Composite matrix used for promoting innervation, osteogenesis and angiogenesis

Abstract

Provided is a composite material used for promoting innervation, osteogenesis and angiogenesis.

Description

[0001] Composite matrix useful for promoting innervation, osteogenesis and angiogenesis

[0002] TECHNICAL FIELD

[0003] The present invention relates to a composite matrix useful for promoting innervation, osteogenesis and angiogenesis.

[0004] TECHNOLOGICAL BACKGROUND

[0005] The team of inventors (Silva et al, Cell Death and Disease, 2017 Dec 13;8(12):3209; Leroux et al., Cell Commun Signal. 2020 Oct 19;18(1):162) demonstrated, using co-culture models of sensory neurons, mesenchymal cells and endothelial cells, the impact of communication between these three cell types and the importance of neurovascular dialogue on osteogenesis. Based on these observations, this team developed a hydrogel based on elastin peptides, capable of stimulating the recruitment of nerve fibers (Paiva dos Santos et al., Acta Biomaterialia, 2019 Nov;99:154-167; PCT / EP2019 / 055075), including sensory neurons, and of hosting other cell types.It is still in this context that the inventors sought to improve various properties of said hydrogel, in particular in terms of homogeneity of cell colonization and increasing the biodegradability of the material, and propose a biocompatible, injectable composite matrix, capable of stimulating innervation, vascularization of tissues and of presenting osteogenic properties for application in bone engineering.

[0006] SUMMARY OF THE INVENTION

[0007] The inventors have developed a new composite material comprising an organic phase and a mineral phase. This material is biocompatible, injectable, capable of stimulating innervation and tissue vascularization and of exhibiting osteogenic properties for application in bone engineering. The invention relates more particularly to a composite matrix combining an organic phase functionalized by bioactive peptides and a mineral phase comprising calcium phosphate, the composite matrix according to the invention is capable of recruiting sensory neurons and hosting bone-forming and endothelial cells.

[0008] DETAILED DESCRIPTION

[0009] The composite matrix according to the invention is characterized in that it comprises an organic phase comprising an elastin-type peptide, at least one bioactive peptide and a mineral phase comprising calcium phosphate. 1. Elastin-type peptides

[0010] The first component of the composite matrix according to the invention is an elastin-like peptide (or ELP for Elastin-Like Polypeptide in English) comprising at least one alkenylated methionine residue before formation of the composite matrix. This type of peptide, their method of manufacturing by genetic engineering, and their purification are known to those skilled in the art who may in particular refer to international application WO2017021334 and to the articles Petitdemange et al. (Biomacromolecules. 2017 Feb 13; 18(2):544-550) and Petitdemange et al. (Bioconjug Chem. 2017 May 17;28(5): 1403-1412). Those skilled in the art may also refer to the examples presented below for the production of the ELP designated ELPM80.

[0011] In the context of the present invention, the term "alkenylated methionine residue" means that the side chain of the methionine residue is covalently linked to a group comprising an alkene group, i.e. comprising at least one double bond between two carbon atoms. Preferably, the term "alkene group" refers to the presence of a -CH=CH2 group in the group linked to the methionine residue. According to a particular embodiment, the methionine group is linked to the group of formula (I):

[0012] According to one embodiment, the synthesis of an alkenylated ELP using the group of formula (I) can be carried out by chemoselective thioalkylation at the level of methionine side chains using an allyl and glycidyl ether according to the procedure described in Petitdemange et al. (Bioconjug Chem. 2017 May 17;28(5): 1403-1412). Those skilled in the art may in particular refer to the examples of the present application to carry out a thioalkylation of the ELP peptide designated ELPM80.

[0013] According to one embodiment, the alkenylated ELP implemented within the scope of the present invention comprises at least one occurrence of the amino acid sequence VPGMG in which the methionine residue is alkenylated.

[0014] According to a particular embodiment, the alkenylated ELP used to produce the composite matrix according to the invention is a high molecular weight ELP, in particular greater than 20 kDa.

[0015] In one embodiment, the alkenylated ELP has a structure of formula (II) Z-[VPGXG] n (II) in which:

[0016] Z is a peptide comprising between 1 and 20 amino acids;

[0017] X represents a glycine residue, a valine residue or an alkenylated methionine residue, in particular an alkenylated methionine residue of formula (III): n is an integer between 40 and 160, even more particularly between 60 and 100, more particularly between 70 and 90; and in which the ratio between the molar ratio of valine / methionine alkenylated in position X is between 0: 1 and 10: 1, more particularly between 1: 1 and 5: 1, in particular between 2: 1 and 4: 1, said ratio being more particularly 3: 1.

[0018] According to one embodiment, X represents a glycine residue or an alkenylated methionine residue, in particular an alkenylated methionine residue of formula (III). According to another preferred embodiment, X represents a valine residue or an alkenylated methionine residue, in particular an alkenylated methionine residue of formula (III).

[0019] According to one embodiment, n is an integer between 60 and 100, in particular between 70 and 90, more particularly between 76 and 84, n being more particularly equal to 76, 77, 78, 79, 80, 81, 82, 83 or 84. More particularly, n is equal to 80.

[0020] According to a particular embodiment, Z is a peptide whose amino acid residue at the amino-terminal end is a methionine. According to another embodiment, the amino acids included in Z immediately upstream of the [VPGXG] motif ncorrespond to the dipeptide MW. Z may in particular consist of the dipeptide MW or comprise this dipeptide. According to a particular embodiment, Z consists of the dipeptide MW.

[0021] According to one embodiment, the ELP employed is a peptide of formula Z-[VPGXG] n in which the ratio between the molar ratio of valine / alkenylated methionine in position X is between 1:1 and 5:1, in particular between 2:1 and 4:1, said ratio being more particularly 3:1. According to another embodiment, the ELP used is a peptide of formula Z- [(VPGVG)(VPGMG)(VPGVG)2] X , in particular MW[(VPGVG)(VPGMG)(VPGVG)2] X in which x is an integer between 15 and 25, in particular between 19 and 21, x being more particularly equal to 20.

[0022] According to one embodiment, the ELP employed is derived from the peptide MW[(VPGVG)(VPGMG)(VPGVG)2]2O (ELPM80) described in the examples, said peptide comprising at least one alkenylated methionine residue. According to another particular embodiment, the ELP employed is of formula MW[(VPGVG)(VPGMaG)(VPGVG)2]2o (ELPM(alkene)-80) described in the examples, where Ma represents the alkenylated methionine residue of formula (III) above.

[0023] According to a particular embodiment, the composite matrix according to the invention comprises from 0.1 to 99.9% (w / v) of ELP. According to another particular embodiment, for a composite matrix with a concentration of between 3 and 5% (w / v) and for a thiol:alkene ratio of 1:1, said matrix comprises from 1 to 4% (w / v) of ELP, in particular ELPM80.

[0024] 2. Bioactive peptides

[0025] The composite matrix may also include bioactive peptides. Bioactive peptides are capable of performing a biological function when the composite matrix is ​​implanted into a tissue or organ of a subject. Such a bioactive peptide may, in particular, be an adhesion peptide, capable of binding a cell of interest, or a peptide enabling calcium fixation.

[0026] These bioactive peptides included in the composite matrix of the invention may comprise cysteine ​​residues at each of their ends. The cysteine ​​residues may be covalently linked directly to the amino acid sequence of the peptide, or via spacers, in particular a peptide or pseudopeptide spacer. The spacer may in particular be an amino acid or an amino acid sequence (in particular a di- or tripeptide), in particular a beta amino acid, more particularly a beta-Ala amino acid.

[0027] According to a particular embodiment, the quantity of bioactive peptides in the composite matrix according to the invention is between 99.9 and 0.1% (w / v). According to another particular embodiment, for a composite matrix with a concentration of between 3 and 5% (w / v) and for a thiol:alkene ratio of 1:1, said matrix comprises from 0.1 to 3% (w / v) of at least one bioactive peptide.

[0028] 2.1. Adhesion peptide A first category of bioactive peptides that can be used in the composite matrix of the invention are adhesion peptides, capable of recruiting neuronal cells, mesenchymal cells and / or endothelial cells. Mention may in particular be made, as peptides capable of recruiting endothelial cells, of peptides derived from laminin, fibronectin or type I collagen, more particularly laminin. Thus, the adhesion peptides may be chosen from the peptides REDV, RGD and GRGDSP derived from fibronectin, IKLLI, IKVAV, PDSGR and YIGSR, derived from laminin, and the peptide DGEA derived from type I collagen. As peptides capable of recruiting neuronal cells, we may in particular cite the peptides YIGSR, RNIAEIIKDI and IKVAV derived from laminin.

[0029] In a preferred embodiment, the composite matrix comprises at least one adhesion peptide selected from IKVAV and YIGSR.

[0030] According to a particular embodiment, the adhesion peptide is an IKVAV peptide. The IKVAV peptide may in particular be an IKVAV peptide of formula Cys-{spacer}-Ile-Lys-Val-Ala-Val-{spacer}-Cys, in particular the peptide of formula Cys-{Beta-Ala}-Ile-Lys-Val-Ala-Val-{Beta-Ala}-Cys.

[0031] According to another particular embodiment, the adhesion peptide is a YIGSR peptide. The YIGSR peptide may in particular be a YIGSR peptide of formula Cys-{spacer}-Tyr-Ile-Gly-Ser-Arg-{spacer}-Cys, in particular the peptide of formula Cys-{Beta-Ala}-Tyr-Ile-Gly-Ser-Arg-{Beta-Ala}-Cys.

[0032] According to an even more preferred embodiment, the composite matrix comprises the IKVAV peptide and the YIGSR peptide.

[0033] According to a preferred embodiment, in order to optimize and control the crosslinking of the composite matrix, the latter comprises a single adhesion peptide comprising at least two adhesion peptides, in particular a biomimetic peptide comprising the adhesion peptides IKVAV and YIGSR. The peptides composing the single adhesion peptide can be included in any order. According to an embodiment, the single adhesion peptide comprises, from the amino-terminal part to the carboxy-terminal part, the peptides YIGSR and IKVAV. According to a preferred embodiment, the single adhesion peptide comprises, from the amino-terminal part to the carboxy-terminal part, the peptides IKVAV and YIGSR. The single adhesion peptide can comprise cysteine ​​residues at each of its ends.The cysteine ​​residues may be covalently linked directly to the amino acid sequence of the single adhesion peptide, or via spacers, in particular a peptide or pseudopeptide spacer. The spacer may in particular be an amino acid or an amino acid sequence (in particular a di- or tripeptide), in particular a beta amino acid, more particularly a beta-Ala amino acid. Furthermore, the peptides composing the single adhesion peptide may be covalently linked directly to each other or via spacers. The spacer between the peptides composing the single adhesion peptide may in particular be a peptide or pseudopeptide spacer, in particular a glycine, diglycine or triglycine amino acid.

[0034] In addition, the single adhesion peptide comprising at least two adhesion peptides may also advantageously include one or more target sequence(s) of one or more metalloproteases, in particular metalloproteases 2 and 9. By way of illustration, we can cite the peptides:

[0035] - PVGLIG;

[0036] - the peptides QPQGLAK, GPLGLSLGK and GPLGMHGK described in Jha et al., Biomaterials, May 2016; 89; p. 136-47; and

[0037] - the peptides GPQGIAGQ, GPQGIWGQ and VPMSMRGG described in Lin et al., Acta Biomaterialia, 10(12), December 2014, p. 5106-5115.

[0038] Such a metalloprotease target peptide may be introduced to enable cleavage between adhesion peptides, which remain bound to the matrix of the invention while being available for adhesion to cells. Furthermore, a metalloprotease target peptide may be useful for triggering matrix degradation, thereby facilitating cell colonization. A metalloprotease target peptide may be included in the single adhesion peptide at any position, including at its amino terminus, between two adhesion peptides comprised in the single adhesion peptide, or at its carboxy terminus. Preferably, the metalloprotease target peptide(s) are positioned between two adhesion peptides in the single adhesion peptide.

[0039] According to a particular embodiment, the unique adhesion peptide includes at least one occurrence, in particular a single occurrence, of the target sequence of metalloproteases 2 and 9 PVGLIG.

[0040] According to a particular embodiment, the composite matrix comprises a single adhesion peptide comprising the peptides IKVAV, PVGLIG and YIGSR. According to a particular embodiment, said single adhesion peptide is of formula:

[0041] IKVAV(spacer)PVGLIG(spacer) YIGSR;

[0042] YIGSR(spacer)PVGLIG(spacer) IKVAV ;

[0043] PVGLIG(spacer)YIGSR(spacer)IKVAV;

[0044] PVGLIG(spacer)IKVAV(spacer) YIGSR;

[0045] IKVAV(spacer)YIGSR(spacer)PVGLIG; or

[0046] YIGSR(spacer)IKVAV(spacer)PVGLIG. Preferably, the single adhesion peptide is of the formula IKVAV(spacer)PVGLIG(spacer)YIGSR.

[0047] The spacers designated "(spacer)" in the unique sequences listed above may be of the same or different sequence, including identical. In particular, a spacer may be selected from a glycine residue, a diglycine dipeptide, or a triglycine tripeptide.

[0048] According to an alternative embodiment, said single adhesion peptide is of formula:

[0049] IKVAV-GGG-PVGLIG-GGG-YIGSR;

[0050] YIGSR-GGG-PVGLIG-GGG- IKVAV;

[0051] PVGLIG-GGG-YIGSR-GGG-IKVAV;

[0052] PVGLIG-GGG-IKVAV-GGG-YIGSR;

[0053] IKVAV-GGG-YIGSR-GGG-PVGLIG; Or

[0054] YIGSR-GGG-IKVAV-GGG-PVGLI.

[0055] Preferably, said unique adhesion peptide is of the formula IKVAV-GGG-PVGLIG-GGG-YIGSR.

[0056] Before crosslinking and formation of the composite matrix, the peptide allowing the introduction of the single peptide into said composite matrix may be of the formula: CpA-IKVAV(spacer)PVGLIG(spacer)YIGSR- PAC;

[0057] CpA-YIGSR(spacer)PVGLIG(spacer) IKVAV- PAC;

[0058] CpA-PVGLIG(spacer)YIGSR(spacer)IKVAV- PAC;

[0059] CpA-PVGLIG(spacer)IKVAV(spacer)YIGSR- PAC;

[0060] CpA-IKVAV(spacer)YIGSR(spacer)PVGLIG- PAC; or CpA-YIGSR(spacer)IKVAV(spacer)PVGLIG- PAC.

[0061] Preferably, before crosslinking and formation of the composite matrix, the peptide allowing the introduction of the single peptide into said hydrogel may be of formula CpA- IKVA V (e spacer)PV GLIG(spacer) YIGSR- PAC .

[0062] In a preferred embodiment, before crosslinking and formation of the composite matrix, the peptide allowing the introduction of the single peptide into said composite matrix is ​​of formula: CPA-IKVAV-GGG-PVGLIG-GGG-YIGSR-PAC;

[0063] CPA-YIGSR-GGG-PVGLIG-GGG-IKVAV-pAC;

[0064] CPA-PVGLIG-GGG-YIGSR-GGG-IKVAV-PAC;

[0065] CPA-PVGLIG-GGG-IKVAV-GGG-YIGSR-PAC; C(3A-IKVAV-GGG-YIGSR-GGG-PVGLIG-(3AC; or

[0066] CPA-YIGSR-GGG-IKVAV-GGG-PVGLIG-PAC.

[0067] Particularly preferably, the peptide before crosslinking and formation of the composite matrix, the peptide allowing the introduction of the single peptide into said composite matrix is ​​of formula CpA-IKVAV-GGG-PVGLIG-GGG-YIGSR-PAC.

[0068] According to a particular embodiment, the quantity of peptide allowing the introduction of the single peptide into the composite matrix according to the invention is between 99.9 and 0.1% (w / v). Depending on the composition of the single peptide, and the presence of other bioactive peptides, and for a given concentration of composite matrix, the person skilled in the art will of course be able to adjust the quantity of the single peptide.

[0069] 2.2. Peptides capable of inducing calcium phosphate nucleation

[0070] Peptides capable of inducing the nucleation of calcium phosphate constitute a second category of bioactive peptides capable of being introduced into the composite matrix according to the invention. The experiments presented below show that in a composite matrix according to the invention, the retention and homogeneity of distribution of calcium phosphate are improved in the presence of such a peptide capable of inducing the nucleation of calcium phosphate. It is notably shown that, unexpectedly, in the presence of the SNA 15 peptide approximately 10 times more hydroxyapatite particles are found in the matrices, which can be advantageously reflected in the osteoconductive and / or osteoinductive properties of the composite matrices.

[0071] For the purposes of forming the composite matrix, they may be included in a single peptide further comprising at least one adhesion peptide, or constitute an independent peptide. According to a preferred embodiment, the peptide capable of inducing the nucleation of calcium phosphate is a peptide which is not linked to an adhesion peptide, before formation of the composite matrix.

[0072] As an illustration, we can cite among the peptides capable of inducing the nucleation of calcium phosphate:

[0073] - statherin-derived peptides, including DDDEEKFLRRIGRFG (SNA 15), and SNA15 analogues, more particularly the peptides DSSEEKFLRRIGRFG (SNS15) and EFLRRIGRFG (SN11) described in Raj et al. JBC, volume 267(9), March 25, 1992, Pages 5968-5976;

[0074] - the DHTKE peptide (Sun et al. Journal of Agricultural and Food Chemistry 2017 65 (44), 9782-9789);

[0075] - the peptide SSSEEIVPN (Meisel et al. Biol Chem Hoppe Seyler. December 1988;369(12):1275-9); - the peptide DEGEQPRPFPFP (Lv et al. Food Chemistry, Volume 141(3), 2013, Pages 1645-1650);

[0076] - the WEWLHYW peptide (Charoenphun et al. Eur Food Res Technol 236, 57-63 (2013));

[0077] - the peptide DGDDGEAGKIG (Chen et al. Journal of Functional Foods, Volume 6, 2014, Pages 575-584);

[0078] - the GPAGPHGPPG peptide (Guo et al. Food Chemistry, Volume 173, 2015, Pages 536-542);

[0079] - the peptide VLSGGTTMYASLYAG (Jung et al. Eur Food Res Technol 224, 763-767 (2007));

[0080] - TCH peptide (Huang et al. Eur Food Res Technol 232, 281-287 (2011));

[0081] - the peptide VLGYIQIR. (Hou et al. Food Chemistry, Volume 243, 2018, Pages 389-395); and

[0082] - the peptide DNLPNPEDNKNYQ (Choi et al. Food Sci Biotechnol 21, 1663-1667 (2012))

[0083] According to a preferred embodiment, the peptide capable of inducing the nucleation of calcium phosphate is a peptide derived from statherin, more particularly an analog of SNA 15, even more particularly a peptide chosen from SNA15, SNS15 and SN11. Preferably, the peptide capable of inducing the nucleation of calcium phosphate is the peptide SNA15.

[0084] The peptide capable of inducing the nucleation of calcium phosphate may comprise cysteine ​​residues at each of its ends. The cysteine ​​residues may be covalently linked directly to the amino acid sequence of the peptide, or via spacers, in particular a peptide or pseudopeptide spacer. The spacer may in particular be an amino acid or an amino acid sequence (in particular a di- or tripeptide), in particular a beta amino acid, more particularly a beta-Ala amino acid. According to a preferred embodiment, before crosslinking and formation of the hydrogel, the peptide allowing the introduction of the peptide capable of inducing the nucleation of calcium phosphate into the composite matrix of the invention may be of formula CpA-DDDEEKFLRRIGRFG-PAC.

[0085] According to a particular embodiment, the quantity of peptide capable of inducing the nucleation of calcium phosphate in the composite matrix according to the invention is between 99.9 and 0.1% (w / v). As noted above, according to a particular embodiment, for a composite matrix with a concentration of between 3 and 5% (w / v) and for a thiobalcene ratio of 1: 1, said matrix may in particular comprise from 0.1 to 3% (w / v) of at least one bioactive peptide.

[0086] 3. Calcium phosphate

[0087] The composite matrix according to the invention further comprises a mineral phase comprising calcium phosphate. By way of illustration, we can cite calcium phosphate in the form of apatite, tri-calcium phosphate, bi-calcium phosphate and mixtures thereof. These sources of calcium phosphates can be used in any available crystalline form. Furthermore, derivatives of these sources of calcium phosphate, in particular carbon derivatives, can also be used. According to a particular embodiment, said calcium phosphate is in the form of apatite, more particularly hydroxyapatite. Advantageously, such a source of calcium phosphate makes it possible to increase the osteogenic properties of the composite matrix.

[0088] According to a particular embodiment, the composite matrix according to the invention comprises between 0.1 and 50% (w / v) of calcium phosphate. According to a particular embodiment, the composite matrix according to the invention comprises between 0.1 and 50% (w / v) of hydroxyapatite. More particularly, the composite matrix may comprise between 0.2 and 20% (w / v) of hydroxyapatite, more particularly between 0.5 and 4% (w / v), in particular between 2 and 3% (w / v). According to a particular embodiment, the composite matrix comprises 2.5% (w / v) of hydroxyapatite.

[0089] According to another particular embodiment, the composite matrix according to the invention comprises between 1 and 3% (w / v), more particularly 2% (w / v), of hydroxyapatite.

[0090] 4. Illustrative embodiments of the composite matrix according to the invention

[0091] The composite matrix according to the invention may in particular comprise an elastin-type peptide, at least one bioactive peptide, and calcium phosphate.

[0092] According to one embodiment, the concentration of composite matrix is ​​between 1 and 10% by volume (w / v), in particular between 3 and 5% (w / v).

[0093] According to another embodiment, the thiol:alcohol ratio is between 3:1 and 1:3 in moles, more particularly between 2:1 and 1:2, this ratio being more particularly equal to 1:1.

[0094] According to a particular embodiment, the composite matrix comprises:

[0095] (i) an elastin-like peptide;

[0096] (ii) at least one adhesion peptide;

[0097] (iii) at least one peptide capable of inducing the nucleation of calcium phosphate; and

[0098] (iv) calcium phosphate.

[0099] Components (i) to (iv) may be selected from the components described in parts 1 to 3 above.

[0100] More particularly, the composite matrix according to the invention may comprise:

[0101] (i) a peptide derived from the elastin-like peptide ELPM80; (ii) an IKVAV peptide and a YIGSR peptide;

[0102] (iii) SNA 15 peptide or an analogous peptide of SNA15; and

[0103] (iv) hydroxyapatite.

[0104] Preferably, the composite matrix according to the invention comprises:

[0105] (i) an ELPM80 peptide;

[0106] (ii) a peptide IKVAV(spacer)PVGLIG(spacer) YIGSR;

[0107] (iii) the SNA15 peptide; and

[0108] (iv) hydroxyapatite.

[0109] In a particularly preferred manner, the composite matrix according to the invention comprises:

[0110] (i) an ELPM80 peptide;

[0111] (ii) an IKVAV-GGG-PVGLIG-GGG-YIGSR peptide;

[0112] (iii) the SNA15 peptide; and

[0113] (iv) hydroxyapatite.

[0114] 5, Process for preparing the composite matrix

[0115] The composite matrix of the invention can be manufactured by mixing its various components, and any other optional element. The components of the composite matrix and the quantity of these components are chosen in order to prepare a composite matrix having physical and support properties adapted to the problem of its user.

[0116] The person skilled in the art is familiar with the crosslinking techniques used in the state of the art to manufacture a composite matrix by crosslinking. Thus, the formation of the composite matrix by crosslinking can in particular be carried out under the action of a stimulus such as a change in temperature, pH, or by means of a crosslinking agent, in particular a photosensitive crosslinking agent (or photoinitiator). Mention may thus be made, by way of illustration, of the induction of photopolymerization by means of a photoinitiator such as the compound Irgacure 2959, in particular used at a density of 0.5% (w / v) in the mixture, and activated by UV-visible light (Z=305 - 405 nm, in particular at 305 nm) for 5 to 12 minutes, more particularly between 6 and 10 minutes, in particular for approximately 8 minutes. In another variant, the photoinitiator may be chosen in particular from lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and riboflavin.The concentration of LAP may vary from 0.005% to 0.5% (w / v) in the mixture, and its photoinitiation may be triggered at a wavelength between 365 and 475 nm for 5 to 12 minutes, more particularly between 6 and 10 minutes, in particular for approximately 8 minutes. Advantageously, those skilled in the art may also refer to the preparation method described in the examples of the present application, in which the hydrogel is produced by formation of a cryomatrix. According to a variant of this embodiment, a prematrix solution, comprising the various components of the matrix, is frozen to form water crystals. As the crosslinking reagents (i.e. the components bearing the thiol and alkenyl groups) surround said water crystals, and when the solution is subjected to UV radiation, the alkene and thiol groups of the various components of the mixture react at the interface of said crystals.When the solution thaws, the space occupied by the water crystals becomes a pore in the matrix. This step can be followed by a freeze-drying step. For example, after crosslinking, the matrices can be rehydrated, for example for 24 hours, and freeze-dried under vacuum after freezing, thus sublimating the ice without melting it, and thus generating new pores. The combination of these two techniques has the advantage of allowing the formation of a wide distribution of pore sizes, allowing the colonization of different cell types useful for the induction of angiogenesis, innervation and osteogenesis.

[0117] Thus, according to a particular embodiment of the method according to the invention, the freezing step is carried out at a temperature between 0 and -80°C, more particularly at a temperature of approximately -20°C, for at least 2 hours, in particular for approximately 24 hours. The prematrix may then be subjected to UV radiation suitable for inducing photopolymerization, in particular for approximately 5 to 12 minutes, more particularly for approximately 6 to 10 minutes, in particular for approximately 8 minutes. After crosslinking, and according to a particular embodiment, a rehydration step may be carried out, in particular for at least 2 hours, in particular for approximately 24 hours. This rehydration step may be followed by a deep-freezing step at approximately -80°C for at least 1 hour, in particular for approximately 24 hours, then by a freeze-drying step lasting approximately 24 to 48 hours.

[0118] As indicated above, these steps allow the formation of a composite matrix comprising pores whose pore size distribution is extended and controlled. The composite matrix according to the invention may in particular have macropores with an average size greater than 100 μm.

[0119] 6. Use of the composite matrix according to the invention

[0120] Advantageously, the properties of the composite matrix can be very finely defined. Furthermore, said composite matrix according to the invention is biodegradable and has a porous structure suitable for colonization by different cell types useful for the induction of angiogenesis, innervation and osteogenesis. The inventors were also able to show that the composite matrix according to the invention is not cytotoxic. It thus brings together all the advantageous properties useful for the development of a tool suitable for tissue regeneration.

[0121] The composite matrix according to the invention is therefore capable of effectively supporting the in vitro culture of different cell types. Consequently, according to a particular embodiment, the invention relates to a new three-dimensional support capable of hosting in vitro different cells of interest for bone regeneration, in particular neuronal, bone or endothelial. The invention therefore provides those skilled in the art with a particularly advantageous 3D cell culture system allowing the cells to develop in a favorable environment, but also to study the interactions of different cell types between them. This parameter is important for studying regeneration phenomena which may require complex dialogues between different cell types.The support of the invention can in particular be used to host bone-forming and endothelial cells, and to study the angiogenic, osteogenic and innervation effect in an in vitro cell culture method, comprising the culturing of cells in a support as defined above. The use of the support according to the invention can further comprise the addition of an agent to the culture, such as a growth factor or any other agent having a biological effect or capable of having a biological effect (candidate agent) to determine its effect on one or more cellular parameters and responses such as cell growth, induction of quiescence, cell death, secretion of protein, or other molecules, or ions (in particular calcium ions, potassium), or the expression of certain genes.

[0122] According to another aspect, the composite matrix of the invention is used in a treatment method, in particular as an implant. The composite matrix is ​​in particular used in a regenerative medicine treatment method. It can in particular be used to stimulate the innervation of a tissue, in particular bone tissue, and can in particular be used in bone engineering. Advantageously, the composite matrix according to the invention promotes innervation, in particular in a regeneration context. More particularly, the composite matrix according to the invention can advantageously be used to recruit and stimulate the sensory nervous system, more particularly to promote bone regeneration. The composite matrix according to the invention can also be used to optimize or restore the vascularization and innervation of a tissue.

[0123] According to another embodiment, the composite matrix according to the invention is used for the repair of complex lesions which present vascular and nervous damage.

[0124] Furthermore, the composite matrix according to the invention can also be used to regenerate the interface with the peripheral nervous system, and to control bone development and its repair. According to another embodiment, the composite matrix can be used as a cell therapy vehicle. Thus, a composite matrix as defined above can be previously colonized by cells of therapeutic interest, for example by means of stem cells, in particular stem cells induced in a lineage of interest, hematopoietic stem cells, mesenchymal stromal stem cells derived from bone marrow or adipose tissue, neuronal stem cells, or a mixture of cells of different lineages. Such a composite matrix can be used in a method of treatment by cell or tissue regeneration.

[0125] The composite matrix according to the invention can also be used to modify implant systems, but also to improve their biocompatibility and integration.

[0126] LEGEND OF THE FIGURES

[0127] Figure 1: Fluorescence scattering and pore size quantification of hydrated matrices. A) ELPM80 + Pore size quantification of YIGSR hydrated hydrogels. N=2-5 hydrogels (502 <pores<2100) Kruskal-Wallis; post-hoc de Dunn; p<0,001. C) Classification de la taille des pores des hydrogels ELPM80 + YIGSR en micropores (nécessaires à l’angiogenèse et à l’innervation) et en macropores (nécessaires à la formation osseuse).

[0128] Figure 2: Quantification of vessels and nerve structures formed inside the hydrogel structure of the IKVAV + YIGSR composition and its scramble version VKAIV + GYSRI. Each implantation is represented by a point and the average is represented by a bar (n = 5-6). Note that within the same group, a large variability is observed, confirming heterogeneous cell colonization within the hydrogel structure. No statistical difference was detected.

[0129] Figure 3: Steps in matrix production: (1) preparation of pre-matrix solutions, frozen at -20°C for 24 hours before (2) crosslinking under UV light. (3) The cryogel is hydrated for 24 hours before (4) lyophilization.

[0130] Figure 4: Quantification of pore structure and size of 4% (w / v) dehydrated matrices with 0% or 5% (w / v) HA. Pore size was quantified using ImageL. Data are represented as ± SD, 10 <n<32, avec des différences statistiques indiquées *** p<0,001 (ANOVA avec test de Bonferroni post-hoc). Figure 5: Structure et taille des pores des matrices déshydratées produites à 8 min de temps de réticulation avec différentes concentrations massiques finales (% p / v) et différentes concentrations d’HA (% p / v). Quantification de la taille des pores. Les données sont représentées comme moyennes ± ET, 7<n<26, aucune différence statistique n’a été observée (ANOVA avec test de Bonferroni comme test post-hoc ou test de Mann-Whitney).

[0131] Figure 6: Internal porosity of 3% (w / v) and 4% (w / v) hydrated matrices with different HA concentrations. (B) Pore size was quantified using hnageJ. Data are represented as ± SD, 21 <n<65, avec des différences statistiques indiquées par * p<0,05 et *** p<0,001 (ANOVA avec test de Bonferroni, post-hoc). Barres: 100 pm.

[0132] Figure 7: Mineralization potential of 3% w / v matrices without HA or containing 1%, 2%, and 2.5% w / v HA implanted subcutaneously in mice for 2 and 4 weeks. Quantification of microCT acquisitions using MicroView software at day 0 (the day of implantation), and at 2 and 4 weeks. It should be noted that the volume of mineralized tissue increases over time, and matrices containing 2% and 2.5% w / v HA produced a higher volume of mineralized tissue than matrices without HA and containing 1% w / v HA. Data are represented as mean ± SD, n = 4-5 mice per group, with statistical differences indicated by * p < 0.05 and ** p < 0.01 (ANOVA with Bonferroni test, post-hoc).

[0133] Figure 8: Scanning electron microscopy coupled with EDX analysis on ELPs + IKVAV / YIGSR peptide matrices containing or not the SNA 15 peptide. The ELPs + IKVAV / YIGSR peptide matrices containing 0, 1, 2 and 2.5% w / v HA containing or not the SNA 15 peptide were lyophilized and cut in 2. The interior of the cut matrices was analyzed. The signal in the form of white dots corresponds to the calcium particles constituting the hydroxyapatite. The bars indicated in the figure correspond to 2 mm.

[0134] Figure 9: Longitudinal monitoring of matrix mineralization by micro-CT. Representative micro-CT images of subcutaneous implantation of matrices without HA or containing 1%, 2% and 2.5% (w / v) HA after 0, 15 and 30 days of implantation. Mineral volume / total volume (MV / TV) was measured from reconstructed three-dimensional micro-CT images using Microview® software. ANOVA statistical test followed by Bonferroni post hoc test with * p<0.05 and ** p<0.01 was performed.

[0135] Figure 10: Micro-CT monitoring of mineralization of ELPs matrix containing 2% (w / v) HA implanted subcutaneously with MicroView® software. Figure 11: Histological analysis of matrices implanted in ectopic sites and stained with Masson's Trichrome. Matrices comprising 0%, 1%, 2% and 2.5% HA were implanted subcutaneously and stained with Masson's Trichrome. Quantitative analysis of osteoid tissues formed (15 < n < 23 per sample) with a Kruskal-Wallis multiple comparison test with p ** < 0.01 and p *** < 0.001.

[0136] Figure 12: Immunostaining of vascular networks and nerve structures in composite matrices containing 2% HA after 0, 3, 7, 15 and 30 days of ectopic implantation. (A) Quantification of the surface area occupied by blood vessels relative to the matrix surface area x 1000. (B) Quantification of the surface area occupied by nerve structures relative to the matrix surface area x 1000. Quantitative analysis with a Kruskal-Wallis multiple comparison test.

[0137] Figure 13: Micro-CT analysis of mineralization induced by composite matrices in a rat femoral condyle bone lesion model. (A) Representative micro-CT images of bone lesions after 7, 15, 30 and 60 days of implantation and according to the groups: empty lesion, filled with ELPs matrices containing 2% HA and the positive control Collapat®. (B) Mineral volume / total volume (MV / TV) was measured from three-dimensional micro-CT images reconstructed using Microview® software for the three groups (empty, ELPs, Collapat®) and for all implantation times (D7, D15, D30 and D60). Quantitative analysis of mineralization with a Kruskal-Wallis multiple comparison test with p * < 0.1 and ns = not significant.

[0138] Figure 14: Histological section of a lesion made in the femoral condyle filled with the ELP / peptide matrix containing 2% HA. Masson's Trichrome staining of a bone lesion filled with the composite matrix after 7 days of implantation. The area corresponding to the lesion is delimited by a dotted circle in black.

[0139] Figure 15: Mineralization of matrices in the mandibular lesion model. 3D micro-CT images representing the reconstruction of the mandibular defect in the presence of the composite matrix.

[0140] EXAMPLES

[0141] Example 1: Preparation of a hydrogel with improved properties

[0142] Recently, the inventors' team evaluated a biomaterial considering the importance of vascularization and innervation during bone regeneration (Paiva dos Santos et al., Acta Biomater. 2019, 99, 154). The aim of this work was to develop an acellular, growth factor-free hydrogel that promotes angiogenesis and innervation. Hydrogels comprising ELPM40, polyethylene glycol (PEG), and different concentrations of the adhesion peptide IKVAV (25% w / w and 50% w / w) were produced, characterized, and evaluated. In vitro tests were able to show that the 50% IKVAV composition had greater potential to support osteogenesis, angiogenesis, and innervation. In vivo, this composition did not cause an inflammatory reaction, and was able to induce higher vascular density and the formation of nerve endings in the surrounding tissues, after subcutaneous implantation in mice.This previous study, however, has some limitations. First, the porosity of the hydrogel was not found to be sufficient to allow homogeneous cell colonization in the implant. Furthermore, the hydrogel does not appear to degrade after implantation, probably due to the PEG not being fully biodegradable in the long term.

[0143] The inventors therefore sought to modify the composition and structure of the hydrogel in order to improve the homogeneity of cell colonization and the biodegradability of the material. the induction of improved porosity

[0144] First, the inventors sought the best method to induce porosity in the hydrogel matrices and allow their colonization by cells. These structures were analyzed by scanning electron microscopy (SEM).

[0145] State-of-the-art hydrogels composed of ELPM40 and PEG were produced and their pore sizes were characterized.

[0146] The first pore induction method used, referred to as "CryoUV" below, involves the formation of a cryomatrix. A prematrix solution was frozen to form water crystals. As the crosslinking reagents surrounded the water crystals, and when the solution was subjected to UV radiation, the alkene and thiol groups of the various components of the mixture reacted at the interface of the crystals. When the solution thawed, the space occupied by the water crystals became a pore in the matrix.

[0147] The other method used is based on the production of matrices using the CryoUV method, followed by a lyophilization step (method designated "CryoUV + Lyoph" below). After crosslinking, the matrices were rehydrated for 24 hours, and lyophilized under vacuum after deep freezing for 24 h, thus sublimating the ice without melting it, and thus generating new pores. The combination of these two techniques has the advantage of allowing the formation of a wide distribution of pore sizes, allowing the colonization of different cell types useful for the induction of angiogenesis, innervation and osteogenesis. The inventors evaluated the external structure of the matrices using environmental SEM. CryoUV hydrogels were produced and, before SEM analysis, their water content was evaporated to allow SEM visualization.In the CryoUV + Lyoph method, freeze-drying allowed the three-dimensional structure of the hydrogel to be maintained. The results show that both methods can produce hydrogels with a porous structure.

[0148] The inventors then explored the possibility of modifying the composition of the hydrogels in order to improve the colonization and biodegradability properties mentioned above.

[0149] Production of the peptide ELPM(alkene)-80

[0150] The plasmid ELPM40-pUC19 encoding ELPM40 MW[(VPGVG)(VPGMG)(VPGVG)2] I5 described previously (Petitdemange et al., Biomacromolecules. 2017 Feb 13; 18(2):544-550) was digested with the restriction enzyme BsmFl to linearize it, then dephosphorylated at the 5' end by Antarctic phosphatase. The nucleotide sequence encoding the amino acid sequence [(VPGVG)(VPGMG)(VPGVG)2]5 was extracted from a plasmid pUC19 encoding ELPM20 also described previously, by digestion with the restriction enzymes BsmFl and BtgZI. This sequence was used as an insert introduced by ligation into the linearized ELP M40-pUC19 plasmid, thus obtaining plasmids pUC19-ELPM60 and pUC19-ELPM80 encoding ELPM60 and ELPM80 respectively. The ELPM80 coding sequence was then transferred, after digestion of the plasmid pUC19-ELPM80 with the restriction enzymes NdeI and BamHI, into the expression vector pET44a, allowing IPTG-inducible expression of ELPM80. The article Petitdemange et al.(Biomacromolecules. 2017 Feb 13; 18(2):544-550) describes the construction of the MX[VPGVGVPGMG(VPGVG)2]IO peptide expression vector (ELPM40), its expression in E. coli, its isolation from bacterial lysates, its purification, and its characterization. These conditions were adapted for the production and purification of ELPM80. Correct structure and molecular mass were confirmed by 1H NMR and MALDI mass spectrometry.

[0151] An alkene group was then added to the ELPM80 peptide by chemoselective thioalkylation of the methionine side chains of the ELPM80 peptide, again according to the conditions described in Petitdemange 2017, using allyl glycidyl ether to produce the ELPM(alkene)-80 peptide, designated ELPMa-80 in the following. The structure of the alkenylated peptide thus produced was confirmed by 1H NMR.

[0152] Production and characterization of a composite matrix comprising ELPM80

[0153] To improve the hydrogel composition, the inventors replaced ELPM40 included in the prior art compositions with the elastin-like peptide ELPM80. Furthermore, the adhesion peptide YIGSR was also introduced into the hydrogel composition. Its scramble version GYSRI was used as a control. The compositions of the produced hydrogels are shown in Table 1.

[0154] Table 1: Hydrogel compositions comprising ELPM80 and the adhesion peptide YIGSR

[0155] Diffusion of FITC-dextran 500 kDa into the matrix structure and fluorescence assessment in the core of the material by confocal microscopy were used to quantify pore size and porosity using ImageJ. The smallest measured pore size for both CryoUV and CryoUV + Lyoph methods is 2.16 pm, and the largest size is 382.5 pm with the CryoUV method, and 523.5 pm with the CryoUV = Lyoph method, indicating that the latter allows producing larger pores (Figure 1).

[0156] To satisfy angiogenesis, innervation, and bone formation, the range of pores must be very diverse. Literature data suggest that the optimal size for innervation is 0.005 to 0.3 pm, 5 to 15 pm for fibroblast growth, 50 to 400 pm for rapid vascularization, and 100 to 400 pm for bone regeneration. Considering the pore size to induce bone formation, we evaluated the frequency of pores according to their size, representing them as micropores (smaller than 100 pm) and macropores (larger than 100 pm). In general, macropores were more frequent when the samples were freeze-dried compared to the CryoUV method (Figure 1) which satisfies angiogenesis, innervation, and bone regeneration.

[0157] Based on the previous part, the CryoUV + Lyoph method was standardized to ensure (i) high porosity taking into account the criteria of (ii) large pore sizes to meet the prerequisite of cell colonization by different cells allowing angiogenesis, innervation and osteogenesis; iii) compositional modifications were made to produce a completely natural polymeric material to facilitate matrix degradation, i.e., the production of a PEG-free matrix was possible, while maintaining the alkenelhiol equinolarity; iv) ELPM40 was replaced by ELPM80. The inventors implanted the IKVAV + YIGSR composition as well as its scramble version subcutaneously in mice to evaluate angiogenesis and innervation potential in vivo.After 4 weeks of implantation, samples were collected and analyzed histologically by hematoxylin-eosin (HE) staining to assess tissue architecture, cell colonization, and persistent inflammation. For angiogenesis potential, CD31 immunohistochemistry (IHC) was performed, and for innervation potential, P3 tubulin IHC was used. For both compositions, no persistent inflammation signals were observed, and nerves and vessels were observed inside the hydrogels. However, heterogeneous cell colonization occurred, indicating the need to optimize and standardize material production. Quantification of vessel and nerve densities also confirmed these observations, showing high variability within the same group (Figure 2).

[0158] Based on the data presented above, modifications seemed necessary to increase the functionality of the hydrogel in vivo. Given the limited reproducibility of hydrogels and the resulting cell colonization, the inventors decided to optimize the matrix production protocol and modify the composition in order to combine the osteogenic potential in a new generation of materials. As discussed above, using ELP and adhesion peptide-based hydrogels, the inventors have already shown that the hydrogels are capable of inducing angiogenesis and innervation surrounding the implantation site.

[0159] In order to increase the biological functionality, the inventors considered other biomimetic peptides to increase neural and vascular cell recruitment, matrix degradation, cell colonization and mineral content retention: (i) the basic adhesion peptides CpA-IKVAV-pAC and CpA-YIGSR-pAC were replaced by the sequence CpA-IKVAV-GGG-PVGLIG-GGG-YIGSR-pAC, where the same crosslinking chemistry strategy was retained: the thiol group is present on the flanking cysteines, allowing crosslinking with the alkenes grafted on ELPM80. The inventors also used a control peptide where the scramble versions VKAIV and GYSRI replaced their original sequences IKVAV and YIGSR, respectively. The PVGLIG sequence was retained in this control peptide.Three glycines were used as spacers between the functional motifs and (ii) PVGLIG, a target degradation site of matrix metalloproteinases 2 and 9 was included between the two adhesion sequences. Thus, once cleaved, each adhesion sequence will still be attached to the matrix and available for cell attachment. In addition, this sequence will trigger matrix degradation, and therefore help in cell colonization, (ii) The SNA15 peptide, CpA-DDDEEKLLRRIGRFG-pAC, a calcium phosphate nucleating motif derived from statherin, was used in order to retain and ensure the homogeneous distribution of hydroapatite particles. Therefore, (iii) hydroxyapatite (HA), corresponding to the synthetic bone mineral content, is included in the material composition to provide biochemical signals for osteoconduction properties.The inventors' group has already synthesized and characterized them, having rod-shaped HA crystals of 50-100 nm that form aggregates with an average diameter of 3.26 ± 0.62 pm. Using the CryoUV+Lyoph method, the inventors standardized the incubation time in the matrix production protocol (Figure 3).

[0160] Given the importance of porosity and pore size, the next step in the characterization process consisted of optimizing the matrices based on several factors: crosslinking time, final mass concentration, and HA concentration. Photopolymerization was induced using the photoinitiator Irgacure 2959, specifically used at a density of 0.5% (w / v) in the mixture, and activated by UV-visible light at 305 nm. First, crosslinking time was evaluated as it has a direct effect on matrix creation. 4% (w / v) matrices composed of ELPM80, biomimetic peptide containing the sequences IKVAV, PVGLIG, and YIGSR, SNA 15, and containing or not HA (Table 4) were produced, crosslinked for 5 min, 8 min, and 15 min. Using scanning electron microscopy, images of the matrices were obtained and the pore size could be quantified (Figure 4).

[0161] 4% (w / v) 3% (w / v) 2% (w / v)

[0162] ELPM80 0.67mM 0.51mM 0.34mM

[0163] Biomimetic peptides 3.54 mM 2.65 mM 1.77 mM *

[0164] SNA15 3.54 mM 2.65 mM 1.77 mM

[0165] HA 2.5 - 10% (w / v) 2.5 - 10% (w / v) 2.5 - 10% (w / v)

[0166] Table 4. Compositions of matrices with the concentration of all components.

[0167] *Biomimetic peptides consist of a single peptide containing the motifs IKVAV, PVGLIG, and YIGSR. The scramble version consists of VKAIV and GYSRI without affecting the PVGLIG sequence.

[0168] The results obtained suggest that the shorter the crosslinking time (5 min), the larger the pores. Indeed, with 15 minutes of crosslinking time, few pores were observed. Without wanting to be bound by any theory, the inventors hypothesized that the longer the crosslinking time, the more likely the cryomatrices are to begin to thaw. This would result in stiffer matrices, due to a higher crosslinking rate, and smaller pores due to thawing. Indeed, this may explain why with 15 minutes of crosslinking time, very few pores could be observed. In general, pore size is different depending on the crosslinking time in matrices without HA, but there is no difference in pore size with 5% (w / v) HA matrices. This suggests that HA interferes in pore formation, regardless of the crosslinking time.Considering the pore size in the matrices with and without HA, and the fact that the matrices were dehydrated, the inventors decided to standardize the crosslinking time to 8 minutes for the following experiments. Next, the inventors evaluated the effect of the final matrix concentration on the pore size. They produced 2% (w / v), 3% (w / v), and 4% (w / v) matrices with different HA concentrations and analyzed their microstructure (Figure 5).

[0169] The 3% (w / v) matrices tended to maintain their structure across different HA concentrations, while the 2% (w / v) and 4% (w / v) matrices had powder-like structures, with potentially reduced reproducibility. SEM analysis confirmed the external porosity of the dehydrated matrices produced with 8 min of crosslinking time, and demonstrated that the apparent mesh density increased proportionally to the matrix concentration. The 4% (w / v) matrices appeared to comprise more pores but with smaller diameters, while the 2% (w / v) and 3% (w / v) matrices appeared to comprise fewer pores but with larger dimensions. The pore sizes of the dehydrated matrices with the same HA concentration did not vary with the final concentration.Again, HA content appears to interfere with pore size in matrices with the same final concentration, but this trend does not appear to be statistically significant.

[0170] To confirm these results and to assess the internal porosity, the inventors quantified the pore size in the hydrated matrices by cutting them in the middle and producing histological slices by cryostat. Figure 6 shows that in the 3% (w / v) matrices, the 5% HA matrices have smaller pore sizes than the matrices comprising 0% and 2.5% HA. Furthermore, when comparing the 3% (w / v) and 4% (w / v) matrices, the 3% (w / v) matrices with 0% HA, 2.5% HA, and 5% HA have significantly larger pores than the 4% (w / v) matrices with the same HA concentration. It is important to note that in the 3% (w / v) matrices, there is no difference in pore size for the HA-free and 2.5% (w / v) HA matrices, and the pore size is larger than 100 pm, the minimum pore size described in the literature to satisfy osteogenesis.From there, work focused on 3% (w / v) matrices containing a maximum of 2.5% (w / v) HA.

[0171] Next, the inventors evaluated the overall porosity of the HA-free and 2.5% (w / v) HA matrices after rehydration. They used the methodology described by Ma and Zang, based on the overall densities of the fibrous matrix and the skeletal density (Ma and Zhang, J. Biomed. Mater. Res. 1999, 46, 60). The skeletal density is considered as the density of the polymers and HA. The HA-free and 2.5% w / v HA matrices did not differ in terms of porosity. The HA-free matrices were 98.9 ± 0.26% porous compared to 98.9 ± 0.49% for 2.5% w / v HA. These results suggest that the presence of HA does not interfere with the overall porosity of the hydrated matrices. The retention of HA in the matrix structure was then studied by thermogravimetric analysis (TGA). The content of HA retained in the matrix structure reached a plateau at 2.5% (w / v).The inventors therefore decided to use a maximum final HA concentration of 2.5% (w / v) for the rest of the tests, without excluding the possibility of using matrices containing 1 or 2% HA given the results obtained at 2.5%.

[0172] Next, the inventors analyzed the HA distribution by EDX on the surface and inside the matrices. SEM images confirmed the surface and internal porosity of the matrices with or without HA. EDX analysis is an X-ray technique used to identify the elemental composition of materials, in this case the calcium content is visible as white dots. The experiments were able to show that hydroxyapatite is distributed homogeneously on the surface and in the matrices.

[0173] In parallel, biological assessments were performed to analyze cell survival and distribution within the matrices. Primary rat endothelial cells (EC)-RFP and bone marrow-derived mesenchymal stem cells (BMSCs) were cocultured with the matrices and observed by confocal microscopy. The acquisitions made by the latter had a z-volume of 130 to 190 pm and were analyzed using Imaris for 3D representation. After 7 days of coculture, EC-RFPs were observed at the periphery and within the matrices, indicating that the matrices can support cell culture. To assess whether the cells colonize the matrix core, they were cut in the middle, and then histological sections were prepared using a cryostat.The core of the samples was counterstained with DAPI (40,60-diamidino-2-phenylindole) and images were captured to detect RFP and DAPI, to assess cell colonization in the matrix core. For matrices without HA, no RFP or DAPI staining was observed in the core of the matrices, but only at their periphery. On the other hand, RFP+ cells were homogeneously distributed inside matrices containing 2.5% HA.

[0174] Following extensive matrix characterization, the inventors subcutaneously implanted 3% w / v matrices without HA or containing 1%, 2%, and 2.5% w / v HA into mice. The inventors then monitored the implantations on the day of implantation (Day 0), 2 (2W), and 4 weeks (4W) to assess the mineralization potential of the matrices using microCT. MicroCT confirmed the formation of ectopic mineralized tissue within the week 2 and week 4 implantations (Figure 7). Once quantified, the volume of mineralized neotissue formed in matrices with 1%, 2%, and 2.5% w / v HA increased over time. Matrices containing 2% and 2.5% w / v HA did not differ on the induction of mineralized neotissue formed after 2 or 4 weeks of implantation. Matrices containing 2% and 2.5% produced a higher mineralized tissue volume than matrices containing 1% w / v HA after 2 weeks.A p = 0.059 was achieved when comparing the volume of mineralized tissue produced by matrices containing 1% and 2% w / v HA. Matrices without HA were not visible and did not produce significant mineralized tissue over time. A small amount of mineralized tissue was observed at 2 weeks in some animals, but it was not detectable by week 4, likely reabsorbed. These data suggest that matrices containing 1-2.5% HA have the potential for ectopic mineralization, and that 2% and 2.5% w / v HA performed best after 2 weeks.

[0175] In conclusion, the inventors described a novel cell-free and growth factor-free matrix composite material capable of inducing the formation of ectopic mineralized tissue in mice. This matrix is ​​composed of EEPM80 and functionalized with biomimetic peptides responsible for recruiting nerve and vascular cells, stimulating cell colonization and retaining HA. The functionalized matrices combined with 1 - 2.5% (w / v) of HA have osteoinductive properties. of the interest of the SNA 15 in the retention and distribution of

[0176] The inventors produced a composite matrix, according to the methods set out above, composed for its organic part of ELPM80-alkene, of biomimetic peptides containing the adhesion sequences IKVAV, YIGSR, the proteolytic cleavage motif PVGLIG, as well as the calcium nucleation peptide SNA15, and for its mineral phase of hydroxyapatite microparticles.

[0177] The calcium phosphate nucleating peptide SNA15, with the sequence DDDEEKFLRRIGREG, is derived from the salivary protein statherin. A modified SNA 15 peptide with the sequence CpA-DDDEEKPLRRIGREG-PAC was used.

[0178] Composite matrices containing or not containing this SNA 15 peptide were produced with the same concentration of ELPs matrix and varying the HA concentrations from 0% to 2.5%. An Energy Dispersive X-Ray (EDX) analysis was performed to determine the surface elemental composition of the materials, and more specifically the calcium element (identified as white dots in Figure 8). It can be seen that the matrices without HA contain little or no calcium. As expected, the more the matrices are supplemented with HA particles, the more the signal increases. This phenomenon is even more visible when the gels contain the SNA15 peptide.

[0179] Quantitative analysis shows that the atomic percentage of calcium in matrices containing SNA 15 is higher than in those that do not: compared to matrices not containing the SNA15 peptide, there is 8 times more calcium in 1% HA + SNA15 matrices, 8.9 times more in 2% HA + SNA15 matrices and 12.6 times more in 2.5% HA + SNA15 matrices.

[0180] It can be concluded that the SNA 15 peptide advantageously allows to substantially improve the retention of calcium phosphate particles since they are between 8 and 12.6 more numerous in its presence.

[0181] Example 3: In vivo evaluation of ELP-based composite matrices in ectopic sites and in bone lesions

[0182] I. Ectopic site implantations of ELP matrices supplemented with different concentrations of hydroxyapatite

[0183] ELP and peptide matrices containing different concentrations of HA particles were implanted subcutaneously to select the one with the best capacities in terms of mineralization, vascularization and innervation. The four matrices differed only in their HA content (0%, 1%, 2% and 2.5% of particles).

[0184] 1.1. Study of the mineralization potential of composite matrices

[0185] Mineralization of the matrices implanted subcutaneously was monitored by X-ray microtomography (micro-CT) from the time of implantation (D0) of the matrix, then after 15 and 30 days (D15 and D30) (Figure 9). On day 0, no mineralization was observed in the 0%, 1% and 2% HA implants, while very low mineralization was observed for the 2.5% HA matrix (Figure 8). These results indicate that HA particles at the concentrations studied are only slightly visible by the micro-CT technique.

[0186] After 15 and 30 days of implantation, ectopic mineralized tissue formed in the matrices containing HA particles. The volume of mineralized tissue formed in these composite matrices increased over time (Figure 8). However, no mineralization was detected after implantation of matrices lacking HA, demonstrating the importance of these particles in the mineralization process. After 1 month of implantation (D30), matrices containing 1% HA induced an increase in mineral volume of 21 ± 10%, 42 ± 7% for those containing 2% HA, and 37 ± 5% for matrices containing 2.5% particles. We did not measure a significant difference in the amount of mineralized tissue formed between matrices containing 2% and 2.5% HA after 30 days of implantation. The monitoring of the mineralization of the 2% HA matrix is ​​presented in figure 10. 1.2. Study of the osteoinductive properties of the composite matrices

[0187] After 4 weeks of implantation, the matrices were excised, embedded in paraffin, and analyzed histologically by Masson trichrome staining to assess tissue architecture, cell colonization, inflammation, and osteoid tissue formation.

[0188] Histological sections of the materials associated with the surrounding epidermal, adipose, and muscle tissues show that after one month of implantation, very little fibrosis occurs around the ELP matrices, regardless of the formulation. This indicates that the matrices induce little inflammation and are not rejected. These results are consistent with the biocompatibility demonstrated in vitro. We find on these sections a sheet-like structure, as well as the presence of pores.

[0189] The presence of cell nuclei within the implanted matrices was also observed, indicating that cells were able to penetrate inside these matrices.

[0190] Since the tissues and matrices were not decalcified, some areas of high mineralization were damaged during microtome sections. However, mineralization is present in the form of dark purple crystals. Osteoid tissue can also be observed at the periphery and within the matrices.

[0191] The amount of newly formed osteoid tissue (Figure 11) increases significantly depending on the amount of HA in the matrix from 0 to 2%. On the other hand, the values ​​measured in matrices containing 2.5% mineral fraction are not significantly different from those containing 2%. Thus, the matrix containing 2% HA particles seems to be an advantageous formulation to stimulate a mineralization and osteoinduction process.

[0192] 1.3. Study of the angiogenic and innervation potential of ELP matrices

[0193] The formation of vascular and nervous structures was observed by immunostaining of a vascular marker (Endomucin) and a neuronal marker (P III tubulin). During this study, histological sections of the matrices were taken at 0, 3, 7, 15 days and 1 month post-implantation to determine the kinetics of formation of these structures.

[0194] Regarding the formation of vascular structures on day 0, approximately 3 to 4 hours after implantation, no blood vessels were observed in the matrices. Three days after implantation, blood vessels appear mainly at the periphery of the matrices, and then we observe their infiltration on day 7 eme day. After 15 days of implantation, the vessels are present both at the periphery and inside the matrices. At 30 eme day, the vascular network within the matrices has become denser.

[0195] These observations are confirmed by the quantification of blood vessels within the studied matrices (Figure 1A). After 1 month of implantation, vascular structures increase by approximately 10% in all matrices, regardless of the HA concentration. Only a significant difference is observed between matrices without HA (0%) and those containing 1% HA.

[0196] The matrix's ability to stimulate innervation was also demonstrated by the presence of nerves at the periphery of the material, particularly in the early stages after implantation. During bone regeneration, nerve structures are the first to appear, and this is already in the early stages following bone injury. This explains the presence of several nerve fibers visible at the periphery of the composite matrix from the 3 eme day of implantation. After 1 month, the nerve structures are still detected at the periphery of the matrix, but their diameter appears to have increased.

[0197] Quantitative results obtained by measuring the surface area occupied by nerve structures located at the periphery of the matrices relative to the total surface area of ​​the matrices confirmed previous observations (Figure 12B). In terms of formulation, it appears that composite matrices containing 1% HA significantly stimulate the formation of nerve structures after one month of implantation. This is particularly visible when comparing these results with those obtained with matrices without HA, or supplemented with 2.5% HA.

[0198] Since the measured density of nerves or vessels can vary depending on the location and / or angle of tissue sections, we also used a technique to visualize the entire tissue in 3 dimensions after immunofluorescence labeling of neuronal networks, without having to cut the sample. This technique allows us to observe in 3 dimensions the autofluorescence of the matrix and the presence of networks of nerve structures expressing P III tubulin. In particular, a strong presence of nerve structures can be seen near the matrix. Furthermore, the vascular networks appear to be homogeneously distributed.

[0199] 1.4. Study of the inflammatory reaction after subcutaneous implantation of composite matrices. The inflammatory reaction, when moderate, is an essential process for tissue regeneration and tissue vascularization. On the other hand, an excessively strong response from the immune system can lead to implant rejection.

[0200] In the case of composite matrices, histological analysis by Masson trichrome (TM) staining did not reveal inflammatory fibrotic capsules around the implants, regardless of their composition.

[0201] To confirm these data, we performed immunostaining of immune cell populations. We used CD11b, which is expressed by the entire myeloid cell line (monocytes, macrophages, neutrophils), and CD45, which is a more specific marker for lymphocytes, as inflammation markers. We observed that there is a recruitment of immune cells to the periphery of the matrix a few hours after implantation. These cells are detected up to the 7 eme post-implantation day. Subsequently, after 3 months and 6 months, immune cells are only present inside the matrix. These could be macrophages that play a role in the degradation of the matrix.

[0202] 1.5. In vivo study of the fate of the composite matrix

[0203] To determine the lifespan of the matrices, subcutaneous implantations were performed and we monitored their development up to 6 months after implantation.

[0204] Immediately after implantation (IO), the composite matrix containing 2% HA particles is clearly visible (reddish color), and blue-colored HA crystals can be seen. After 1 month of implantation, the free spaces left by the pores are filled following cell colonization. We can identify several nuclei inside the matrix. After 3 months of implantation, the structure of the matrix is ​​no longer visible, which is probably due to its degradation by the immune system. We can note the presence of a strong cellular component and an extracellular matrix that fills the spaces previously occupied by the composite material. Similar results are observed after 6 months, with however an increase in the mineralization of the entire area previously occupied by the composite matrix.

[0205] To conclude on these studies carried out in ectopic site, the matrices developed from ELPs, IKVAV / YIGSR peptides and SNA15 revealed their capacity to promote angiogenesis and innervation. The supplementation of matrices with HA gives them osteoinductive properties since they are then capable of forming mineralized tissue and osteoid tissue. In conclusion, the composite matrix containing 2% HA being the most efficient in terms of new bone formation, neovascularization and generation of nerve structures, we chose it for implantations in bone site.

[0206] II. Implantation of the composite matrix in bone site

[0207] The regenerative capacity of the composite matrix containing 2% HA was evaluated in rats in models of femoral condylar defect and mandibular defect.

[0208] II. 1. Implantation in the rat femoral condyle

[0209] Circular defects of 3 mm diameter were made in the femoral condyles of rats. Three conditions were tested: the unfilled defect which corresponds to the negative control, the defect filled with our composite matrix, and the defect filled with the commercial matrix Collapat® which served as a positive control.

[0210] Micro-CT scans were performed at different times after implantation (7, 15, 30, and 60 days) to monitor the mineralization of condylar defects. 2D images of the femoral condyles reconstructed by 3D micro-CT are shown in Figure 13A, and the quantification of mineralized tissue volume relative to the total volume (MV / TV) is shown in Figure 13B.

[0211] Images observed by micro-CT reveal the formation of a mineralized volume in the lesions filled with composite matrices and Collapat®, while less mineralization is detected for the unfilled defects (Figure 13A).

[0212] The results of the quantitative analysis (Figure 13B) show that there is a partial mineralization of the unfilled defects with a non-significant increase in MV / TV as a function of time. At 7 days, this ratio is 8.6% ± 4.8%; then it increases to reach 10.3% ± 5.6% at 15 days, 14.7% ± 5.9% at 30 days and 37.5% ± 3.0% at 60 days. However, we observed fractures of the site in some animals, which complicated the quantification of MV / TV. Our previous work had shown that bone lesions of 2.8 mm in diameter could repair naturally (Schlaubitz et al. 2014). In our case, lesions of 3 mm in diameter were made, probably weakening the lesion site and causing fractures that delayed or altered the kinetics of repair. Micro-CT images nevertheless reveal mineralization at the periphery of the lesion, which explains the high MV / TV ratio of the area of ​​interest (ROI) (Figure 13B).In all cases, these images observed for unfilled lesions are different from those obtained after implantation of composite matrices. Indeed, in this case, we observe a mineralization that begins at the internal edges of the lesion and increases as a function of post-implantation time. The mineralization is initially essentially peripheral, then evolves over time towards the interior of the lesion. After 60 days of implantation, we observe that the cortical part has reformed, unlike what is observed with the unfilled defect (Figure 13A). Quantitative analyses show that MV / TV values ​​increase significantly over time (Figure 13B). They are 8.0 ± 3.9% after 7 days of implantation; 15.6 ± 7.0% after 15 days; 28.8 ± 14.2% after 30 days to reach 56.0 ± 6.9% after 60 days.

[0213] Regarding the Collapat® control material, the MV / TV is 40.2 ± 9.4% from the 7 eme day of implantation, which is much higher than that measured with the ELP / peptides / HA composite matrix. This result was expected since this commercial material contains a high content of calcium phosphate particles, particles that induce an X-ray signal (Figure 13A). We did not observe a significant increase in mineralization from D7 to D30. The MV / TV ratio varies little or not at all, the MV / TV ratio is 67.3 ± 6.3% after 60 days.

[0214] Thus, if we compare the three conditions studied, we observe that the quantity of mineralized tissue measured between days 7 and 60 is multiplied by 4.4 in the unfilled defects, by 7.1 with our composite matrix and by 1.7 in the case of Collapat®.

[0215] We then performed a histological analysis of the new bone formation induced by the composite matrices.

[0216] 7 days, 15 days, 1 month and 2 months after implantation, the femurs were explanted, decalcified and histological sections were taken. These sections were then stained with Masson's Trichrome (Figure 14).

[0217] Histological sections of the lesion areas were observed. For unfilled lesions, the defect boundary was still clearly visible 7 days after surgery. After 15 days, the lesion had been filled with primarily collagenous tissue. On day 30, the lesion boundaries were difficult to distinguish, and organized bone-like tissue appeared at the periphery.

[0218] Histological sections show that the ELP / peptide / HA composite matrices were well integrated into the bone defects. There is no visible demarcation between the lesion area and the material. The presence of numerous cell nuclei inside the matrix confirms cell colonization. In addition, the absence of fibrosis is a sign of the good biocompatibility of the matrices used. Quantification of the newly formed osteoid tissue shows an increase from the 15 eme day, the maximum quantity being measured on the 30th eme day. At two months, however, the amount of osteoid tissue had decreased. Analysis of histological sections shows that bone formation occurs from the periphery towards the center of the defect, which confirms the images observed by micro-CT (Figure 13A).

[0219] Regarding the Collapat® material, good osseointegration was also observed as well as bone tissue formation from the periphery to the center of the defect. Quantification of newly formed osteoid tissue also showed a maximum of neoformation after 30 days, which is comparable to the kinetics observed with our composite matrices.

[0220] To specifically observe the presence of bone cells, we performed immunostaining of osteocalcin, a specific marker of bone tissue. The images show that the unfilled lesions have few osteoblastic cells 7 and 15 days after the intervention. After 30 days, we detect a few osteoblastic cells at the edge of the area delimiting the bone lesion.

[0221] In the case of composite matrices and Collapat®, osteocalcin is detected from the 15 eme day, and its presence is proven at 30 eme and 60 emedays, confirming the presence of osteoblastic cells in the newly formed tissues.

[0222] To assess vascularization and innervation in condylar lesions, we performed an analysis of vascular (Endomucin, Podocalyxin, and Meca32) and neuronal (P III tubulin) markers using the so-called whole bone transparency technique. This analysis allowed us to visualize the organization of vascular and neuronal networks in the initially injured area. We used multiple labeling to facilitate visualization of thick samples, as is the case for whole bones (Kirst et al. 2020, Cell 180, 780-795, e25).

[0223] 2D sections of the lesion filled with the composite matrix containing 2% HA were analyzed after 30 days of implantation. The bone tissue appears in green thanks to its autofluorescence, the vascular networks are in cyan blue and the neuronal extensions in red. 2D images obtained for each condition, 15 and 30 days after implantation were also analyzed: unfilled lesions, lesions filled with composite matrices or with Collapat®.

[0224] In one lesion that was not filled, a vascular network developed. It appeared to originate primarily from peripheral muscle tissue. After 30 days, we detected blood vessels and nerve structures within the injured area. However, histological analysis by TM staining showed that the injured part had been filled with a soft, disorganized, collagen-rich tissue.

[0225] For lesions filled with composite matrices, from the fifteenth day we observed the formation of a network of blood vessels throughout the matrix.

[0226] In contrast, for defects filled with Collapat®, vascularization was mainly present at the external periphery of the defect. Moreover, this vascularization was only visible from the 30 eme day following implantation. These qualitative results therefore show that the Collapat® matrix is ​​less effective than our composite matrix in promoting implant vascularization.

[0227] Regarding innervation, although the majority of fibers are present in the periosteum covering the surface of the cortical bone, some fibers can be seen inside the lesions for defects not filled or filled with the composite matrix. In contrast, no nerve fibers were detected when the condylar defects had been filled with Collapat®.

[0228] IL Implantation of composite matrix in a mandibular defect

[0229] To evaluate the use of the developed material for craniofacial bone reconstruction, we chose to create a mandibular bone defect model. Among the bones of the skull, only two are mobile: the hyoid bone and the mandible. The mandible has the advantage of being a larger and easily accessible bone.

[0230] The mandibular defect also offers the advantage, compared to calvaria defects classically used to test bone repair materials, of being subjected to high mechanical stresses. These mechanical stresses are necessary for effective bone repair. In the mandible, the stresses are mainly related to chewing. We therefore chose this mandibular lesion model to test bone repair materials, because it is more suited to physiological reality than the calvaria defect.

[0231] To validate this model, a preliminary experiment was conducted. A circular defect of 3 mm diameter was created, then its repair was monitored by micro-CT. After 3 months, no bone repair was observed, indicating that the defect created was indeed of critical size.

[0232] Preliminary micro-CT observations were performed at different times after implantation (7, 15, 30 days) to monitor the mineralization of mandibular defects. The 3D images of the micro-CT reconstructed mandibles filled with the composite matrix are shown in Figure 15.

[0233] A partial reconstruction at the periphery of the defect is observed at 30 eme day. The quantification of MV / TV was 0.037 ± 0.010 on day 7 and 0.040 ± 0.018 on day 15, therefore comparable. At 30 eme day that we measured an increasing MV / TV ratio (0.094 ± 0.049).

[0234] TM stainings of histological sections obtained after 15 days of implantation of the composite matrix were analyzed.

[0235] Interestingly, we observed at the junction of bone and matrix, an area highlighting the presence of newly formed bone tissue with the presence of bordering osteoblastic cells, and osteocyte cells within a bone structure that appears lamellar. Blood vessels were also seen in the newly formed tissue.

[0236] Furthermore, if we consider the reconstruction of the tissue from the periphery to the center of the lesion, it is important to note the tissue invasion which is accompanied by vascularization but also by the formation of bone tissue in contact with the matrix, confirming their osteoconductive potential.

[0237] Example 4: Conclusion on in vivo studies

[0238] In conclusion, different composite materials were tested in vivo in small animals in ectopic and orthotopic sites.

[0239] All tested matrices integrated well into the surrounding tissues and did not induce a major inflammatory reaction. We also showed that the HA-containing matrices induced mineralization. Vascular structures invaded the implants, demonstrating their angiogenic potential. The matrices' ability to stimulate innervation was also demonstrated by the presence of nerves at the periphery of the material, particularly in the early stages following implantation.

[0240] The matrix containing 2% HA was selected for its ability to promote mineralization as well as for its angiogenic potential. It was implanted into condylar and mandibular bone defects.

[0241] Bone reconstruction was improved after implantation of composite matrices in a condylar defect compared to what occurred in an unfilled defect. Organized bone structures expressing osteocalcin were identified in these hydrogels. An organized vascular network developed throughout the bone lesion, which was not the case for the Collapat® control material. The results obtained in the mandibular defect model show very satisfactory results on the osteoconductive and angiogenic properties of the composite matrices. Histological analyses showed that the materials integrated well. Bone and vascular structures were found after 15 days of implantation. These results therefore demonstrate the therapeutic interest of the composite matrices according to the invention.

Claims

DEMANDS 1. Composite material comprising: at least one elastin-type peptide, at least one bioactive peptide, at least one peptide capable of inducing calcium phosphate nucleation; and calcium phosphate.

2. Composite material according to claim 1, the elastin-type peptide comprising at least one alkenylated residue prior to formation of the composite material.

3. Composite material according to claim 1 or 2, the elastin-type peptide having the formula MW[VPGVGVPGMG(VPGVG)2] X in which x is an integer between 15 and 25, in particular between 19 and 21, x being more particularly equal to 20, and whose methionine residues are alkenylated before the formation of the composite material.

4. Composite material according to any one of claims 1 to 3, the elastin-type peptide being the ELPM80 peptide of formula MW[(VPGVG)(VPGMaG)(VPGVG)2]2o where Ma represents the alkenylated methionine residue of formula (III): before the composite material is formed.

5. Composite material according to any one of claims 1 to 4, said at least one bioactive peptide being an adhesion peptide capable of recruiting cells of interest.

6. Composite material according to any one of claims 1 to 5, said at least one bioactive peptide being an adhesion peptide of formula IKVAV-GGG-PVGLIG-GGG-YIGSR.

7. Composite material according to any one of claims 1 to 6, the peptide capable of inducing calcium phosphate nucleation being a peptide of sequence DSSEEKLFRRIGRFG 8. Composite material according to any one of claims 1 to 6, the peptide capable of inducing calcium phosphate nucleation being a peptide of sequence DDDEEKFLRRIGRFG.

9. Composite material according to any one of claims 1 to 8, calcium phosphate corresponding to hydroxyapatite.

10. Composite material according to claim 9, comprising between 1 and 3% (w / v) of hydroxyapatite.

11. Composite material according to claim 9, comprising 2% (w / v) of hydroxyapatite.

12. Composite material according to any one of claims 1 to 11, for its use in promoting innervation, osteogenesis and angiogenesis.

13. Composite material according to any one of claims 1 to 11, for use in controlling bone development.

14. Composite material according to any one of claims 1 to 11, for use in bone tissue repair.

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