/well were seeded onto 24-well plate. The next day, 1.875µg GFP/0.625µg NS1 mRNAs were formulated into LPRs, and delivered to C2C12 cells.

Lipofectamine TM MessengerMax (LfM, Invitrogen) complexing same amount of mRNA was set as control. 24h post-transfection, the GFP expression was quantified by flow cytometry (FACSort, Becton Dickinson).

For in vivo transfection, LPRs containing 3.75μg Fluc/1.25μg NS1 mRNAs were intradermally injected into mouse skin. LfM/mRNA complexes intradermal injection was set as control. 24h post-injection, the in vivo luciferase expression was observed with IVIS Lumina LT In Vivo Imaging System (PerkinElmer).

Quantification of BMP-2 released by transfected hMSCs

Lipofectamine TM MessengerMax (LfM) was used for hMSCs transfection according to the manufacturer's protocol.

hMSCs were seeded in a 12-well plate at 1x10 5 cells/well. The next day, cells were transfected with either 1µg BMP2 mRNA or 0.75µg BMP2 mRNA/0.25µg NS1. Every 12h, half culture medium (containing the released BMP-2) was collected and replaced with fresh growth medium. Collected media were stored at -80℃ before BMP-2 quantification. BMP-2 contents were determined by ELISA (Abcam kit) following the manufacturer's instruction. The absorbance was measured at 450nm ( 3 V spectrophotometer, PerkinElmer) and BMP-2 contents were calculated based on a standard curve (range: 0-4000pg/ml human BMP-2).

Collagen-nanohydroxyapatite scaffolds preparation and characterization

The collagen-nanohydroxyapatite scaffolds (CoLL-nHA) were prepared as reported previously [START_REF] Cunniffe | Development and characterisation of a collagen nano-hydroxyapatite composite scaffold for bone tissue engineering[END_REF][START_REF] Offeddu | Multi-scale mechanical response of freeze-dried collagen scaffolds for tissue engineering applications[END_REF] with some modifications. Briefly, the collagen (Calf skin type I collagen, MP Biomedicals) was first hydrated in 0.1M acetic acid overnight at 4℃. The nano-sized hydroxyapatite (<50nm, Sigma) was dispersed in 0.1M acetic acid by applying two runs of sonication, for 30min each. The hydroxyapatite suspension was then dropped into the collagen slurry under vortexing (final collagen concentration was 0.75% w/v). The resultant CoLL-nHA mix was further homogenized overnight at 4℃ using a roller mixer (Cat Ingenieurbuero™ RM540), then degassed under vacuum for 1min, and dispatched into polypropylene containers. The CoLL-nHA suspension was frozen at -80℃ for 2h with a cooling rate of 1℃/min and lyophilized by sublimation at -80℃ and 0.1mbar for at least 16h (Bioblock Scientific). The CoLL-nHA scaffolds were then crosslinked by incubating the foams with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) [START_REF] Olde Damink | Cross-linking of dermal sheep collagen using a water-soluble carbodiimide[END_REF] in 95% ethanol at RT for 3h. The scaffolds were then washed five times in distilled water for 15min each and freeze-dried by described above Three types of scaffold were prepared: collagen scaffold without nHA (CoLL); scaffold containing collagen and nHA at the mass ratio of 1:1 (CoLL-1nHA), and 1:3 (CoLL-3nHA).

Microstructure

The scaffold microstructures were observed with a scanning electron microscope (SEM) (Zeiss Ultra Plus, Carl Zeiss) after mounting on SEM stubs and coating with gold-palladium.

Pore size and porosity

Pore size was measured using the ImageJ software (1.52a, National Institutes of Health) according to previously reported study [START_REF] Bartos | Note on the use of different approaches to determine the pore sizes of tissue engineering scaffolds: what do we measure?[END_REF]. Briefly, 50 randomly selected pores from each of 8 SEM images (from two different samples) were analyzed. The circularities were assumed as 1 for pore size calculation.

Porosity was assayed by the liquid displacement method [START_REF] Karageorgiou | Porosity of 3D biomaterial scaffolds and osteogenesis[END_REF][START_REF] Nazarov | Porous 3-D scaffolds from regenerated silk fibroin[END_REF]. Briefly, the scaffold was immersed in 1ml (V1) distilled water in a cylindrical container, and a vacuum was applied for 1min allowing water 8/28 fully infiltrate into the scaffold. After that, the total volume of water plus scaffold was defined as V2.

Then, the water-impregnated scaffold was removed from the container, the remaining water volume was V3 and the porosity was calculated as follows:

Porosity = (V1-V3) / (V2-V3)

Mechanical strength

The mechanical measurements were performed at room temperature using a tensile testing machine (AG-X, Shimadzu) with a crosshead speed of 2 mm/min and a load cell of 5 kN. The compressive strength (Cs) was determined with the following Equation:

Cs=F/A
where F corresponds to the load strength (N) at the time of fracture and A represents the scaffold contact area (mm).

Cytocompatibility

1 x 10 4 C2C12 cells suspended in 100 µl medium were dropped onto each type of scaffold (cylindrical in shape, 6 mm diameter × 2.5 mm height), and incubated for 2h to allow cell adhesion, then cultured after addition of culture medium Cell proliferation inside the scaffolds was measured with Cell Proliferation Kit II (XTT) (Sigma-Aldrich) according to the manufacturer's instruction with minor modifications. Briefly, at defined time points, cell constructs were washed twice with DPBS with gentle squeezing for complete medium removal.

After removing the remnant DPBS with an absorbent tissue, the cell constructs were immerged into the reaction mix (200 µl culture medium, 50 µl XTT solution, and 1 µl electron-coupling reagent) and maintained for 4h. From each well, 100µl of solution was transferred into 96-well plate, and the absorbance was read at 450nm and 650nm with Victor 3 V spectrophotometer (PerkinElmer).

Cell density and morphology were observed with confocal laser scanning microscopy (LSM510, Zeiss).

The constructs were washed as described above, and then fixed with methanol (90%) at -20 ℃ for 30min; the cells containing constructs were blocked with PBS-1% BSA at RT for 1h, incubated with anti-actin antibody (Sigma) at RT for 2h; after washing, the constructs were incubated with Alexa Fluor® 488 conjugated secondary antibody (Invitrogen) at RT for 2h before observation.

mRNA activated matrices (RAMs) preparation and characterization

The LPRs were prepared as described above, except that the total volume was decreased 10-fold and containing 5% sucrose. Concentrated LPRs containing 20 µg mRNA were loaded into each CoLL-1nHA scaffold disc (6mm × 2.5mm), which were then freeze-dried. The mRNA activated matrices (RAMs) were stored at -20 ℃ for further usage.

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The SEM observation and mechanical test of RAMs were performed as described in section 2.3.1 and 2.3.3.

LPRs release kinetics

The RAMs were immerged in 1ml RNase-free TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH7.5) and incubated at 37℃. At scheduled time points, 800μl TE buffer was collected and replaced with fresh RNase-free TE buffer. All collected samples were stored at -80 ℃ for further measurement. The released LPRs were labeled with Quant-iT TM RiboGreen (Invitrogen) following the manufacturer's instruction, and then quantified by a spectrophotometer (excitation: 490nm; emission: 525nm) (Victor 3 V, PerkinElmer).

In vitro LPRs release and transfection

1 x 10 5 C2C12-BRE/Luc cells were seeded into each well of 24-well plate. The next day, RAMs either containing 20µg BMP2 mRNA (RAM-BMP2) or 15µg BMP2/ 5µg NS1 mRNAs (RAM-BMP2/NS1) were added into each well. At the defined time points, cells were lysed (RIPA, Thermofisher) and stored at -80 ℃. Luciferase activity was measured as previously reported [START_REF] Perche | Enhancement of dendritic cells transfection in vivo and of vaccination against B16F10 melanoma with mannosylated histidylated lipopolyplexes loaded with tumor antigen messenger RNA[END_REF]. Cells transfected with fresh prepared LPRs containing 1μg BMP2 mRNA or 0.75μg BMP2/0.25μg NS1 mRNAs were set as control.

2.5

In vivo de novo osteogenesis

In vivo implantation

The osteoinductive potential of scaffolds was assessed in vivo in a mouse ectopic model. Four-week-old, male, BALB/c mice were purchased from Janvier, and handled in accordance with the European Directive 2010/63/EU regarding the protection of animals used for scientific purposes. All animal experiments were performed with the approval of the French ministry of research (approval number:

15910-2020030213573920). The RAM-BMP2 and RAM-BMP2/NS1 were subcutaneously implanted into mice back as previously described [START_REF] Monfoulet | The pH in the microenvironment of human mesenchymal stem cells is a critical factor for optimal osteogenesis in tissue-engineered constructs[END_REF] (one RAM per mouse, n=8 per group). Empty CoLL-1nHA scaffolds were used as control (n=3). Eight weeks post-implantation, the mice were sacrificed through cervical dislocation. The RAMs were excised and fixed in 4% paraformaldehyde.

Micro-computed tomography (µCT) analysis

The new bone formation induced by the BMP-2-expressing RAMs was determined by µ-CT analysis using a Skyscan 1172 high resolution µ-CT scanner (Bruker). Images were acquired using the following settings:

voltage 80 kV, current 100 μA, exposure for 810 ms, and 0.4-degree rotation-step settings, through a 0.5 mm-thick aluminium filter. The initial pixel size at these settings was 6 μm. The scanned images of each implant were reconstructed as a stack of slices using Nrecon software (Bruker). The new bone volume per each scaffold was determined using the CTAn software (v1.15.4.0; Bruker; grayscale threshold 10/28 values within 123-240). The binarization threshold was determined by Otsu's method, based on histograms of 3D µCT scans of bone containing scaffolds.

Histological analysis

Retrieved implants were processed for decalcified histology. Samples were decalcified in ethylenediaminetetraacetic acid (14.5% w/v) at 4 °C for 2 weeks, and embedded in paraffin. Some sequential sections of each specimen were stained with Masson's Trichrome and TRAP stainings.

Statistical analysis

Unless otherwise indicated, the numerical data are expressed as mean ± standard deviation with n=3.

Statistical analysis was performed using GraphPad Prism version 6.07 (GraphPad software). Any p-value less than 0.05 was considered statistically significant. Specifically, * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001; **** represents P < 0.0001.

Results

LPR generates higher in vivo mRNA delivery efficacy than in vitro

To efficiently deliver BMP2 and NS1 mRNAs, we compared the LPR with a commercial vector Lipofectamine TM MessengerMax (LfM). The LPR formulation were prepared by a well-established two-step approach. First, mRNA was complexed with His-lPEI (1/3, mRNA/His-lPEI weight ratio), and the resulting polyplexes were mixed with Lip100 liposomes (1/2, mRNA/Lip100 weight ratio) [START_REF] Gonçalves | Lipopolyplexes comprising imidazole/imidazolium lipophosphoramidate, histidinylated polyethyleneimine and siRNA as efficient formulation for siRNA transfection[END_REF][START_REF] Perche | Enhancement of dendritic cells transfection in vivo and of vaccination against B16F10 melanoma with mannosylated histidylated lipopolyplexes loaded with tumor antigen messenger RNA[END_REF] (Fig. 1A). The agarose gel electrophoretic mobility shift assay, in which no free mRNA migration was observed (Fig. 1B), confirmed the complexation of mRNA. The LPRs had a mean hydrodynamic diameter of 208nm

with the core morphology of mRNA/His-lPEI complex surrounded by the lipid bilayer structure of Lip100 liposome (Fig. 1C-insert). The mean zeta potential of LPRs were slightly positively charged (+ 18.9 ± 0.6 mV) as shown in Fig. 1D.

As the main goal of our study was to evaluate the efficiency of our strategy for de novo bone formation following subcutaneous implantation of RAM and myoblasts from back muscle could be one of the sources of cells that can colonize the implanted scaffold, we checked their ability of LPR to transfect murine C2C12 cells, a myoblast cell line. As shown in Figure 1E, LPRs transfection resulted in only 27% of transfected cells whilst LfM led to 83% of positive cells . For clinical relevance, hMSC transfection was performed as well. Similarly, LPRs yielded 9% transfection efficiency which was 73% for LfM (Fig. 1F).However, the trend was completely reversed in vivo following intradermal injection, a strong bioluminescence around the injection site was observed following LPRs injection, while, no detectable signal was found on the site injected with LfM/mRNA complexes (Fig. 1G). 12/28

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BMP2/NS1 mRNAs delivery favors BMP-2 expression in hMSC

Before evaluating the efficiency of our strategy for RAMs implantation in vivo, we first sought to assess whether the delivery of BMP2 mRNA and NS1 mRNA is also able to enhance BMP2 production in human mesenchymal stem cells. LfM was used as commercial standard for the mRNA delivery based on the LPR and LfM comparative results. We performed the transfection at the same BMP2: NS1 mass ratio of 3:1 based on our previous study [START_REF] Wang | Co-delivery of NS1 and BMP2 mRNAs to murine pluripotent stem cells leads to enhanced BMP-2 expression and osteogenic differentiation[END_REF]. The BMP-2 content was quantified in culture media collected every 12h (Fig. 2). Compared to the transfection of BMP2 mRNA alone, co-transfection with NS1 mRNA increased significantly the BMP-2 production from the early (12h-24h) to the later (48h-60h) time points.

Following BMP2/NS1 mRNAs transfection, the peak of BMP-2 production started at 24h and maintained up to 36h. The BMP-2 expression steadily increased up to 36h. By contrast, following the transfection of BMP-2 mRNA alone, the maximal BMP-2 content was obtained in the first 12h, and then decreased as a function of time. These data confirm that the platform is potentially translatable in human context at least concerning the ratio between the mRNA of the osteogenic gene and that of NS1.

nanoHydroxyapatite reinforces collagen matrix

In a view of producing RAMs, we decided to prepare biomimetic collagen-based scaffolds that bear different percentage of nanohydroxyapatite, the main inorganic bone component. Add a sentence on collagen-hydroxyapatite scaffolds with refs? Three types of collagen-based scaffolds with 13/28 collagen/nanohydroxyapatite weight ratio of either 1/0 (CoLL), 1/1 (CoLL-1nHA) or 1/3 (CoLL-3nHA)

were produced by a freeze-drying approach using acetic acid as a porogen. The scanning electron microscopy was performed to observe their porous structure (Fig. 3A). CoLL (Fig. 3A-a) and CoLL-1nHA (Fig. 3A-c) exhibited similar microstructure with connected macro-(>100μm) and micro-pores (<50μm), whilst CoLL-3nHA (Fig. 3A-e) was denser and devoid of micropores. The SEM images observed under the high magnification revealed embedded nHA particles (Fig. 3A-d andf). Two key parameters, i.e. pore size and porosity, of porous scaffold were quantified. As shown in Fig. 3B andC, CoLL, CoLL-1nHA and CoLL-3nHA had an average pore size/porosity of 168.7μm/ 91.26%, 151μm/ 89% and 165.9μm/ 76%, respectively. Compared to CoLL, both CoLL-1nHA and CoLL-3nHA demonstrated higher compressive strength in axial and radial orientations (Fig. 3D). The differences between CoLL and CoLL-3nHA are significant.

The cytocompatibility of the three collagen-based scaffolds was evaluated by seeding C2C12 cells (Fig. 4A). One day after cell seeding, XTT assay results reveal that more cells were attached to CoLL-1nHA compared to CoLL-3nHA and CoLL. The OD values significantly increased from day 1 to day 3 and remained steady at day 7, indicating that the cells were proliferating within the scaffolds during the first 3 days post-seeding followed by a stagnation. No significant differences in cell viability were observed among each type of scaffold tested. Actin filaments of cells were labeled to visualize the cell distribution within the CoLL-1nHA scaffold (Fig. 4B). At day 1, faint labeling corresponding to few dispersed cells were observed in the scaffold matrix. One week later, one can observe a dense cellular network filling the field.

14/28 The LPRs loading did not impact the average pore size of CoLL-1nHA (Fig. 5C), while increased its mechanical strength (Fig. 5D). Compared to CoLL-1nHA, the axial compressive strength and radial compressive strength of RAM increased 6.3-fold and 1.7-fold, respectively.

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RAM serves as LPRs release platform

The loading efficiency and release kinetics of LPRs from RAM were then evaluated (Fig. 6. A). The quantification of unloaded LPRs, which leaked into the container during RAM preparation revealed that , which were not adsorbed in RAM revealed that? 74% LPRs were absorbed by the CoLL-1nHA scaffold. The release of LPRs from RAMs was monitored along time as a function of time. The kinetics showed a fast release in the first 120h (37% of loaded LPRs) followed by a slow release ending up to 46% afterwards. In order to assess the activity of released LPRs, the BMP2 mRNA containing-RAMs were incubated with C2C12-BRE/Luc cells, a stable cell line containing a BMP-response element fused with firefly luciferase reporter gene [START_REF] Logeart-Avramoglou | An assay for the determination of biologically active bone morphogenetic proteins using cells transfected with an inhibitor of differentiation promoter-luciferase construct[END_REF] (Fig. 6B), and the luciferase activity was monitored over time (Fig. 6C). Four experimental groups were set for comparison, i.e. RAM containing BMP2 mRNA (RAM-BMP2), RAM containing BMP2/NS1 mRNAs (RAM-BMP2/NS1), LPRs containing either BMP2 mRNA (BMP2) or BMP2/NS1 mRNAs (BMP2/NS1) (Fig. 6C). Maximal luciferase 17/28 expression was observed 24h post-transfection in the presence of freshly prepared LPRs (both BMP2 and BMP2/NS1 groups) and luciferase activity gradually decreased over time until 168h (7 days). In contrast, in the presence of RAMs (both RAM-BMP2 and RAM-BMP2/NS1), the kinetics curve showed a peak of luciferase expression at later time, i.e. after 72h of incubation; although decreasing over time, a noticeable luciferase activity was measured until 336h (14 days) post-incubation. As a result, RAMs promoted luciferase expression for a duration of 2-fold longer than with fresh LPRs. More interestingly, the combination NS1 mRNA with BMP2 mRNA (either in LPR groups or in RAM groups) significantly enhanced the luciferase activity: the areas under the curve (AUC) for BMP2/NS1 and RAM-BMP2/NS1 were 2.4-and 3-fold higher than the ones obtained with BMP2 and RAM-BMP2/NS1 groups, respectively.

These observations are consistent with our previous findings related to the beneficial effect of NS1 mRNA [START_REF] Wang | Co-delivery of NS1 and BMP2 mRNAs to murine pluripotent stem cells leads to enhanced BMP-2 expression and osteogenic differentiation[END_REF]. 2.

RAM-BMP2/NS1 induces de novo bone formation in vivo

RAM-BMP2, RAM-BMP2/NS1 and CoLL-1nHA scaffolds were implanted subcutaneously into the mice back for 8 weeks. Following surgical retrieval, there was no evidence of inflammation or rejection 18/28 around any scaffold; RAM-BMP2/NS1 explants were also found to be well vascularized (Fig. 7A).

Specimens were examined for micro-CT and histological analyses. Representative micro-CT images and quantification of the new bone tissue from these images are shown in Fig. 8. No bone deposit was found in CoLL-1nHA scaffolds proving that these matrices were not osteoinductive per se. Whereas a minimal bone tissue was observed in some RAM-BMP2 scaffolds, bone deposits were observed in 7 out of 8 RAM-BMP2/NS1 samples (Fig. 7B). In four of them, the bone tissue is surrounded the scaffold as a thin peripheral shell connected with internal trabeculae. Quantification of the bone using µCT-analysis confirmed significantly (2.1-fold) enhanced bone formation when BMP2 mRNA was co-delivered with NS1 (Fig. 7C).

Histological examination of the explants was consistent with radiographic findings. Representative gross view of CoLL-1nHA controls confirmed absence of bone tissue and revealed a substantial colonization of the scaffold with fibrous tissue, but only peripheral (< 500 mm of depth) (Fig. 8A andD), the center of the scaffold remained acellular (Fig. 8E). RAM-BMP2 matrices were entirely colonized with highly vascularized fibrous tissue and minimal bone tissue was observed at the periphery of the scaffold (Fig. 8 B, G, F). The Masson's Trichrome staining, which identifies bone and osteoid matrix, revealed the large bony ossicles induced by RAM-BMP2/NS1. The bone tissue was organized as thin and mature bone trabeculae present mainly at the outer part of the implants and forming a shell (Fig. 8C,H); the center of the ossicles was occupied by bone marrow, containing numerous adipocytes, and by dense fibrous tissue (Fig. 8I). Additionally, Tartrate Resistant Acid Phosphatase (TRAP) staining, which reveals the presence of osteoclasts or osteoclast-like multinucleated giant cells (MNGC), was found either on the bone surfaces (Fig. 8L) or in soft tissue close to the bone tissue (Fig. 8K,L), indicating some extent of osteoclastic activity and, therefore, of bone remodeling activity.

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Discussion

These last years, the delivery of mRNA for therapeutic purposes has gained remarkable progress [START_REF] Sahin | Türeci, mRNA-based therapeutics-developing a new class of drugs[END_REF],

with extended application from vaccination to protein replacement therapies, such as cardiovascular regeneration [START_REF] Zangi | Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction[END_REF]. The use of mRNA coding osteogenic proteins for bone regeneration is a field still in its infancy as it started from 2015 [START_REF] Elangovan | Chemically modified RNA activated matrices enhance bone regeneration[END_REF], and most of the studies are based on mRNA bearing modified nucleosides to improve their translatability [START_REF] Khorsand | A Comparative Study of the Bone Regenerative Effect of Chemically Modified RNA Encoding BMP-2 or BMP-9[END_REF][START_REF] Balmayor | Chemically modified RNA induces osteogenesis of stem cells and human tissue explants as well as accelerates bone healing in rats[END_REF][START_REF] Zhang | An Improved, Chemically Modified RNA Encoding BMP-2 Enhances Osteogenesis In Vitro and In Vivo[END_REF]. Today, mRNA therapeutics are still costly due to the complex supply chain of their production including the addition of chemically modified nucleosides.

Unlike previously reported studies using chemically modified mRNA, here we report an alternative strategy using non-modified mRNAs to prepare bone graft substitutes, i.e. mRNA activated matrix (RAM),

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for in vivo osteoinduction. The mRNAs platform contains BMP2 mRNA and NS1 mRNA that works as an adjuvant to enhance BMP-2 expression by inhibiting host cell immune responses [START_REF] Wang | Co-delivery of NS1 and BMP2 mRNAs to murine pluripotent stem cells leads to enhanced BMP-2 expression and osteogenic differentiation[END_REF].

Our data shown in figure 2 indicate that the optimal ratio of NS1 and BMP2 mRNAs determined for murine cells is also efficient for human cells. In line with our previous results, BMP2/NS1 mRNA transfected hMSCs yielded a significantly higher amount of BMP-2, than single BMP2 mRNA transfected cells. Interestingly, in those cells, the produced amount of BMP-2 from the dual mRNAs delivery was stable up to 60h. As a result, one could expect to get a significant expression of osteogenic genes in the transfected hMSCs. However, no significant difference between the BMP2/NS1 group and BMP2 group was observed for most of the osteogenic genes and the deposited extracellular calcium (S. Fig. 2). This phenomenon could be caused by two reasons. First, the transient expression profile of administered mRNA. After 3 days, the BMP2/NS1 group and BMP2 group had similar BMP-2 expression. In contrast, the MSCs osteogenic differentiation needs 2 weeks of continuous stimulation. Second, it has been reported that human derived MSCs have low responsiveness to BMPs [START_REF] Diefenderfer | BMP Responsiveness in Human Mesenchymal Stem Cells[END_REF][START_REF] Osyczka | Different effects of BMP-2 on marrow stromal cells from human and rat bone[END_REF]. Thus, although the BMP2/NS1 group resulted in significantly higher BMP-2 than the BMP2 group in the first 3 days, no significant difference was induced in gene level of osteoblastic markers (except for Runx2 at day 3) (S.

Fig. 2A). Despite those data, the significant enhanced production of BMP2 with the dual mRNAs delivery is encouraging and opens the room for improvement, by addition of mRNA coding additional osteogenic factors.

In clinical practice, bone grafts are critical for reconstructing massive bone loss or complex non-union defects [START_REF] Mobbs | Clinical outcomes and fusion rates following anterior lumbar interbody fusion with bone graft substitute i-FACTOR, an anorganic bone matrix/P-15 composite[END_REF]. According to a recent investigation from Zion market research, the global bone grafts and substitutes market was approximately $ 2,745 million in 2018 and is expected to generate around $ 4,235 million by 2026, registering a compound annual growth rate (CAGR) of 5.6% during the forecast period (accessed on 3 rd January 2020). To meet this increasing demand, the aim of the BTE is to construct bone substitutes (BTE grafts) that as close as possible to the gold standard autografts through components, structure and function mimicking [START_REF] Amini | Bone tissue engineering: recent advances and challenges[END_REF][START_REF] Kesireddy | Approaches for building bioactive elements into synthetic scaffolds for bone tissue engineering[END_REF].

RAM have emerged as an advanced approach for constructing bone graft substitutes with the combination of BTE technology and mRNA-therapy technology [START_REF] Elangovan | Chemically modified RNA activated matrices enhance bone regeneration[END_REF][START_REF] Balmayor | Chemically modified RNA induces osteogenesis of stem cells and human tissue explants as well as accelerates bone healing in rats[END_REF].

Here, the collagen and nanohydroxyapatite (nHA), which represent the primary organic and inorganic compound in natural bone, were chosen to produce the basal porous scaffold (Fig. 3). In preliminary experiments, we compared the type I collagen from bovine Achilles tendon and calf skin. As shown in S.

Fig. 3, the bovine Achilles tendon collagen made scaffold exhibited closed pore structure which decreased its water absorption ability. The low pore interconnection will lead to less fluid exchange and 22/28 cell invasion, and finally less or no new bone formation during in vivo application [START_REF] Karageorgiou | Porosity of 3D biomaterial scaffolds and osteogenesis[END_REF]. The likely explanation of this phenomenon is the solubility and fibrillar level differences between the two kinds of collagen. In Achilles tendon, the collagen fibers and fibrils are highly cross-linked and organized as parallel strands along the loading axis [START_REF] Khayyeri | Achilles tendon compositional and structural properties are altered after unloading by botox[END_REF], which makes it acid-insoluble. In contrast, skin collagen fibrils, especially for young species, are less cross-linked and randomly distributed in the dermal layer of skin [START_REF] Yamauchi | Aging and cross-linking of skin collagen[END_REF]. During scaffold preparation, the collagen from calf skin homogeneously dissolved in acetic acid solution resulting in continuous interpenetrating ice network after freezing, and forming interconnective porous structure after sublimation. While, the bovine Achilles tendon only swelled, even after three times longer incubating time. Thus, the calf skin collagen was selected as basal material for further combination with nHA.

As expected, the nHA reinforced collagen scaffold (CoLL-1nHA, CoLL-3nHA) had an increased mechanical strength, especially in axial (Fig. 3D). During scaffolds freeze-drying, the aqueous solution was sublimated mainly through axial direction leading more pores aligned in the axial direction. It was reported that the mechanical properties of a porous scaffold depend on the direction of the loading along the pore channels [START_REF] Mao | Fabrication of porous titanium scaffolds with centrosymmetric pore channels and improved radial fracture loading[END_REF]. More nHA in CoLL-3nHA decreased its interconnection degree (in terms of porosity) (Fig. 3C). The elimination of micropores from the pore walls (Fig. 3A-e), and the non-embedded nHA, to some extent, resulted in its low porosity (Fig. 3A-f). Both the porosity, the permeability and the mechanical properties are crucial parameters for the BTE scaffold, influencing host cell ingrowth, migration and proliferation [START_REF] Amini | Bone tissue engineering: recent advances and challenges[END_REF][START_REF] Karageorgiou | Porosity of 3D biomaterial scaffolds and osteogenesis[END_REF]. For instance, Lewandrowshi et al., found that the poly(propylene fumarate) scaffold has a higher porosity generating a greater and deeper bone ingrowth in rat tibias defect model [START_REF] Lewandrowski | Developing porosity of poly(propylene glycol-co-fumaric acid) bone graft substitutes and the effect on osteointegration: a preliminary histology study in rats[END_REF] due to higher level of vascularization and fluid permeability [START_REF] Mastrogiacomo | Role of scaffold internal structure on in vivo bone formation in macroporous calcium phosphate bioceramics[END_REF].

In terms of biological performance, the addition of nHA did not show any significant influence on cell growth (Fig. 4A). Compared to CoLL and CoLL-3nHA, the CoLL-1nHA preserved slightly more cells, which could be caused by its high porosity (Fig. 3C), and the rough wall surface (Fig. 3A-d). This is in line with the study from Yuan et al., who showed that the surface roughness enhances the attachment, proliferation of anchorage-dependent bone forming cells [START_REF] Yuan | A preliminary study on osteoinduction of two kinds of calcium phosphate ceramics[END_REF].

To note, all scaffolds prepared in this study underwent chemically crosslinking (EDC: NHS: collagen carboxyl group molar ratio of 10: 4: 1), enabling them more resistance to enzyme degradation during in vivo usage (S. Fig. 4). In addition, the crosslinking resultant mechanical enhancement has been shown to increase osteoblast cell number and cellular distribution within collagen scaffolds [START_REF] Haugh | Crosslinking and mechanical properties significantly influence cell attachment, proliferation, and migration within collagen glycosaminoglycan scaffolds[END_REF]. Overall, the outstanding physical and biological performances of CoLL-1nHA giving it the valuable interesting candidate for preparing RAM.

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IVT mRNA mediated immunogenicity and lacking of efficient in vivo delivery vector are the main obstacles relating to mRNA therapy application. By introducing NS1, we resolved the immunogenicity concerning. Next, we need a proper vector to deliver the therapeutic mRNAs to exert their function in vivo.

We previously reported a lipopolyplex (LPR) formulation to delivery small interfering RNA (siRNA) in vitro [START_REF] Gonçalves | Lipopolyplexes comprising imidazole/imidazolium lipophosphoramidate, histidinylated polyethyleneimine and siRNA as efficient formulation for siRNA transfection[END_REF]. The Lip100/His-lPEI/siRNA formulated LPRs were more efficient than formulations made with commercial vectors, i.e. JetPRIME TM , INTERFERin® and Lipofectamine TM 2000. The formulation was then transferred to this study to complex and deliver mRNA. During in vitro and in vivo transfection studies (Fig. 1E andF), LPRs showed a reversed trend compared to the commercial vector (Lipofectamine TM MessengerMax, LfM). We are not surprised by the results, as LfM is designed and optimized for in vitro cultured cell transfection, and LPRs for in vivo environment. Indeed, in our previous studies, although different in polymer and liposome, the LPRs formulation has been successfully used for in vivo vaccination, against tumors and influenza A virus [START_REF] Van Der Jeught | Dendritic Cell Targeting mRNA Lipopolyplexes Combine Strong Antitumor T-Cell Immunity with Improved Inflammatory Safety[END_REF][START_REF] Perche | Neutral Lipopolyplexes for In Vivo Delivery of Conventional and Replicative RNA Vaccine[END_REF].

On the other hand, from the aspect of surgical handling and cost of transport and storage, a ready-to-use RAM is more in line with market demand. After freeze-drying, within the RAM, the majority of LPR particles maintained a spherical shape, while LfM/mRNA particles collapsed (Fig. 5A-bottom), highly due to lack of supportive polyplex cores. The structure integrity helped the LPRs preserve their mRNA delivery ability (Fig. 6C). Compared to one-time transfection, the protein expression from the RAM groups was increased over 72h prior to decrease. From the LPRs release profile (Fig. 6A), we conclude that this phenomenon could be generated by multiple transfection of cells thanks to continuously released LPRs.

Compared to the basal scaffold (CoLL-1nHA), the LPRs loading enhanced compressive strength, especially in the axial direction (Fig. 6C). The LPRs were prepared in HEPES buffer containing 5% sucrose, thus, after sublimation, the HEPES and sucrose molecules homogeneously deposit on pore walls, which may contribute to the mechanical changes as tougheners.

The observed ectopic bone formation in mice induced by the BMP2 mRNA containing-matrices confirmed the osteoinductive performance of the RAM system. The co-delivery of NS1 mRNA with BMP2 mRNA significantly (2-fold) improved the potential of RAMs to promote new bone formation. The 3D µ-CT images and histology revealed that BMP2/NS1 mRNA RAMs induced large bony ossicles, typical of BMP-2-induced ectopic bone induction with the formation of a shell enclosing thin bone trabeculae and fatty bone marrow. The single delivery of BMP2 mRNA promoted minimal bone tissue but the matrices were entirely invaded by highly vascularized fibrous tissue. In contrast, CoLL-1nHA scaffolds prepared 24/28 without mRNA not only did not promote bone tissue but were also minimally invaded by host tissue at the periphery of the constructs. It is known that BMP-2 promotes bone formation in a dose-dependent manner [START_REF] Wang | Recombinant human bone morphogenetic protein induces bone formation[END_REF]. As a result, the higher newly-formed bone in RAM-BMP2/NS1 scaffolds was explained by the higher expected amount of BMP-2 produced within these matrices. At lower dose, BMP-2 did not induce bone formation but displayed other functions including chemotaxis of several cell types and angiogenesis [START_REF] Lind | Bone morphogenetic protein-2 but not bone morphogenetic protein-4 and -6 stimulates chemotactic migration of human osteoblasts, human marrow osteoblasts, and U2-OS cells[END_REF][START_REF] Finkenzeller | Effects of bone morphogenetic protein 2 on human umbilical vein endothelial cells[END_REF]. Considering the environmental differences between subcutaneous tissue and bone tissue, we believe that, when applied in-situ for bone defect repair, the RAM-BMP2/NS1 can further exert its function and induce as higher new bone tissue as previously reported [START_REF] Elangovan | Chemically modified RNA activated matrices enhance bone regeneration[END_REF].

Conclusion

In this study, we demonstrated the non-modified dual mRNAs (BMP2/NS1 mRNAs) system was capable to remarkably improve the production of BMP2 when applied to hMSCs in vitro. The main results concern the use of Lip100/His-lPEI based LPR that can serve as an mRNA delivery formulation for the production of RAMs made with collagen-nanohydroxyapaptite scaffold. RAMs-BMP2/NS1 were able to mediate a high and prolonged in vitro BMP-2 expression and, excitingly, in vivo new bone induction.

Taken together, these results provided the basis for translating this RAM-BMP2/NS1 matrix into in vivo bone defect intervention. Moreover, as an advanced combination of mRNA therapy and tissue engineering, the RAM technology has broad potential applications, such as for wound healing, skin, cartilage and vertebral disc regeneration.

Fig. 1 .

 1 Fig. 1. Lipopolyplexes formulation and transfection efficiency. (A) Illustration of LPR preparation. mRNA was first complexed with His-lPEI to form polyplexes, which are mixed with Lip100 liposomes to form LPR nanoparticles. (B) Agarose gel electrophoresis of LPRs and eGFP mRNA. (C, D) LPR physico-chemical characterization. The representative diagram of LPRs (complexing with eGFP mRNA) hydrodynamic diameter distribution (inset: TEM image of LPR with scale bar = 100 nm), and zeta potential. (E, F) In vitro transfection efficiency. hMSC and C2C12 Cells were transfected either with LPRs containing eGFP/NS1 mRNAs (3:1 weight ratio, 2.5 µg mRNA in total) or with Lipofectamine Messengermax (LfM) containing the same mRNAs content. 24h post-transfection, the eGFP positive population was quantified via flow cytometry. t test was used for statistical analysis. (G) In vivo transfection efficiency. LPRs or LfM formulations comprising firefly luciferase mRNA and NS1 mRNA (3:1, weight ratio, 5 µg in total) were intradermally injected. One-day post-injection, in vivo luciferase expression was observed with IVIS Lumina LT In Vivo Imaging System.

Fig. 2 .

 2 Fig.2. BMP-2 released from transfected hMSCs. The secreted BMP-2 from hMSCs transfected with either 1µg BMP2 mRNA (BMP2) or 0.75µg BMP2/0.25µg NS1 mRNAs. Secreted BMP-2 was quantified by ELISA every 12h post-transfection. 2way ANOVA, Sidak's multiple comparisons test, was used for statistical analysis.

Fig. 3 . 28 Fig. 4 . 3 . 4

 328434 Fig. 3. Characterization of collagen and collagen-nanohydroxyapatite scaffolds. (A) The representative SEM images of collagen scaffold (CoLL: a, b), collagen-nanohydroxyapatite scaffold with 1: 1 mass ratio of collagen to nanohydroxyapatite (CoLL-1nHA: c, d), and 1: 3 mass ratio of collagen to nanohydroxyapatite (CoLL-3nHA: e, f). n=4. (B) Relative measurement of CoLL, CoLL-1nHA and CoLL-3nHA pore sizes, calculated by ImageJ software based on SEM images (n=8). (C) The open porosities of CoLL, CoLL-1nHA and CoLL-3nHA calculated by the liquid displacement method (n=4).Ordinary one-way ANOVA, Tukey's multiple comparisons test, was applied for statistical analysis. (D) The scaffolds' compressive strength. The axial and radial mechanical performances of the three types of scaffold (n=4). Statistical analysis was calculated with 2way ANOVA, Tukey's multiple comparisons test.

Fig. 5 .

 5 Fig. 5. Characterization of RAMs. The RAMs were prepared based on CoLL-1nHA scaffold by loading with LfM/mRNA complexes (RAM-LfM) or with LPRs (RAM-LPR). (A, B) Representative SEM images of RAMs (upper) with magnifications corresponding to vector/mRNA complexes (lower). B-insert shows a photograph of RAM-LPR. (C) Relative pore sizes of RAM-LPR calculated from SEM images, and normalized to the mean pore size of CoLL-1nHA (n=8). (D) The axial and radial compressive strengths of RAM-LPR normalized to the mean compressive strengths of CoLL-1nHA (n=4). Multiple t tests was used for statistical analysis.

Fig. 6 .

 6 Fig. 6. LPRs release and activity. (A) The release kinetic of LPRs from RAM containing 20µg mRNA. RAMs were incubated in TE buffer at 37℃, and the released mRNA were quantified after labeling with RiboGreen TM . The right column bar indicates the percentage of non-loaded LPRs. (B, C) The transfection activity of released LPRs. The RAMs containing 20µg BMP2 mRNA (RAM-BMP2) or 15µg BMP2/ 5µg NS1 mRNAs (RAM-BMP2/NS1) were incubated in wells containing cultured C2C12A-BRE/Luc cells that stably express luciferase under BMP-2 stimulation (BMP-2 responses element, BRE). 1µg BMP2 mRNA (BMP2) and 0.75µg BMP2/0.25µg NS1 mRNA (BMP2/NS1), formulated as LPRs, were set as controls. At indicated time points, C2C12-BRE/Luc cells were harvested and lysed for luciferase expression assay. The statistic differences among the groups within each time point are shown in Supplementary Table2.

Fig. 7 . 28 Fig. 8 .

 7288 Fig. 7. Micro-CT analysis of bone induced by RAMs. (A) Representative photos of retrieved mRNA-free (CoLL-1nHA), BMP2 mRNA LPRs containing-RAM (RAM-BMP2) and BMP2/NS1 mRNAs LPRs containing-RAM (RAM-BMP2/NS1) scaffolds 8 weeks post subcutaneous implantation. (B) Representative 2D (left) and 3D (right) µCT images of the retrieved scaffolds. (C) Bone formation quantified using µCT analysis. N= 3 for CoLL-1nHA; N=8 for RAMs. ANOVA with Dunnett's post-hoc test was used for statistical analysis. B: scale = x nm

Acknowledgment

We are grateful to David Gosset from the P@CYFIC platform (CBM) for technical help in confocal laser scanning microscopy observation, Antony Delalande and Cyril Guimpied for mice surgery assistance, and Audrey Sauldubois from the Centre de Microscopie Electronique platform (Université d'Orléans) for TEM study. We also thank Sonia Georgeault from Plateforme IBiSA de Microscopie Electronique de 25/28 l'Université de Tours for SEM observation. Region Centre Val de Loire and CNRS supported this work.

Pinpin Wang was supported by a CSC grant from China.

Author Contributions


L Aveneau
email: aveneau@sic.univ-poitiers.fr

P Combeau

Path finding based on Monte Carlo Techniques Compared with a Full Ray-Tracing Approach in Narrow and Wide Bands

Efficient propagation prediction requires a tool both fast and accurate, which are two opposite qualities. Generally, such a tool is fast using crude approximation, leading to miss some propagation paths. Moreover, its accuracy is usually validated by some comparisons to measurement. Then, only the statistical behavior of such a tool is available, but not its particular one for a given scene.

In this paper we propose a new optimization for finding the propagation paths, based on the Monte Carlo techniques. It allows a theoretical knowledge of the error due to our approximation, so that any engineer can really choose between faster or accurate calculations.

Our approach is compared with a Full Ray Tracing tool : This allows to discuss about the validity of our model, and its gain in terms of computation time. Comparisons are produced both in narrow and wide bands.

I. INTRODUCTION

Ray tracing is an interesting technique for propagation prediction. Based on the Uniform Theory of Diffraction [START_REF] Kouyoumjian | A uniform geometrical theory of diffraction for an edge in a perfectly conducting surface[END_REF], a Full Ray Tracing implementation (or FRT) allows to find all the theoretical contributions between a transmitter and a receiver. Many confrontations with measurements have shown that such a solution gives precise predictions [START_REF] Aveneau | Development and Evaluations of Physical and Computer Optimizations for the 3D UTD Model[END_REF].

Nevertheless, the problem with FRT is the computation time, that may become very large even with a simple scene. Indeed, the calculation consists in testing all the possible contributions. By increasing the number of allowed interactions in path computation, the combinatorial explosion of the number of such tests leads to impracticable tools.

Of course, many optimizations exist, mainly with visibility pre-computation, or using propagation properties [START_REF] Aveneau | Development and Evaluations of Physical and Computer Optimizations for the 3D UTD Model[END_REF]. Moreover, very powerful optimizations are less precise, since they imply to miss valid propagation paths [START_REF] Aguado | Efficient Ray-Tracing Acceleration Techniques for Radio Propagation Modeling[END_REF] [START_REF] Yun | A Ray-Tracing Method Based on the Triangular Grid Approach and Application to Propagation Prediction in Urban Environments[END_REF]. Computation time becomes certainly acceptable for an engineering use, but two questions arise : The validity of the prediction, and the user control on it. While the first one is established with comparisons to measurements, the second one is generally not available, even if the error control is the most important point of any optimization.

In this paper, we present a new approximate approach with error prediction account, and then compare it to a full solution. This error control is achieved using a mathematical framework for the optimization purpose. Our solution is a two pass one, with an oracle construction, followed by a ray tracing computation guided by the oracle.

In the second section, we present our approach, based on Monte Carlo techniques. So, we recall the Monte Carlo basis, and then detail our two pass implementation.

The purpose of the third section is to compare this new approach with a full one. We propose some test scenes, and discuss about the number and the importance of the missed paths with a wide band approach, and the narrow band differences. Next, we show the computation times, which are drastically reduced with our approximate solution.

As a conclusion, we discuss about our approach capacity to tend to the full solution, and about future works.

II. THE MONTE CARLO APPROACH

Monte Carlo techniques are used in many research fields, from nuclear computations to financial simulations. The basis of these techniques is the ability to compute the estimation of integral functions. Of course, this is useful when such functions are non computable in a reasonable time, as it is the case in visibility computations.

This section begins with a short presentation of the Monte Carlo principles, followed by implementation details.

A. Monte Carlo principles

The main idea of Monte Carlo integration [START_REF] Kalos | Basics[END_REF] is to use random sampling for evaluating the following integral :

I = Ω f (x)dx (1)
It is done by computing the estimate :

F N = 1 N N i=1 f (X i ) p(X i ) (2)
where the N independent random values X 1 , ..., X N are sampled using a convenient density function p.

The convergence rate mainly depends on the number of samples N . The standard deviation is

σ[F N ] = 1 √ N V f p (3)
where V denotes the variance. Then, dividing σ by a factor 2 necessitates 4 times more samples, and a fine calculation requires a large number of samples.

To deal with this problem, many variance reduction techniques have been developed. In this paper we use mainly one, the stratified sampling. It consists in partitioning the domain Ω into several non-overlapping regions -or strata -Ω 1 , ..., Ω n . Then, the work is done on each stratum Ω i by sampling n i random variables, according to some given density function p i . This technique greatly reduces the variance on Ω, which becomes the sum of the variances for every stratum.

B. Implementation

Our implementation is quite easy to understand. It is based on a two pass process.

1) Oracle Construction: The first step consists in exploring the visible part from a given transmitter, using Monte Carlo techniques, by sampling each face and each dihedron. The samples are produced using a stratified strategy, by fixing n i = 1. It is similar to a ray launching strategy, but without the reception part.

A sample is a primary ray, that can be reflected or diffracted a given number of times. The reflection is obtained by searching the first face intersecting by the primary ray. The diffraction is more difficult to control: in a ray launching strategy, the diffracting edges are found by testing the intersection of a primary ray with cylinders. But their size must depend on the parameterization of the oracle. Since we search to obtain as many paths as possible, we used the discrete tube technique [START_REF] Aveneau | The discrete tube: an efficient acceleration for diffraction computation[END_REF]. This solution allows to quickly find all the edges included into the n first Fresnel ellipsoids. In order to miss the minimum of edges, the number of ellipsoids directly depends on the number and on the lengths of the primary rays.

Then, each reflection or diffraction fact is stored on the corresponding face or dihedron. We do not keep the localization of diffraction or reflection, but the history of faces or dihedra that have been used to bounce on the current object.

Notice that the final user may specify either the total number of samples, or the standard deviation.

2) Propagation: The propagation step looks like a classical Full Ray Tracing (FRT) solution. The only difference is that we do not explore all the combinations of faces and dihedra, but only those predicted by the oracle. Then, at a given reception location, for each history stored into each face or dihedron, we compute the corresponding path that may add some propagation information.

Since we do not explore all the theoretically possible paths, then the computation time is really reduced. We obtain a better ratio between the combinations that lead to a valid path, and those that are explored for nothing.

Notice that this technique is different from Tan's one [START_REF] Tan | Propagation Model for Microcellular Communications Applied to Path Loss Measurements in Ottawa City Streets[END_REF], mainly in two points:

• We can use standard deviation to ensure a given precision, • We do not use a sphere around a given receiver. In fact, the receiver location does not have any importance in the first step.

C. Error control

As detailed in the next subsection, the oracle construction can be computed with either a number of primary rays or a maximum standard deviation. With this last solution, the oracle must decide itself when to stop the computations.

With a classical Monte-Carlo approach, this is usually implemented in the following way. The computation is made two times, with the same number of samples. The results are compared, in order to verify that their difference is under a given precision. If it is the case, then the calculation is stopped. In the other case, then these two results is merged into one, and another Monte Carlo simulation is made with the same number of samples (i.e. twice the initial number). This process is repeated until the convergence is ensured.

In our oracle construction, the main question is how to compare the results. In fact, the results is the set of faces and dihedra visible from the transmitter, after some reflection and diffraction. So, the convergence of the Monte-Carlo process can be established by comparing these sets: our tests have shown that this can be obtained quickly, and allow an auto parameterization of the oracle construction.

In the ideal case, the computation leads to two identical sets of faces and dihedra. Of course, this do not imply that we obtain all the desired faces and dihedra. Nevertheless, this solution ensure that this kind of error have a small probability to arise.

III. COMPARISON WITH FULL SOLUTION

The comparison of our proposed solution with a Full Ray-Tracing is made with three different test scenes. We give here some results in narrow bands, by comparing the number of covered receivers, and the mean number of paths at each receiver locations. Then, in wide band we propose some comparisons of the power delay profil for some receivers.

All the computation made with the presented solution are performed with a mean of 1024 primary rays for each face or dihedron, and 32 diffracted rays at each diffraction points. The computation was performed on a Pentium IV at 2.6 Mhz, with 512 Mo of RAM, under Linux.

A. The test scenes

The first scene -scene A -represents a small part of the campus at the Poitiers University, in France. It contains 194 faces and 206 dihedra. As it can be observed on the FIG. 1, it is a classical suburban scene, with a poor building density.

As depicted in FIG. 2, the scene B is a classical downtown environment, with only 1352 building faces, and 1956 dihedra. The transmitter is below the rooftop at a street corner.

The last scene -scene C -is an extended view of the scene B, with 4038 building faces and 5999 dihedra, as it is shown in FIG. 3.

B. Narrow band results

Clearly, a good computation of the coverage area for these three scenes requires to process many reflections and diffractions. Then, with a full ray-tracing implementation, the computation time becomes very large and can not be achieved.

Since, with a FRT, the computation times are very important, we sometimes used an accelerated version base on a visibility graph construction -the italic computation times -. For information, the real time with 2 reflections and 0 diffraction for the test scene B is estimated to more than 218 minutes, and to more than 4926 hours with 3 reflections. The results are respectively presented in the table I, II and III for the test scene A, B and C. The number of receiver locations is 15158 for the scene A, 8983 for scene B, and 8024 for the scene C.

Clearly, the computation times are drastically reduced, even with a small number of interactions. The mean number of computed paths is not so reduced. This is verified by comparing the coverage area obtained. Moreover, since we obtain almost all the main paths, the full path loss is not very different. For instance, with the test scene A, the total path losses for the 5 receiver locations depicted in FIG. 1 -where Rx1 and Rx2 are in LOS, and Rx3, Rx4 and Rx5 in NLOS -are 64, 76, 107, 112 and 99 dB with the FRT, and 64, 80, 109, 114 and 99 dB with the oracle method.

The computation with a FRT implementation are often impossible to obtain, due to the very long time needed. Nevertheless, the results presented show that our solution is faster and valid in term of number of paths obtained.

As a general conclusion on the narrow band results, it is shown that the Monte Carlo construction produces an important computation time optimisation, without missing too many paths. Moreover, the missed contributions generaly imply small faces or dihedra, and so are neglictable in the final results.

C. Wide band results

The results presented here are produced only with the scene test A, since the results are roughly the sames with the other scenes. They are computed with the same receiver locations than those presented in the previous section, since they illustrate LOS and NLOS configurations. The FIG. 4 shows the impulse responses at Rx1, and the FIG. 5 at Rx3, for the FRT algorithm and the oracle one. It seems obvious that the two curves are very similar, which is in accordance with the narrow band results. The Delay Spread parameter is one of the most popular for characterizing the channel impulse response. The table IV shows such Delay Spread results for two reflexions and one diffraction, with both the FRT and the oracle method. These results seem to be different even if the impulse responses are very closed. This can be explained by the fact that the delay spread parameter is very sensible to the low variations of the impulse response. Furthermore, one can note that, as it is shown in the litterature, the considered dynamic for the impulse response computation play an important role in the determination of the Delay Spread. Thus, we constate a difference of about 80 ns for the RX2 location between when we use a dynamic of 15 dB, which is the recommanted value by the IUT.

IV. CONCLUSION

We have presented here a propagation prediction tool, with an oracle construction based on Monte Carlo techniques. It allows to control the trade-off between the rapidity and the accuracy of the calculations. We have shown some results and discussed about them, showing the reduction of the computation times and the low prediction error obtained, compared to a full ray tracing software.

Of course, many aspect can be improved: The first one is the computation time, which may be reduced using some visibility precomputation from the receiver locations. The second problem concerns the missed paths, due to the Monte Carlo approach. A solution may be to perform the oracle construction using 3D volume propagation with a discrete approach. Nevertheless, this may imply to find too much contribution, and so to increase the global computation times.

Fig. 1 .Fig. 2 .Fig. 3 .

 123 Fig. 1. The test scene A, for 1 diffraction and 2 reflections

Fig. 4 .Fig. 5 .

 45 Fig. 4. Power Delay profi les at Rx1: Red line for FRT, and dark line for Oracle construction

TABLE I NARROW

 I BAND RESULTS FOR THE SCENE A : THE COMPUTATION TIMES AND THE MEAN PATH NUMBERS

	Inter.	Classical FRT	With oracle
		Comp. Time Mean PN Comp. Time Mean PN
	1R 0D	2"93	2.73	2"31	2.73
	2R 0D	3'21	4.36	5"20	3.90
	3R 0D	2h19'31"	5.84	8"46	4.64
	4R 0D			11"24	4.73
	0R 1D	16"87	11.90	13"45	11.90
	1R 1D	17'36"	52.89	1'31"	44.74
	2R 1D	3h 18'	122.22	4'35"	83.94
	3R 1D			9'22"	109.41
	Inter.	Classical FRT	With oracle
		Comp. Time Mean PN Comp. Time Mean PN
	1R 0D	9"71	3.06	3"58	3.06
	2R 0D	51'04"	5.85	6"04	5.60
	3R 0D			11"41	7.04
	4R 0D			19"03	8.17
	0R 1D	16"17	11.90	13"14	11.80
	1R 1D	1h 09' 49"	62.61	1'35"	56.60
	2R 1D			6'08"	116.08
	3R 1D			18'59"	171.32

TABLE II NARROW

 II BAND RESULTS FOR THE SCENE B : THE COMPUTATION TIMES AND THE MEAN PATH NUMBERS

	Inter.	Classical FRT	With oracle
		Comp. Time Mean PN Comp. Time Mean PN
	1R 0D	22"73	3.12	4"83	3.12
	2R 0D			7"74	5.13
	3R 0D			12"74	6.47
	4R 0D			19"82	7.83
	0R 1D	1'34"	14.71	15"89	14.71
	1R 1D			2'30"	53.10
	2R 1D			11'46"	113.17

TABLE III NARROW

 III BAND

RESULTS FOR THE SCENE C : THE COMPUTATION TIMES AND THE MEAN PATH NUMBERS

TABLE IV DELAY

 IV SPREAD RESULTS IN NANO SECONDS